ALLIUM, CUCUMIS, AND DAUCUS GERMPLASM ENHANCEMENT, GENETICS, AND BIOCHEMISTRY

Goals / Objectives
Objective 1: Continue research to characterize Daucus, Allium, and Cucurbit germplasms for diversity and beneficial traits. Objective 2: Continue research to determine the genetic basis of, and initiate or continue selection for, carrot, onion, and cucumber quality attributes influencing human nutrition and health (genetics of pigment bioavailability in carrot and onion and antiplatelet activity and fructan synthesis in onion) and flavor (carrot terpenoid and carbohydrate genetics and onion pungency). Objective 3: Continue research to determine the genetic basis of, and initiate or continue selection for, disease resistances in carrot, onion, and cucumber, including root-knot nematodes and Alternaria leaf blight in carrot, smut in onion, and potyviral resistances in cucumber and other cucurbits. Objective 4: Continue research to determine the genetic basis of yield components including multiple lateral branching in cucurbits and performing yield trials for carrot, onion, and cucumber. Objective 5: Continue or initiate biotechnology research in Daucus, Allium, and Cucurbit germplasms and development of genetic stocks including map construction using candidate genes, SCARs, SNPs, cytogenetic stocks, and the organellar genomes; genomics and fine mapping of pigment and carbohydrate genetics in carrot and onion, nematode resistance genes in carrot, and epistasis and yield in cucumber; marker-assisted selection of carrot nematode resistance, onion male sterility, and cucumber yield; and transgene escape in Cucurbits.

Project Methods
Continue research to characterize Daucus, Allium, and Cucurbit germplasms for diversity and beneficial traits for the development for improved germplasms and management strategies for genetic resources. Continue research to determine the genetic basis of, and initiate or continue selection for, carrot, onion, and cucumber quality attributes influencing human nutrition and health (genetics of pigment bioavailability in carrot and onion and antiplatelet activity and fructan synthesis in onion) and flavor (carrot terpenoid and carbohydrate genetics and onion pungency). Continue research to determine the genetic basis of, and initiate or continue selection for, disease resistances in carrot, onion, and cucumber, including root-knot nematodes and Alternaria leaf blight in carrot, smut in onion, and potyviral resistances in cucumber and other cucurbits. Continue research to determine the genetic basis of yield components including multiple lateral branching in Cucurbits and performing yield trials for carrot, onion, and cucumber. Continue or initiate biotechnology research in Daucus, Allium, and Cucurbit germplasms and development of genetic stocks including map construction using candidate genes, SCARs, SNPs, and the organellar genomes; genomics and fine mapping of pigment and carbohydrate genetics in carrot and onion, nematode resistance genes in carrot, and epistasis and yield in cucumber; marker-assisted selection of carrot nematode resistance, onion and carrot male sterility, and cucumber yield; cucurbit organellar genetics; transgene escape in Cucurbits; and transgene escape in Cucurbits. BSL-1; Recertified through June 7, 2009. Certificate #SC06-116R.

Progress 07/03/03 to 03/11/08

Outputs
Progress Report Objectives (from AD-416) Objective 1: Continue research to characterize Daucus, Allium, and Cucurbit germplasms for diversity and beneficial traits. Objective 2: Continue research to determine the genetic basis of, and initiate or continue selection for, carrot, onion, and cucumber quality attributes influencing human nutrition and health (genetics of pigment bioavailability in carrot and onion and antiplatelet activity and fructan synthesis in onion) and flavor (carrot terpenoid and carbohydrate genetics and onion pungency). Objective 3: Continue research to determine the genetic basis of, and initiate or continue selection for, disease resistances in carrot, onion, and cucumber, including root-knot nematodes and Alternaria leaf blight in carrot, smut in onion, and potyviral resistances in cucumber and other cucurbits. Objective 4: Continue research to determine the genetic basis of yield components including multiple lateral branching in cucurbits and performing yield trials for carrot, onion, and cucumber. Objective 5: Continue or initiate biotechnology research in Daucus, Allium, and Cucurbit germplasms and development of genetic stocks including map construction using candidate genes, SCARs, SNPs, cytogenetic stocks, and the organellar genomes; genomics and fine mapping of pigment and carbohydrate genetics in carrot and onion, nematode resistance genes in carrot, and epistasis and yield in cucumber; marker- assisted selection of carrot nematode resistance, onion male sterility, and cucumber yield; and transgene escape in Cucurbits. Approach (from AD-416) Continue research to characterize Daucus, Allium, and Cucurbit germplasms for diversity and beneficial traits for the development for improved germplasms and management strategies for genetic resources. Continue research to determine the genetic basis of, and initiate or continue selection for, carrot, onion, and cucumber quality attributes influencing human nutrition and health (genetics of pigment bioavailability in carrot and onion and antiplatelet activity and fructan synthesis in onion) and flavor (carrot terpenoid and carbohydrate genetics and onion pungency). Continue research to determine the genetic basis of, and initiate or continue selection for, disease resistances in carrot, onion, and cucumber, including root-knot nematodes and Alternaria leaf blight in carrot, smut in onion, and potyviral resistances in cucumber and other cucurbits. Continue research to determine the genetic basis of yield components including multiple lateral branching in Cucurbits and performing yield trials for carrot, onion, and cucumber. Continue or initiate biotechnology research in Daucus, Allium, and Cucurbit germplasms and development of genetic stocks including map construction using candidate genes, SCARs, SNPs, and the organellar genomes; genomics and fine mapping of pigment and carbohydrate genetics in carrot and onion, nematode resistance genes in carrot, and epistasis and yield in cucumber; marker-assisted selection of carrot nematode resistance, onion and carrot male sterility, and cucumber yield; cucurbit organellar genetics; transgene escape in Cucurbits; and transgene escape in Cucurbits. Significant Activities that Support Special Target Populations Germplasm was collected and evaluation of phenotypic and molecular relationshiops was made of Allium Cucumis, and Daucus germplasm, and reported in Germplasm Resources Information Network (GRIN). Melon diversity groups were identified, garlic variation was characterized with markers, and germplasm with unique pigment and carbohydrate profiles was released of onion and carrot. Field evaluation of carrot, onion, and cucumber breeding stocks and experimental hybrids developed by this program was carried out in CA, MI, OR, WA, and WI. Genetic resistance for nematodes and alternaria in carrots, and multiple viruses in cucumber was identified. Identification of QTL for cucumber yield and carrot nematode resistance and sugar type were identified and applied with marker-assisted selection (MAS). Genetic models explaining variation in onion carbohydrates and pungency; carrot carbohydrates, carotenoids, and anthocyanins; and cucumber plant architecture and fruiting were developed. Linkage maps for onion, cucumber, melon, and carrot were enriched several-fold during the course of this project. Unique plant breeding germplasm and methods useful for plant breeders were developed including onion haploids, cucumber mitochondrial mutants and interspecific crosses, and carrot transposable elements and a transposon tagging system. Genomic information was derived from expressed sequence tags (ESTs), bacterial artificial chromosomes (BACs), select tag sequences (STSs), and synteny evaluation. This research is relevant to the NP 301 Action Plan, Component 2, Problem Statement 2C: Genetic Analyses and Mapping of Important Traits; and Component 3, Problem Statement 3B: Capitalizing on Untapped Genetic Diversity. Significant Activities that Support Special Target Populations Efforts were made attracting under graduate and graduate minorities to the agricultural sciences by participating in annual meetings of the Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) through mentoring and career counseling (ongoing effort started in 1993). Assistance is given for judging student presentations oral and poster sessions, and support is provided to USDA, ARS recruitment in the form of poster displays, organized workshops and symposia, and provided mentoring for undergraduate and graduate students. Symposia, workshops, and field trips have been regularly organized. In 2007, a leadership workshop was conducted relating to team-building in science. These are the first such USDA, ARS-promoted activities at SACNAS (the most recognized society for minority scientists). SACNAS recruiting activities have led to attraction of minority students and funding (competitive minority-based fellowship) for graduate education [3 Ph.D. and 1 M.S. candidates] in Plant Genetics and Plant Breeding (PGPB) curriculum at the University of Wisconsin. These are the first minority graduate students to receive training in horticulture PGPB. Likewise, similar programs have been developed in the location for training of elementary school students to encourage their consideration of agricultural sciences as a career target.

Impacts
(N/A)

Publications

Peloquin, S.J., Boiteux, L.S., Simon, P.W., Jansky, S.H. 2008. A chromosome-specific estimate of transmission of heterozygosity by 2n gametes in potato. Journal of Heredity. 99(2):177-181.

Progress 10/01/06 to 09/30/07

Outputs
Progress Report Objectives (from AD-416) Objective 1: Continue research to characterize Daucus, Allium, and Cucurbit germplasms for diversity and beneficial traits. Objective 2: Continue research to determine the genetic basis of, and initiate or continue selection for, carrot, onion, and cucumber quality attributes influencing human nutrition and health (genetics of pigment bioavailability in carrot and onion and antiplatelet activity and fructan synthesis in onion) and flavor (carrot terpenoid and carbohydrate genetics and onion pungency). Objective 3: Continue research to determine the genetic basis of, and initiate or continue selection for, disease resistances in carrot, onion, and cucumber, including root-knot nematodes and Alternaria leaf blight in carrot, smut in onion, and potyviral resistances in cucumber and other cucurbits. Objective 4: Continue research to determine the genetic basis of yield components including multiple lateral branching in cucurbits and performing yield trials for carrot, onion, and cucumber. Objective 5: Continue or initiate biotechnology research in Daucus, Allium, and Cucurbit germplasms and development of genetic stocks including map construction using candidate genes, SCARs, SNPs, cytogenetic stocks, and the organellar genomes; genomics and fine mapping of pigment and carbohydrate genetics in carrot and onion, nematode resistance genes in carrot, and epistasis and yield in cucumber; marker- assisted selection of carrot nematode resistance, onion male sterility, and cucumber yield; and transgene escape in Cucurbits. Approach (from AD-416) Continue research to characterize Daucus, Allium, and Cucurbit germplasms for diversity and beneficial traits for the development for improved germplasms and management strategies for genetic resources. Continue research to determine the genetic basis of, and initiate or continue selection for, carrot, onion, and cucumber quality attributes influencing human nutrition and health (genetics of pigment bioavailability in carrot and onion and antiplatelet activity and fructan synthesis in onion) and flavor (carrot terpenoid and carbohydrate genetics and onion pungency). Continue research to determine the genetic basis of, and initiate or continue selection for, disease resistances in carrot, onion, and cucumber, including root-knot nematodes and Alternaria leaf blight in carrot, smut in onion, and potyviral resistances in cucumber and other cucurbits. Continue research to determine the genetic basis of yield components including multiple lateral branching in Cucurbits and performing yield trials for carrot, onion, and cucumber. Continue or initiate biotechnology research in Daucus, Allium, and Cucurbit germplasms and development of genetic stocks including map construction using candidate genes, SCARs, SNPs, and the organellar genomes; genomics and fine mapping of pigment and carbohydrate genetics in carrot and onion, nematode resistance genes in carrot, and epistasis and yield in cucumber; marker-assisted selection of carrot nematode resistance, onion and carrot male sterility, and cucumber yield; cucurbit organellar genetics; transgene escape in Cucurbits; and transgene escape in Cucurbits. Significant Activities that Support Special Target Populations Objectives include continued multidisciplinary efforts to develop and evaluate multiple disease resistant cucumber hybrids and inbred lines, and identify appropriate cultural practices for genotypes with unique vining and fruiting habits. Evaluation of Cucumis and Cucurbita germplasm for diversity will continue to facilitate the construction of core collections, the enhancement of germplasm management methodologies, the identification of tolerance to suboptimal conditions (i.e., water and heat stress), and the development of efficient breeding methodologies. Continued progress will be made to identify linkage associations between molecular markers and economically important traits in order to identify technologies which will increase the efficiency of cucumber breeding. This will require the identification, inheritance and mapping of traits for disease resistance and fruit yield and quality using isozymes, restriction fragment length polymorphisms (RFLPs), DNA fragments obtained from random amplification polymorphism detection (RAPDs), amplified fragment length polymorphisms (AFLPs), single sequence repeats (SSRs), and single nucleotide polymorphisms (SNP) technologies. These markers will be used to more clearly understand the physiology and genetics of horticulturally important characters in cucumber and melon to allow for more efficient ways to develop lines and hybrids for commercial production. Such increase will shorten time for hybrid development to reduce development costs and increase grower competitiveness. Customers are: Pickle Packers International, Inc., Midwest Pickle Packers, Vegetable Seed Companies, and Public Research Scientists. Accomplishments Accomplishment: Phenotypic selection with subsequent marker-assisted selection for line extraction in cucumber. The genetic map position of quantitative trait loci (QTL) conditioning of quantitatively inherited yield component traits is known, and linked molecular markers may have utility in marker-assisted selection (MAS) programs to increase selection efficiency and effectiveness. This project was designed to determine if gain from selection was realized after MAS and, if so, whether there was concomitant changes in frequencies of markers linked to QTL associated with those traits under selection. Marker-assisted backcrossing produced families for comparative analysis of gain from selection by phenotypic and marker-assisted selection. Similar gain from selection was detected as a result of phenotypic and MAS selection for branching (~0.3 branches/cycle), and fruit length (~0.1 unit increase/cycle) with concomitant changes in frequency at linked marker loci. Although genetic gain was not realized for femaleness during phenotypic selection, the percentage of female flowers of plants subjected to MAS was increased (5.6 to 9.8% per cycle) depending upon the BC1 population examined. Selection-dependent changes in frequency were also detected at marker loci linked to female sex expression during MAS. MAS operated to fix favorable alleles that were not exploited by phenotypic selection in this population, indicating that MAS could be applied for altering plant architecture in cucumber to improve its yield potential. Such information would allow for the development of strategies for more efficient incorporation of exotic traits into commercial cucumber germplasm. Research supports NP #301 by furnishing plant breeding information and genetic raw materials to create new plant types (new uses) to enhance American agricultural productivity. Accomplishment: Variance component analysis of parthenocarpy in elite U. S. processing type cucumber lines. Parthenocarpic (seedless) U.S. processing type cucumber germplasm can bear more high quality fruit when compared to their seeded counterparts. The inheritance of parthenocarpy in this market type is not fully understood, and thus understanding genetic components of variation would assist cucumber breeders when incorporating this economically important trait into commercial varieties. The inheritance of parthenocarpy in elite U.S. processing type cucumber was investigated in F3 progeny derived from a mating between parthenocarpic lines and non-parthenocarpic inbred lines. A variance component analysis was applied to once-over harvest parthenocarpic yield data collected from F3 generation at two locations in Wisconsin. There was a difference in the relative importance of additive genetic variance compared to dominance genetic variance across growing environments. The minimum number of effective factors controlling parthenocarpy was estimated to be between 5 to 13 genes. While narrow-sense heritability of individual plants within F3 family ranged from 0.02 to 0.07, broad- sense heritability of individual plants within F3 family ranged from 0.07 to 0.09. The environmental variance accounted for about 90% of total phenotypic variance in both locations. In contrast, narrow-sense heritability estimates based on F3 family mean performance ranged between 0.33 and 0.62, and broad-sense heritability ranged between 0.53 and 0.67. These results provide for the development and implementation of breeding strategies for the efficient and effective incorporation of parthenocarpy into commercial cultivars. More specifically, advanced generation selection for parthenocarpy based on F3 family mean performance will be more effective than selection of individual plants within F3 family. Research supports NP #301 by furnishing plant breeding information and genetic raw material to create new plant types (new uses) to enhance American agricultural productivity. Accomplishment: Identification of quantitative trait loci (QTL) associated with parthenocarpy in processing cucumber. Parthenocarpy (seedless fruit) is an economically important yield- related trait in cucumber. However, the genomic locations of factors controlling parthenocarpic fruit development in this species are not known. Therefore, an F2:3 mating design was utilized to map quantitative trait loci (QTLs) for parthenocarpy using a narrow cross employing two gynoecious, indeterminate, and normal leaf lines. Single-marker analysis, simple interval mapping, composite interval mapping, a two-QTL model, and bulk segregant analysis were employed for QTL detection, comparison, and confirmation. QTL detection was performed using the parthenocarpic yield of 120 F3 families grown at two locations in Wisconsin. There were ten QTLs for parthenocarpy detected; seven operating in one location and three in the other. Four of seven QTL in Linkage Groups (LG) 1 and 4 and one of three QTL in LG 1 detected by QTL mapping analysis were confirmed by BSA using E-block and G-block data, respectively. Three genomic regions conditioning parthenocarpic QTLs were also mapped to the same genomic regions as QTLs detected for fruit yield at first-harvest as reported in a previous study. These results provide for the development and implementation of breeding strategies for the efficient and effective incorporation of parthenocarpy into commercial cultivars. More specifically, the eight molecular markers linked to parthenocarpy through QTL mapping defined herein are candidates for use in marker-assisted selection programs where breeding for increased levels of parthenocarpy is an objective in the elite processing cucumber populations. Research supports NP #301 by furnishing genomic information and genetic raw materials to create new plant types (new uses) to enhance American agricultural productivity. Accomplishment: Sequencing of cucumber chloroplast genome to identify putative candidate genes for chilling tolerance. Chilling temperature stress (above 0 deg C but lower than 12 deg C) is a major abiotic stress in cucumber (Cucumis sativus L.) during vegetative and reproductive growth. Chilling temperature response in cucumber is maternally inherited. Previous results suggest that chloroplast and not mitochondrial factors are responsible for chilling tolerance in cucumber, and that chilling tolerance is most likely controlled by gene(s) located in the chloroplast genome, or perhaps in the cpDNA itself. It would be useful to identify candidate genes for chilling in cucumber and thus an experiment was designed to sequence the chloroplast genome of cucumber and identify candidate cucumber chloroplast genomic regions associated with chilling tolerance using this methodology. Chloroplast sequencing detected sequence differences at three cpDNA sites and these sites were also detected in a genetically diverse array of cucumber germplasm with differing chilling responses. These and previously reported results suggest that one or several of these sequences could be responsible for the observed response to chilling injury in cucumber. This study identified candidate genes for chilling that if confirmed could have value for further elucidating chilling resistance factors in cucumber and thereby increasing the breeding efficiency and effectiveness for this trait. Research supports NP #301 by furnishing genomic information to create new plant types (new uses) to enhance American agricultural productivity. Accomplishment: Rice has been proposed as a genomic model for the monocots. Sequencing of bacterial artificial chromosomes (BACs) carrying asparagus DNA and comparisons with rice DNA revealed essentially no similarities, indicating that the grasses are not appropriate models for other major groups of monocots. Accomplishment: The inheritance of orange carotenoid pigments has not been well-characterized, so this study evaluated heritability of that trait and important candidate genes. Heritability of carrot carotenoid content was determined and specific biosynthetic genes were mapped and sequenced. This combines phenotypic and molecular genetic evaluations of this important trait. Carrot carotenes are the single largest source of dietary vitamin A from vegetables in the U.S. diet and with this knowledge carrot breeders will be able to more efficiently select for improved nutritional quality and better understand mechanisms of carotenoid biosynthesis. This study provides a foundation for further genetic study and more efficient breeding of this trait. Significant Activities that Support Special Target Populations Efforts were made attracting under graduate and graduate minorities to the agricultural sciences by participating in annual meetings of the Society for the Advancement of Chicanos and Native Americans in Science (SACNAS) through mentoring and career counseling (ongoing effort started in 1993). Assistance is given for judging student presentations oral and poster sessions, and support is provided to USDA, ARS recruitment in the form of poster displays, organized workshops and symposia, and provided mentoring for undergraduate and graduate students. Symposia (3; genetics/genomics, reclimation, career opportunities in the USDA, ARS) and workshops (4; grant writing, leadership development), and field trips (1; ARS National Water and Cotton Laboratories) have been regularly organized. In 2007, a leadership workshop was conducted relating to team- building in science. These are the first such USDA, ARS-promoted activities at SACNAS (the most recognized society for minority scientists) . SACNAS recruiting activities have led to attraction of minority students and funding (competitive minority-based fellowship) for graduate education [3 Ph.D. (1 graduated) and 1 M.S. (graduated) candidates] in Plant Genetics and Plant Breeding curriculum at the University of Wisconsin. These are the first minority graduate students to receive training in horticulture PGPB. Likewise, similar programs have been developed in the location for training of elementary school students to encourage their consideration of agricultural sciences as a career target. Technology Transfer Number of Invention Disclosures submitted: 4 Number of Non-Peer Reviewed Presentations and Proceedings: 6 Number of Newspaper Articles,Presentations for NonScience Audiences: 5

Impacts
(N/A)

Publications

Zalapa, J.E., Staub, J.E., McCreight, J.D. 2006. Generation means analysis of plant architectural traits and fruit yield in melon (Cucumis melo l.). Plant Breeding. 125:482-487.
Staub, J.E., Sun, Z., Chung, S., Lower, R.L. 2007. Colinearity among genetic linkage maps in cucumber. HortScience. 40:20-27.
Zalapa, J., Staub, J.E., McCreight, J.D. 2007. Mapping and QTL analysis of plant architecture and fruit yield in melon. Theoretical and Applied Genetics. 114:1185-1201.
Sun, Z., Lower, R.L., Staub, J.E. 2006. Identification and comparative analysis of quantitative trait loci (QTL) associated with parthenocarpy in processing cucumber (Cucumis sativus l.). Plant Breeding. 125:281-287.
Gokce, A., Havey, M.J. 2005. Selection favors the dominant MS allele in open pollinated onion populations possessing S-Cytoplasm. Genetic Resources and Crop Evolution. 53:1495-1499. Available: http://www. springerlink.com/(hb4hbqzcpsbiyi45hb1lw145)/app/home/contribution.asp? referrer=parent&backto=issue,81,87;searcharticlesresults,2,2;
Jakse, J., Suzuki, G., Cheung, F., Town, C.D., Mccallum, J., Havey, M.J. 2006. Comparative-sequence and genetic analyses of asparagus reveal no microsynteny with rice or onion. Journal of Theoretical and Applied Genetics. 114:31-39. Available: http://www.springerlink. com/content/h3617u2315348n08/?p=9d65c800a75a46d8ad2ce2053a432b43&pi=15.
Santos, C., Simon, P.W. 2006. Heritabilities and minimum gene number estimates of carrot carotenoids. Euphytica. 151:79-86.
Porter Dosti, M., Mills, J.P., Simon, P.W., Tanumihardjo, S.A. 2006. Bioavailability of beta-carotene (betaC) from purple carrots is the same as typical orange carrots while high-betaC carrots increase betaC stores in Mongolian gerbils (Meriones unguiculatus). British Journal of Nutrition. 96(2):260-269.
Simon, P.W., Goldman, I.L. 2007. Carrot. In: Singh, R.J. Genetic Resources, Chromosome Engineering, and Crop Improvement Series, Volume 3. Boca Raton: CRC Press. p. 497-517.
Ipek, M., Ipek, A., Simon, P.W. 2006. Sequence homology of polymorphic AFLP markers in garlic (Allium sativum l.). Genome. 49:1246-1255.
Grzebelus, D., Jagosz, B., Simon, P.W. 2007. The DcMaster Transposon Display maps polymorphic insertion sites in the carrot (Daucus carota L.) genome. Gene. 390:67-74.
Cavagnaro, P.F., Camargo, A., Galmarini, C.R., Simon, P.W. 2007. Effect of cooking on garlic (Allium sativum) antiplatelet activity and thiosulfinates content. Journal of Agricultural and Food Chemistry. 55:1280-1288.
Zhuang, F., Chen, J., Staub, J.E., Qian, C. 2006. Taxonomic relationships of a rare cucumis species (C. hystrix chakr.) and its interspecific hybrid with cucumber. HortScience. 41:571-574.
Chung, S., Staub, J.E., Chen, J. 2006. Molecular phylogeny of cucumis species as revealed by ccSSR marker length and sequence variation. Genome. 49:219-229.
Chung, S., Decker-Walters, D.S., Staub, J.E. 2006. Cultivar to wild population introgression in Cucurbita pepo subsp. ojiegra. Journal of New Seeds. 8:1-18.
Chen, J., Ren, G., Luo, X., Staub, J.E., Jahn, M. 2006. Inheritance of aspartate aminotransferase (AAT) in cucumis species as revealed by interspecific hybridization. Canadian Journal of Botany. 84:1503-1507.
Chung, S., Gordon, V., Staub, J.E. 2006. Sequencing of cucumber (Cucumis sativus l.) chloroplast genomes identifies putative candidate genes for chilling tolerance. Genome. 50:215-225.
Ipek, A., Masson, P., Simon, P.W. 2006. Genetic transformation of an Ac/Ds - based transposon tagging system in carrot (Daucus carota). European Journal of Horticultural Science. 71:245-251.
Just, B.J., Santos, C., Boiteux, L.S., Oloizia, B.B., Simon, P.W. 2006. Carotenoid biosynthesis structural genes in carrot (Daucus carota): isolation, sequence-characterization, single nucleotide polymorphism (SNP) markers and genome mapping. Journal of Theoretical and Applied Genetics. 114:693-704.
Mccallum, J., Clarke, A., Sheffer, J., Sims, I., Heusden, S., Shigyo, M., Havey, M.J. 2006. Genetic mapping of a major gene affecting onion bulb fructan content. Journal of Theoretical and Applied Genetics. 112:958-967.
Abou-Jawdah, S.Y., Havey, M.J. 2006. Tolerance in cucumber to cucurbit yellow stunting disorder virus. Plant Disease. 90:645-649.
Zicheng, F., Robbins, M.D., Staub, J.E. 2006. Population development by phenotypic selection with subsequent marker-assisted selection for line extraction in cucumber (Cucumis sativus l.). Journal of Theoretical and Applied Genetics. 112:843-855.
Sun, Z., Lower, R.L., Staub, J.E. 2006. Variance component analysis of parthenocarpy in elite U.S. processing type cucumber (Cucumis sativus l.) lines. Euphytica. 148:333-341.
Sun, Z., Lower, R.L., Staub, J.E. 2006. Analysis of generation means and components of variance for parthenocarpy in cucumber (Cucumis sativus l.). Plant Breeding. 125:277-280.

Progress 10/01/05 to 09/30/06

Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is aligned with National Program 301, Plant Genetic Resources, Genomics and Genetic Improvement. Allium, carrot and cucurbit crops are important to the agriculture economy and more germplasm and information are needed to sustain production. This project will characterize germplasm of these crops, determine the genetics and select genetic stocks of important onion, carrot, and cucumber nutritional components, disease resistances, and yield components; and continue or initiate biotechnology research in Daucus, Allium, and Cucurbit germplasms. Superior inbreds and populations for producers and consumers will be released. This is the only USDA project focusing on germplasm enhancement and genetics of these important vegetable crops. Economical agricultural production of carrots, onions, and cucumbers requires new germplasm on an ongoing basis with improved traits, as well as genetic and other information to meet grower and consumer and research needs. Genetic and production information and new germplasm are urgently needed for these crops. We will identify and characterize molecular markers in cucumber and melon for diversity analysis to enhance germplasm management of these crops. The study of cucumber and melon genetics and physiology leading to the development of unique cultivars for use by the consuming public requires the use of classical plant breeding and plant genetic methods as well as the deployment of biotechnological tools where appropriate. Genetic mapping, trait inheritance, and application of this information for plant improvement are facilitated through classical and biotechnological methods. The application of this information in field and laboratory experiments with melon and cucumber results in an increase in the efficiency of cultivar development of these crops for the consuming public. Unique lines and populations have increased disease resistance, improved fruit yield and quality and tolerance to water stress. Many viruses infect cucumber and numerous sources of resistance are known. Breeding cucumber for resistance to many viruses is a time consuming and expensive process because of numerous independently inherited resistance loci, the need to inoculate plants with different viruses with similar phenotypes, and the cost of independently maintaining several viruses in plant materials. Cucumber is unique among the angiosperms in that it has one of the largest mitochondrial genomes among all plants, the mitochondria are paternally transmitted, and passage through cell culture produces unique rearrangments in the mitochondrial DNA conditioning a mosaic phenotype. Chemical inputs threaten the environment and profitability of carrots. Consumers rely on carrots as the single most important source of dietary vitamin A in the U.S. and for vegetables in their diet. Nematodes, alternaria leaf blight, and bacterial blight, generate significant production losses for carrot growers. Consumers would prefer better tasting, more nutritious carrots. General genetic information regarding carrots is not very extensive. Genetic mapping and inheritance patterns are being developed. Onion is a high value vegetable crop that is slow to breed by classical methods because of a long generation time. This project focuses research on development of superior male sterile inbred lines to be used by commercial seed companies to produce hybrid onion and technologies making onion breeding more difficult. For garlic, seed production and tissue culture are difficult, genetic transformation has not been successful and knowledge of genetics is minimal. Research to improve the techniques and knowledge of these areas is underway. Garlic breeding has only been possible for the last decade and progress toward solving production, disease, and quality problems is just underway. Without continued effort no improvements to garlic will be realized. 2. List by year the currently approved milestones (indicators of research progress) Milestones for Objective 1 The evaluation of germplasms will result in the identification of accessions with unique characteristics useful for germplasm enhancement, the description of phenotypic correlations between traits, and the assessment of genetic differences among adapted and exotic germplasms. The accessions identified and the accompanying information will result in the release of unique inbred lines and germplasm pools. The assessment of melon germplasms in the U.S. NPGS will allow for the development of a core collection and test arrays in melon. For carrots this will include assessment of ~ 150 entries of new germplasm from Europe in year 1,2, and 3; and release of disease resistant/high quality inbreds and populations in years 3 and 5. For onion, new low-pungency and red inbreds will be testcrossed and hybrid performance evaluated with release of new inbreds by year 5. For melon, the phenotypic (years 1 and 2) and molecular assessment (years 3 and 5) of the collection (2,185 accessions available) will be performed to form test arrays and a core collection. Milestones for Objective 2 The identification of genes conditioning nutritional value and flavor in these vegetable crops will provide the information and germplasm for increasing the farm value and improving the nutritional value of the food supply for consumers. For carrot this will include genes for carotenoid biosynthesis year 1, anthocyanin and sugar biosynthesis years 2-3, high pigment germplasm will be released years 4 and 5. For onion, evaluations for low pungency and high fructan content will occur in years 1 through 5 by selection of these traits in elite germplasms. For cucumber, the development of a high carotene parthenocarpic population (years 1 to 3), and the extraction of gynoecious lines for release years 4 and 5. Milestones for Objective 3 The identification of disease resistance in these vegetable crops will reduce the cost of production and pesticide applications. Because of our vegetable breeding programs, we are in excellent positions to incorporate these resistances from unadapted germplasms into elite genetic backgrounds for release to public and private vegetable breeders. For carrot this will include nematode resistance germplasm years 2 and 5; alternaria resistance germplasm years 3-4; and markers for resistance year 3. For onion, evaluations for smut resistance will occur in years 1 through 3. If resistance is identified, incorporation of resistance into elite germplasms will begin in years 4 and 5. For melon, the development and assessment of segregating populations (F2, BC, and F3) will be conducted to determine the linkage relationships between molecular markers and melon aphid resistance genes (years 1 to 3) as a precursor to studies of marker efficacy in marker-assisted selection experiments (years 4 and 5). Milestones for Objective 4 Stepwise recurrent selection and line development for yield components will result in improved cucumber and melon lines and germplasm pools. Line and germplasm pool development will require the fixation of quantitative trait loci controlling yield and quality components (milestone 1) and the subsequent evaluation of unique selections to determine the appropriate cultural conditions that allow for yield optimization (milestone 2). In addition, the determination of the genetics of water stress in cucumber will constitute a significant outcome (milestone 3). Populations of onions and carrots with improved yield will provide growers with higher economic returns. For carrot this will include field performance trials in California, Wisconsin, and Washington every year. For onion, inbreds and populations will be selected for low pungency and high quality, but because of the biennial generation time of onion progress will be slow over the 5 years. The evaluation of and phenotypic selection in melon and cucumber populations for improved yield to include plant reduced plant architecture, gynoecy, multiple lateral branching, and early flowering (years 1 to 3), and then the extraction and evaluation of lines in preparation for release (years 4 and 5). Milestones for Objective 5: The identification of molecular markers closely linked to major economically important loci in these vegetable crops will reduce the cost to develop elite inbred populations and germplasms. Haploid development avoids the time-consuming and labor-intensive inbreeding cycles and allows for the relatively quick production of completely homozygous inbred lines. For carrot this will include markers for pigments years 2 and 3, for nematode and alternaria resistance year 3 and 4; and transformation of pigment genes year 4. For onion, molecular markers revealing nuclear genotypes at the fertility-restoration locus and cytoplasms of onion will be developed in years 1 through 3 and released for use by year 5. The comparative analysis of phenotypic and marker- assisted selection schemes for multiple trait improvement in melon and cucumber populations developed by recurrent selection (years 1 to 5). 4a List the single most significant research accomplishment during FY 2006. This project is aligned with NP 301, Component II, Genomic Characterization and Genetic Improvement. Allium genetic markers. Most monocot genomics and genetic marker evaluation has been done in the grasses, such as rice and wheat, but not onion or other alliums. We completed the first comparative map between rice and onion, a monocot outside of the grasses. Scant collinearity of genetic markers was observed, indicating that the enormous genetic and genomic resources developed for the grasses are not directly applicable to other major monocots. Therefore, this research indicates that genomic resources, such as deep coverage libraries or large collections of expressed sequences, must be independently generated for major monocots outside of the grasses when studies of diverse germplasm are initiated. 4b List other significant research accomplishment(s), if any. Accomplishments are aligned with NP 301,Component II, Genomic Characterization and Genetic Improvement. Cucumber marker assisted selection. Cucumber yield improvement has been difficult with classical breeding approaches. We have used cucumber as a model species to evaluate the efficacy of marker-assisted selection (MAS) for yield component traits (i.e., flowering date, sex expression, multiple lateral branching, and fruit size) to determine that MAS can be used to increase selection efficiency during inbred line extraction. This is the first report of gain from selection for multiple quantitatively inherited traits in vegetable crops, and provides for methodological approaches which will improve breeding efficiency in a broad array of crop species. Cucurbit chloroplast genomic relationships. Very few intercrosses have been successful between cucumber and related cucurbits, and molecular markers reflecting genetic relationships among cucurbits have not been well-developed. Chloroplast markers developed by the USDA were used to examine the genetic relationships between melon (2n = 24), cucumber (2n = 14) and an exotic wild free-living species, Cucumis hystrix (2n = 24) which is cross compatible with cucumber, but not with melon. C. hystrix possesses genes for Gummy Stem Blight and Belly Rot Resistance which are not present in cucumber. Chloroplast markers indicate that C. hystrix is genetically closer to cucumber than melon, and thus provides information to allow for strategic crosses for the incorporation of economically important traits in cucumber. Onion germplasm markers. Tracking genetic stocks in onion breeding programs is difficult with visual examination and few molecular markers have been developed to facilitate this process. A set of codominant molecular markers was developed that confidently distinguish among onion germplasms and cultivars, which will be useful for maintenance of genetic diversity and quality control of onion-seed production. Segregation of these molecular markers was also demonstrated in garlic and onion, demonstrating their genetic bases. Asparagus-onion relationships. Asparagus is related to the Alliums, but the nature of the relationship is not well understood. Asparagus species have different amounts of DNA. European Asparagus species has twice the DNA of South-African Asparagus species. We undertook genetic and sequence analyses and demonstrated that the European species have not undergone a recent genome duplication as compared to related species with one-half the DNA. This is important because it means that vegetable asparagus may be a good genomic model for economically important plants in the Alliaceae (onion, garlic, leek, and chive) and agavaceae (agave and yucca) . Mobile genetic elements of carrot. Mobile genetic elements are known to be key genomic building blocks in many plants, but they have not been studied much in carrot. Two new families of transposable elements, DcMaster and DcMITEs, were identified in the carrot genome. Initial discovery of these transposons resulted from the observation of a naturally-occurring "knock-out" mutant in invertase activity in 1979. Two members of the DcMaster family were thoroughly characterized and differed by ~1.9 kb deletion. DcMaster-a, contains an open reading frame coding for a putative transposase with characteristics of plant class II transposable elements belonging to PIF/Harbinger superfamily. DcMaster is the first PIF/Harbinger-like element identified and characterized in any dicot species other than Arabidopsis thaliana. Under ten copies of the DcMaster element occur in genomes of carrot and other Apiaceae, but more copies with internal deletions or insertions may occur. DcMaster elements were associated with putative coding regions in several insertion sites. We speculate that these may all represent a MITE-like family of transposable elements that we named DcMITEs. To our knowledge this is the first report on identification of a novel MITE family based directly on sequence information from class II transposable elements. This basic knowledge expands our understanding of the carrot genome. Molecular basis of carrot Rs mutant. The Rs mutant of carrot controls the type of sugar stored in roots, but the tract has not been evaluated in breeding materials. The original "knock-out" mutation that we eventually attributed to the DcMaster element, called Rs, was able to be used to track this mutation in the USDA carrot breeding program with simple PCR-based plant evaluation at as early as 1-week-old seedlings. Among several thousand seedlings evaluated 100% accuracy was realized and heterozygotes could be distinguished from homozygotes in all cases where progeny testing was done. The codominant PCR-based markers developed are being used in carrot research programs for selection, for identifying seed mixtures, and for studying the distribution and origins of this trait in cultivated and wild carrots. 5. Describe the major accomplishments to date and their predicted or actual impact. Accomplishments are aligned with NP 301, Component II, Genomic Characterization and Genetic Improvement. Objectives include continued multidisciplinary efforts to develop and evaluate multiple disease resistant cucumber hybrids and inbred lines, and identify appropriate cultural practices for genotypes with unique vining and fruiting habits. Evaluation of Cucumis and Cucurbita germplasm for diversity will continue to facilitate the construction of core collections, the enhancement of germplasm management methodologies, the identification of tolerance to suboptimal conditions (i.e., water and heat stress), and the development of efficient breeding methodologies. Continued progress will be made to identify linkage associations between molecular markers and economically important traits in order to identify technologies which will increase the efficiency of cucumber breeding. This will require the identification, inheritance and mapping of traits for disease resistance and fruit yield and quality using isozymes, restriction fragment length polymorphisms (RFLPs), DNA fragments obtained from random amplification polymorphism detection (RAPDs), amplified fragment length polymorphisms (AFLPs), single sequence repeats (SSRs), and single nucleotide polymorphisms (SNP) technologies. These markers will be used to more clearly understand the physiology and genetics of horticulturally important characters in cucumber and melon to allow for more efficient ways to develop lines and hybrids for commercial production. Molecular marker systems are now being developed that will increase breeding efficiency. Such information will shorten time for hybrid development to reduce development costs and increase grower competitiveness. Customers are: Pickle Packers International, Inc., Midwest Pickle Packers, Vegetable Seed Companies, and Public Research Scientists. Numerous germplasm releases have been made to carrot growers for development of new carrots with improved production, less pesticide application and with improved flavor and nutritional value for consumers. New germplasm with nematode resistance and better quality provide expanded market opportunities. The understanding of carrot pigment genetics, disease resistance, genetic map, and germplasm diversity has been provided. New markers for sugar types, nematode resistance, orange and purple color, and male sterility are in use. Garlic seed production is now routinely possible because of this research. Garlic tissue culture and transformation are now feasible and genetic variation of the crop is better understood. Most, if not essentially all, hybrid onion cultivars grown in the U.S. are produced using USDA germplasm from this program. We continue to breed and develop superior germplasm and develop new technologies to make this process quicker and less costly. We identified differences in the DNA flanking the Ms locus, a major gene that determines whether male sterile inbreds can be developed from a population or family. To make these DNA differences useful in applied breeding programs, we converted these DNA differences to genetic markers revealed by the polymerase chain reaction (PCR). It now takes only hours, as opposed to years, to establish the cytoplasm and estimate the nuclear genotype of individual onion plants and improved markers are being developed and tested. We continued a major field and laboratory study to establish the genetic bases of correlated flavor, production, and health enhancing attributes of onion. These correlated traits appear to be under the control of genes conditioning the synthesis of inulins, which are complex carbohydrates with well established health benefits in humans. When onion bulbs synthesis inulins, they take up less water at maturity causing the concentration of carbohydrates, as well as the compounds associated with higher pungencies and blood thinning attributes. Plants and bulbs were scored for genetic attributes or selected for commercially acceptable types. We are in the final seed increase of the first male sterile and male fertile maintainer of a red inbred onion population to be released from the public sector in the US. The development of genomic resources for onion will be a major accomplishment, allowing breeders and geneticists to find target onion genes. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The investigations being carried out that seek to increase the yield and quality of cucumber and melon will positively effect growers, processors and consumers. These investigations are part of a broad research program to develop multiple disease resistant, high fruit number cucumber germplasm that is basic to the need of private industry and the consuming public. The release of multiple resistant germplasm decreases product wastage and the use of pesticides, thus directly improving profitability of growers and processors, the food safety of consumers, and harmony between agriculture and the environment. Data concerning cultural practices to optimize germplasm with unique fruiting and vining habits accompanies releases of germplasm to allow for optimized yield. Since many of the growers (~60%) reside in small rural communities and manage relatively small production areas (~1 to 25 acres), such information enhances their economic opportunity and quality of life. For instance, each year processors and growers evaluate commercial and experimental hybrids for fruit quality that were harvested from USDA replicated simulated machine and hand-harvest trials. This activity which includes from 15 to 30 individuals from the Midwest U.S. is hosted by the USDA. Attendees evaluate new lines and hybrids being considered for release to the public. This activity heightened the awareness of processors and growers to new technologies and varieties that would increase their competitiveness. The information obtained from diversity analyses used by the National Plant Germplasm System to increase its managerial efficiency and effectiveness. During the refinement of this germplasm, breeding methodologies will be developed to assist plant geneticists in the subsequent use of this or similar plant material, and provide information potentially useful to other cucurbit species. Properly identified and characterized biochemical markers will be used as tools to determine physiological processes of horticulturally important characters in onion, carrot, cucumber, and melon. Description and characterization of economically important physiological fruit disorders will lead to the establishment of better crop management practices, and thus decrease postharvest losses to increase profitability of growers and processors. Most, if not essentially all, hybrid onion cultivars grown in the U.S. are produced using USDA germplasm from this program. We continue to breed and develop superior germplasm and develop new technologies to make this process quicker and less costly. We identified differences in the DNA flanking the Ms locus, a major gene that determines whether male sterile inbreds can be developed from a population or family. To make these DNA differences useful in applied breeding programs, we converted these DNA differences to genetic markers revealed by the polymerase chain reaction (PCR). It now takes only hours, as opposed to years, to establish the cytoplasm and estimate the nuclear genotype of individual onion plants. Tagging of the Ms locus has provided the seed industry with a quick and cheap alternative to classical genetic studies to determine whether male sterile inbred lines can be developed from populations or families. We continued a major field and laboratory study to establish the genetic bases of correlated flavor, production, and health enhancing attributes of onion. These correlated traits appear to be under the control of genes conditioning the synthesis of inulins, which are complex carbohydrates with well established health benefits in humans. When onion bulbs synthesis inulins, they take up less water at maturity causing the concentration of carbohydrates, as well as the compounds associated with higher pungencies and blood thinning attributes. The genetic bases of fructan accumulation in onion will be important for breeders to select health-enhancing onion cultivars. Our research on the flavor, production, and health enhancing attributes of onion will provide basic genetic information imperative to the development of new value added onion hybrids for growers and consumers. Plants and bulbs were scored for genetic attributes or selected for commercially acceptable types. Numerous germplasm releases have been made to carrot growers for development of new carrots with improved production, less pesticide application and with improved flavor and nutritional value for consumers. The understanding of carrot pigment genetics, disease resistance, genetic mapping, and germplasm diversity has been provided. Garlic seed production is now routinely possible because of this research. Garlic tissue culture and transformation are now feasible and genetic ariation of the crop is better understood. These projects will enable the seed industry to react quickly to changes in consumer preferences, production environments, or exploit new markets.

Impacts
(N/A)

Publications

Kuhl, J.C., Sink, K.C., Havey, M.J., Martin, W.J., Cheung, F., Yuan, Q., Town, C.D., Landherr, L., Hu, L., Leebens-Mack, J. 2005. Comparative genomic analyses of asparagus officinalis L. Genome. 48:1052-1060.
Santos, C.A., Senalik, D.A., Simon, P.W. 2005. Path analysis suggests phytoene accumulation as the key step limiting the carotenoid pathway in roots of white carrot. Genetics and Molecular Biology. 28:287-293.
Simon, P.W. 2005. Realizing value from Central Asian Allium germplasm collections. HortScience. 40:309-310.
Grzebelus, D., Yau, Y., Simon, P.W. 2006. Master - a novel family of PIF/Harbinger-like transposable elements identified in carrot (Daucus carota L.). Molecular Genetics and Genomics. 275:450-459.
Baranski, R., Baranska, M., Schulz, H., Simon, P.W., Nothnagel, T. 2006. Single seed Raman measurements allow taxonomical discrimination of Apiaceae accessions collected in gene banks. Biopolymers. 81:497-505.
Jakse, J., Mccallum, J., Martin, W., Havey, M.J. 2005. Single nucleotide polymorphisms, indels, and simple sequence repeats for onion cultivar identification. Journal of the American Society for Horticultural Science. 130:912-917.
Nam, Y., Lee, J., Song, K., Lee, M., Staub, J.E. 2006. Construction of two BAC libraries from cucumber (Cucumis sativus L.) and identification of clones linked to yield component quantitative trait loci. Theoretical and Applied Genetics. 111:150-161.
Zewdie, Y., Havey, M.J., Prince, J.P., Jenderek, M.M. 2005. The first genetic linkages among molecular markers and morphological traits in garlic (allium sativum l.). Journal of the American Society for Horticultural Science. 130(4):569-574.

Progress 10/01/04 to 09/30/05

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Allium, carrot and cucurbit crops are important to the agriculture economy and more germplasm and information are needed to sustain production. This project will characterize germplasm of these crops, determine the genetics and select genetic stocks of important onion, carrot, and cucumber nutritional components, disease resistances, and yield components; and continue or initiate biotechnology research in Daucus, Allium, and Cucurbit germplasms. Superior inbreds and populations for producers and consumers will be released. This is the only USDA project focusing on germplasm enhancement and genetics of these important vegetable crops. Economical agricultural production of carrots, onions, and cucumbers requires new germplasm on an ongoing basis with improved traits, as well as genetic and other information to meet grower and consumer and research needs. Genetic and production information and new germplasm are urgently needed for these crops. We will identify and characterize molecular markers in cucumber and melon for diversity analysis to enhance germplasm management of these crops. The study of cucumber and melon genetics and physiology leading to the development of unique cultivars for use by the consuming public requires the use of classical plant breeding and plant genetic methods as well as the deployment of biotechnological tools where appropriate. Genetic mapping, trait inheritance, and application of this information for plant improvement are facilitated through classical and biotechnological methods. The application of this information in field and laboratory experiments with melon and cucumber results in an increase in the efficiency of cultivar development of these crops for the consuming public. Unique lines and populations have increased disease resistance, improved fruit yield and quality and tolerance to water stress. Many viruses infect cucumber and numerous sources of resistance are known. Breeding cucumber for resistance to many viruses is a time consuming and expensive process because of numerous independently inherited resistance loci, the need to inoculate plants with different viruses with similar phenotypes, and the cost of independently maintaining several viruses in plant materials. Cucumber is unique among the angiosperms in that it has one of the largest mitochondrial genomes among all plants, the mitochondria are paternally transmitted, and passage through cell culture produces unique rearrangments in the mitochondrial DNA conditioning a mosaic phenotype. Chemical inputs threaten the environment and profitability of carrots. Consumers rely on carrots as the single most important source of dietary vitamin A in the U.S. and for vegetables in their diet. Nematodes, alternaria leaf blight, and bacterial blight, generate significant production losses for carrot growers. Consumers would prefer better tasting, more nutritious carrots. General genetic information regarding carrots is not very extensive. Genetic mapping and inheritance patterns are being developed. Onion is a high value vegetable crop that is slow to breed by classical methods because of a long generation time. This project focuses research on development of superior male sterile inbred lines to be used by commercial seed companies to produce hybrid onion and technologies making onion breeding more difficult. For garlic, seed production and tissue culture are difficult, genetic transformation has not been successful and knowledge of genetics is minimal. Research to improve the techniques and knowledge of these areas is underway. Garlic breeding has only been possible for the last decade and progress toward solving production, disease, and quality problems is just underway. Without continued effort no improvements to garlic will be realized. 2. List the milestones (indicators of progress) from your Project Plan. Milestones for Objective 1 The evaluation of germplasms will result in the identification of accessions with unique characteristics useful for germplasm enhancement, the description of phenotypic correlations between traits, and the assessment of genetic differences among adapted and exotic germplasms. The accessions identified and the accompanying information will result in the release of unique inbred lines and germplasm pools. The assessment of melon germplasms in the U.S. NPGS will allow for the development of a core collection and test arrays in melon. For carrots this will include assessment of 150 entries of new germplasm from Europe in year 1,2, and 3; and release of disease resistant/high quality inbreds and populations in years 3 and 5. For onion, new low-pungency and red inbreds will be testcrossed and hybrid performance evaluated with release of new inbreds by year 5. For melon, the phenotypic (years 1 and 2) and molecular assessment (years 3 and 5) of the collection (2,185 accessions available) to form test arrays and a core collection. Milestones for Objective 2 The identification of genes conditioning nutritional value and flavor in these vegetable crops will provide the information and germplasm for increasing the farm value and improving the nutritional value of the food supply for consumers. For carrot this will include genes for carotenoid biosynthesis year 1, anthocyanin and sugar biosynthesis years 2-3, high pigment germplasm will be released years 4 and 5. For onion, evaluations for low pungency and high fructan content will occur in years 1 through 5 by selection of these traits in elite germplasms. For cucumber, the development of a high carotene parthenocarpic population (years 1 to 3), and the extraction of gynoecious lines for release years 4 and 5. Milestones for Objective 3 The identification of disease resistance in these vegetable crops will reduce the cost of production and pesticide applications. Because of our vegetable breeding programs, we are in excellent positions to incorporate these resistances from unadapted germplasms into elite genetic backgrounds for release to public and private vegetable breeders. For carrot this will include nematode resistance germplasm years 2 and 5; alternaria resistance germplasm years 3-4; and markers for resistance year 3. For onion, evaluations for smut resistance will occur in years 1 through 3. If resistance is identified, incorporation of resistance into elite germplasms will begin in years 4 and 5. For melon, the development and assessment of segregating populations (F2, BC, and F3) to determine the linkage relationships between molecular markers and melon aphid resistance genes (years 1 to 3) as a precursor to studies of marker efficacy in marker-assisted selection experiments (years 4 and 5). Milestones for Objective 4 Stepwise recurrent selection and line development for yield components will result in improved cucumber and melon lines and germplasm pools. Line and germplasm pool development will require the fixation of quantitative trait loci controlling yield and quality components (milestone 1) and the subsequent evaluation of unique selections to determine the appropriate cultural conditions that allow for yield optimization (milestone 2). In addition, the determination of the genetics of water stress in cucumber will constitute a significant outcome (milestone 3). Populations of onions and carrots with improved yield will provide growers with higher economic returns. For carrot this will include field performance trials in California, Wisconsin, and Washington every year. For onion, inbreds and populations will be selected for low pungency and high quality, but because of the biennial generation time of onion progress will be slow over the 5 years. The evaluation of and phenotypic selection in melon and cucumber populations for improved yield to include plant reduced plant architecture, gynoecy, multiple lateral branching, and early flowering (years 1 to 3), and then the extraction and evaluation of lines in preparation for release (years 4 and 5). Milestones for Objective 5: The identification of molecular markers closely linked to major economically important loci in these vegetable crops will reduce the cost to develop elite inbred populations and germplasms. Haploid development avoids the time-consuming and labor-intensive inbreeding cycles and allows for the relatively quick production of completely homozygous inbred lines. For carrot this will include markers for pigments years 2 and 3, for nematode and alternaria resistance year 3 and 4; and transformation of pigment genes year 4. For onion, molecular markers revealing nuclear genotypes at the fertility-restoration locus and cytoplasms of onion will be developed in years 1 through 3 and released for use by year 5. The comparative analysis of phenotypic and marker- assisted selection schemes for multiple trait improvement in melon and cucumber populations developed by recurrent selection (years 1 to 5). 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Assessment of new carrot germplasm from Europe. New low-pungency and red onion inbreds testcrosses and hybrid performance evaluations. Phenotypic and molecular assessment of the melon collection. Milestone Substantially Met 2. Germplasm for carrot carotenoid, anthocyanin and sugar biosynthesis releases. Evaluations on onion for low pungency and high fructan content. Develop a high carotene parthenocarpic population and gynoecious lines on cucumber. Milestone Substantially Met 3. Evaluations for nematode resistance, alternaria resistance, and markers for resistance on carrots. Evaluations for smut resistance and resistance introgressed into elite onion germplasm. Development and assessment of segregating populations (F2, BC, and F3) to determine the linkage relationships between molecular markers and melon aphid resistance genes. Marker efficacy in marker-assisted selection evaluations on melons. Milestone Substantially Met 4. Carrot field performance trials in California, Wisconsin, and Washington. Inbreds and populations for selections for low pungency and high quality in onions. Evaluation of and phenotypic selection in melon and cucumber populations for improved yield to include plant reduced plant architecture, gynoecy, multiple lateral branching, and early flowering. Milestone Substantially Met 5. Evaluations of markers for carrot pigments,and for nematode and alternaria resistance. Molecular markers revealing nuclear genotypes at the fertility-restoration locus and cytoplasms of onion will be developed and evaluated. A comparative analysis of phenotypic and marker-assisted selection schemes for multiple trait improvement in melon and cucumber populations developed by recurrent selection. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? FY2006 For Objective 1: Assessment of 50 entries of new carrot germplasm from Europe. New low-pungency and red onion inbreds will be testcrossed and hybrid performance evaluated. The phenotypic and molecular assessment of the melon collection will be repeated. For Objective 2: Germplasm for carrot carotenoid, anthocyanin and sugar biosynthesis will be re-evalauted (in F3 and BC1 generations). For onion, evaluations for low pungency and high fructan content will be made. For cucumber, a high carotene parthenocarpic population and gynoecious lineswill be developed. For Objective 3: Carrot with nematode resistance, alternaria resistance, and markers for resistance will be evaluated. For onion, evaluations for smut resistance will occur and resistance introgressed into elite germplasm. For melon, the development and assessment of segregating populations (F2, BC, and F3) to determine the linkage relationships between molecular markers and melon aphid resistance genes will be made andf marker efficacy in marker-assisted selection evaluated. For Objective 4: For carrot field performance trials in California, Wisconsin, and Washington will be made. For onion, inbreds and populations will be selected for low pungency and high quality. The evaluation of and phenotypic selection in melon and cucumber populations for improved yield to include plant reduced plant architecture, gynoecy, multiple lateral branching, and early flowering will be made and the extraction of lines will be made. For Objective 5: Markers for carrot pigments,and for nematode and alternaria resistance will be evaluated. For onion, molecular markers revealing nuclear genotypes at the fertility-restoration locus and cytoplasms of onion will be developed and evaluated. The comparative analysis of phenotypic and marker-assisted selection schemes for multiple trait improvement in melon and cucumber populations developed by recurrent selection will be repeated. FY2007 For Objective 1: Assessment of 50 entries of new carrot germplasm from South America and Europe. New low-pungency and red onion inbreds will be testcrossed and hybrid performance evaluated. The phenotypic and molecular assessment of the melon collection will be repeated. For Objective 2: Germplasm for carrot carotenoid, anthocyanin and sugar biosynthesis will be released. For onion, evaluations for low pungency and high fructan content will be made. For cucumber, a high carotene parthenocarpic population and gynoecious lines will be developed. For Objective 3: Carrot with nematode resistance, alternaria resistance, and markers for resistance will be evaluated. For onion, evaluations for smut resistance will occur and resistance introgressed into elite germplasm. For melon, the development and assessment of segregating populations (BC2, and F4) to determine the linkage relationships between molecular markers and melon aphid resistance genes will be made and marker efficacy in marker-assisted selection evaluated. For Objective 4: For carrot field performance trials in California, Wisconsin, and Washington will be made. For onion, inbreds and populations will be selected for low pungency and high quality. The evaluation of and phenotypic selection in melon and cucumber populations for improved yield to include plant reduced plant architecture, gynoecy, multiple lateral branching, and early flowering will be made and the extraction of lines will be made. For Objective 5: Markers for carrot pigments,and for nematode and alternaria resistance will be evaluated. For onion, molecular markers revealing nuclear genotypes at the fertility-restoration locus and cytoplasms of onion will be developed and evaluated. The comparative analysis of phenotypic and marker-assisted selection schemes for multiple trait improvement in melon and cucumber populations developed by recurrent selection will be repeated. FY2008 For Objective 1: Assessment of 35 entries of new carrot germplasm from Europe and Asia. New low-pungency and red onion inbreds will be testcrossed and hybrid performance evaluated. The phenotypic and molecular assessment of the melon collection will be repeated. For Objective 2: Germplasm for carrot carotenoid, anthocyanin and sugar biosynthesis will be evaluated in Wisconsin. For onion, evaluations for low pungency and high fructan content will be made. For cucumber, a high carotene parthenocarpic population and gynoecious lines will be developed. For Objective 3: Carrot with nematode resistance, alternaria resistance, and markers for resistance will be evaluated. For onion, evaluations for smut resistance will occur and resistance introgressed into elite germplasm. For melon, the development and assessment of segregating populations (F2, BC, and F3) to determine the linkage relationships between molecular markers and melon aphid resistance genes will be made and marker efficacy in marker-assisted selection evaluated. For Objective 4: For carrot field performance trials in California, Wisconsin, and Washington will be made. For onion, inbreds and populations will be selected for low pungency and high quality. The evaluation of and phenotypic selection in melon and cucumber populations for improved yield to include plant reduced plant architecture, gynoecy, multiple lateral branching, and early flowering will be made and the extraction of lines will be made. For Objective 5: Markers for carrot pigments,and for nematode and alternaria resistance will be evaluated. For onion, molecular markers revealing nuclear genotypes at the fertility-restoration locus and cytoplasms of onion will be developed and evaluated. The comparative analysis of phenotypic and marker-assisted selection schemes for multiple trait improvement in melon and cucumber populations developed by recurrent selection will be repeated. 4a What was the single most significant accomplishment this past year? We established the first genetic linkage maps for garlic, a crop which has only been brought into sexual reproduction in the last 2 decades. This demonstrated that gamete and progeny formation was strictly sexual, not asexual, and proved that the diversity stemming from that sexual reproduction can be utilized for classical breeding of garlic. Before this, only mutagenesis could be used to generate variation and traits in two parents could not be combined into recombinant progeny. 4b List other significant accomplishments, if any. We identified a genetic locus in cucumber that controls sorting of the paternally transmitted mitochondrial DNA. Genetic anlaysis of this unique locus should provide insights about the important interaction of the nuclear and organellar DNAs. Genetic markers tightly linked to major disease resistances will increase the efficienty of breeding of multiple disease resistant cucumber populations. We fine mapped a molecular marker near a major resistance gene cluster in cucumber conditioning resistance to multiple potyviruses. The evaluation of the efficiency and effectiveness of molecular markers linked to quantitative trait loci for yield in cucumber. This research investigates the value of molecular markers for plant breeding of yield components in cucumber. Crosses were made in-house to produce segregating progeny which were then selected for yield components using linked molecular markers. It was determined that linked markers could be used effectively for selection of superior plants. Since resources in plant improvement programs are limiting, marker-assisted selection can be applied to reduce costs. It was determined that three candidate chloroplast genes exist may control tolerance to chilling temperatures in cucumber. Examination of response to selection for yield components in melon indicated that molecular marker technologies could be used effectively to identify high yielding melon germplasm at reduced costs. Chloroplast markers were created and applied to define genetic relationships between exotic cucurbit species and commercial cucumber in order to make strategic crosses for the incorporation of economically important traits in cucumber. We established the fact that yellow, red, and purple carrots are, in fact, good dietary sources of lutein, lycopene, and anthocyanins. This is important for carrot growers, nutritionists, and the consuming public since it provides the basis for publicizing these unusually colored carrots as "functional foods" able to enrich the US diet with these nutritionally important compounds that enhance human health by reducing the risks of several forms of cancer and heart disease. This USDA program has developed the genetic basis of hybrid onion production and transferred this technology to the hybrid vegetable seed industry. We are now concentrating on new technologies to produce new generations of onion hybrids. Onion breeding is a slow process because two years are required per generation. Molecular markers closely associated with major traits in onion will provide the seed industry with a quick and cheap alternative to classical genetic studies and selection. These projects will enable the seed industry to react quickly to changes in consumer preferences, production environments, or exploit new markets. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Objectives include continued multidisciplinary efforts to develop and evaluate multiple disease resistant cucumber hybrids and inbred lines, and identify appropriate cultural practices for genotypes with unique vining and fruiting habits. Evaluation of Cucumis and Cucurbita germplasm for diversity will continue to facilitate the construction of core collections, the enhancement of germplasm management methodologies, the identification of tolerance to suboptimal conditions (i.e., water and heat stress), and the development of efficient breeding methodologies. Continued progress will be made to identify linkage associations between molecular markers and economically important traits in order to identify technologies which will increase the efficiency of cucumber breeding. This will require the identification, inheritance and mapping of traits for disease resistance and fruit yield and quality using isozymes, restriction fragment length polymorphisms (RFLPs), DNA fragments obtained from random amplification polymorphism detection (RAPDs), amplified fragment length polymorphisms (AFLPs), single sequence repeats (SSRs), and single nucleotide polymorphisms (SNP) technologies. These markers will be used to more clearly understand the physiology and genetics of horticulturally important characters in cucumber and melon to allow for more efficient ways to develop lines and hybrids for commercial production. Such increase will shorten time for hybrid development to reduce development costs and increase grower competitiveness. Customers are: Pickle Packers International, Inc., Midwest Pickle Packers, Vegetable Seed Companies, and Public Research Scientists. Numerous germplasm releases have been made to carrot growers for development of new carrots with improved production, less pesticide application and with improved flavor and nutritional value for consumers. The understanding of carrot pigment genetics, disease resistance, genetic map, and germplasm diversity has been provided. Garlic seed production is now routinely possible because of this research. Garlic tissue culture and transformation are now feasible and genetic variation of the crop is better understood. Most, if not essentially all, hybrid onion cultivars grown in the U.S. are produced using USDA germplasm from this program. We continue to breed and develop superior germplasm and develop new technologies to make this process quicker and less costly. We identified differences in the DNA flanking the Ms locus, a major gene that determines whether male sterile inbreds can be developed from a population or family. To make these DNA differences useful in applied breeding programs, we converted these DNA differences to genetic markers revealed by the polymerase chain reaction (PCR). It now takes only hours, as opposed to years, to establish the cytoplasm and estimate the nuclear genotype of individual onion plants. We continued a major field and laboratory study to establish the genetic bases of correlated flavor, production, and health enhancing attributes of onion. These correlated traits appear to be under the control of genes conditioning the synthesis of inulins, which are complex carbohydrates with well established health benefits in humans. When onion bulbs synthesis inulins, they take up less water at maturity causing the concentration of carbohydrates, as well as the compounds associated with higher pungencies and blood thinning attributes. Plants and bulbs were scored for genetic attributes or selected for commercially acceptable types. We are in the final seed increase of the first male sterile and male fertile maintainer of a red inbred onion population to be released from the public sector in the US. The development of genomic resources for onion will be a major accomplishment, allowing breeders and geneticists to find target onion genes. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The investigations being carried out that seek to increase the yield and quality of cucumber and melon will positively effect growers, processors and consumers. These investigations are part of a broad research program to develop multiple disease resistant, high fruit number cucumber germplasm that is basic to the need of private industry and the consuming public. The release of multiple resistant germplasm will decrease product wastage and the use of pesticides, thus directly improving profitability of growers and processors, the food safety of consumers, and harmony between agriculture and the environment. Data concerning cultural practices to optimize germplasm with unique fruiting and vining habits will accompany releases of germplasm to allow for optimized yield. Since many of the growers (60%) reside in small rural communities and manage relatively small production areas (1 to 25 acres), such information will enhance their economic opportunity and quality of life. For instance, in August 2004 the USDA cucumber project successfully planned and executed "Wisconsin Pickle Day" held at the University of Wisconsin Experimental Farm, Hancock, WI. About 80 local growers and processors attended and were instructed on new cultural practices and the development of new lines and hybrids being evaluated for release to the public. This activity heightened the awareness of processors and growers to new technologies and varieties that would increase their competitiveness. The information obtained from diversity analyses will be used by the National Plant Germplasm System to increase its managerial efficiency and effectiveness. During the refinement of this germplasm, breeding methodologies will be developed to assist plant geneticists in the subsequent use of this or similar plant material, and provide information potentially useful to other cucurbit species. Properly identified and characterized biochemical markers will be used as tools to determine physiological processes of horticulturally important characters in cucumber and melon. Description and characterization of economically important physiological fruit disorders will lead to the establishment of better crop management practices, and thus decrease postharvest losses to increase profitability of growers and processors. Most, if not essentially all, hybrid onion cultivars grown in the U.S. are produced using USDA germplasm from this program. We continue to breed and develop superior germplasm and develop new technologies to make this process quicker and less costly. We identified differences in the DNA flanking the Ms locus, a major gene that determines whether male sterile inbreds can be developed from a population or family. To make these DNA differences useful in applied breeding programs, we converted these DNA differences to genetic markers revealed by the polymerase chain reaction (PCR). It now takes only hours, as opposed to years, to establish the cytoplasm and estimate the nuclear genotype of individual onion plants. Tagging of the Ms locus has provided the seed industry with a quick and cheap alternative to classical genetic studies to determine whether male sterile inbred lines can be developed from populations or families. We continued a major field and laboratory study to establish the genetic bases of correlated flavor, production, and health enhancing attributes of onion. These correlated traits appear to be under the control of genes conditioning the synthesis of inulins, which are complex carbohydrates with well established health benefits in humans. When onion bulbs synthesis inulins, they take up less water at maturity causing the concentration of carbohydrates, as well as the compounds associated with higher pungencies and blood thinning attributes. The genetic bases of fructan accumulation in onion will be important for breeders to select health-enhancing onion cultivars. Our research on the flavor, production, and health enhancing attributes of onion will provide basic genetic information imperative to the development of new value added onion hybrids for growers and consumers. Plants and bulbs were scored for genetic attributes or selected for commercially acceptable types. Numerous germplasm releases have been made to carrot growers for development of new carrots with improved production, less pesticide application and with improved flavor and nutritional value for consumers. The understanding of carrot pigment genetics, disease resistance, genetic mapping, and germplasm diversity has been provided. Garlic seed production is now routinely possible because of this research. Garlic tissue culture and transformation are now feasible and genetic ariation of the crop is better understood. These projects will enable the seed industry to react quickly to changes in consumer preferences, production environments, or exploit new markets. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). STAUB Decker-Walters, D.S., S.M. Chung, and J. E. Staub. 2004. Chloroplast sequence evolution: A new pattern of nucleotide substitutions in the Cucurbitaceae. J. Mol. Evol. 58:606-614. Zhuang, F.Y., J.F. Chen, J.E. Staub, and C.T. Qian. 2004. Assessment of genetic relationships in Cucumis species by SSR and RADP analysis. Plant Breeding 123:167-172. Staub, J.E., N. Fanourakis, and A. Lopez-Sese. 2004. Genetic diversity in melon (Cucumis melo L.) landraces from the island of Crete as assessed by random amplified polymorphic DNA and simple sequence repeat markers. Euphytica 136:151-166. Chen, J.F., X.D. Luo, C.T. Qian, M.M. Jahn, J.E. Staub, F.Y. Zhuang, Q.F. Lou and G. Ren. 2004. Cucumis monosomic alien addition lines: morphological, cytological, and genotypic analyses. Theor Appl Genet. 108:7:1343-1348. McCreight, J.D, J.E. Staub, A. Lopez-Sese, and S.M. Chung. 2004. Isozyme variation in Indian and Chinese melon (Cucumis melo L.) germplasm collections. J. Am. Soc. Hort. Sci. 129:811-818.

Impacts
(N/A)

Publications

Havey, M.J., Park, Y., Bartoszewski, G. 2004. The psm locus controls paternal sorting of the cucumber mitochondrial genome. Journal of Heredity. 95:492-497.
Surles, R., Weng, N., Simon, P.W., Tanumihardjo, S. 2004. Carotenoid profiles and consumer sensory evaluation of specialty carrots. Journal of Agriculture and Food Chemistry. 52:3417-3421.
Ipek, M., Ipek, A., Almquist, S.G., Simon, P.W. 2005. Demonstration of linkage and development of the first low-density genetic map of garlic based on aflptm markers. Theoretical and Applied Genetics. 110:228-236.

Progress 10/01/03 to 09/30/04

Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Economical agricultural production of carrots, onions, and cucumbers requires new germplasm on an ongoing basis with improved traits, as well as genetic and other information to meet grower and consumer and research needs. Nematodes, alternaria leaf blight, and bacterial blight, generate significant production losses for carrot growers. Consumers would prefer better tasting, more nutritious carrots. General genetic information regarding carrots is not very extensive. Genetic mapping and inheritance patterns are being developed. For garlic, seed production and tissue culture are difficult, genetic transformation has not been successful and knowledge of genetics is minimal. Research to improve the techniques and knowledge of these areas is underway. Allium, carrot and Cucurbit crops are important to the agriculture economy and more germplasm and information are needed to sustain production. This project will characterize germplasm of these crops, determine the genetics and select genetic stocks of important onion, carrot, and cucumber nutritional components, disease resistances, and yield components; and continue or initiate biotechnology research in Daucus, Allium, and Cucurbit germplasms. Superior inbreds and populations for producers and consumers will be released. This is the only USDA project focusing on germplasm enhancement and genetics of these important vegetable crops. We identify and characterize molecular markers in cucumber and melon for diversity analysis to enhance germplasm management of these crops. The study of cucumber and melon genetics and physiology leading to the development of unique cultivars for use by the consuming public requires the use of classical plant breeding and plant genetic methods as well as the deployment of biotechnological tools where appropriate. Genetic mapping, trait inheritance, and application of this information for plant improvement are facilitated through classical and biotechnological methods. The application of this information in field and laboratory experiments with melon and cucumber results in an increase in the efficiency of cultivar development of these crops for the consuming public. Unique lines and populations have increased disease resistance, improved fruit yield and quality and tolerance to water stress. Many viruses infect cucumber and numerous sources of resistance are known. Breeding cucumber for resistance to many viruses is a time consuming and expensive process because of numerous independently inherited resistance loci, the need to inoculate plants with different viruses with similar phenotypes, and the cost of independently maintaining several viruses in plant materials. Cucumber is unique among the angiosperms in that it has one of the largest mitochondrial genomes among all plants, the mitochondria are paternally transmitted, and passage through cell culture produces unique rearrangments in the mitochondrial DNA conditioning a mosaic phenotype. Chemical inputs threaten the environment and profitability of carrots. Consumers rely on carrots as the single most important source of dietary vitamin A in the U.S. and for vegetables in their diet. Genetic and production information and new germplasm are urgently needed for these crops. Garlic breeding has only been possible for the last decade and progress toward solving production, disease, and quality problems is just underway. Without continued effort no improvements to garlic will be realized. Onion is a high value vegetable crop that is slow to breed by classical methods because of a long generation time. This project focuses research on development of superior male sterile inbred lines to be used by commercial seed companies to produce hybrid onion and technologies making onion breeding more difficult. 2. List the milestones (indicators of progress) from your Project Plan. Milestones for Objective 1 The evaluation of germplasms will result in the identification of accessions with unique characteristics useful for germplasm enhancement, the description of phenotypic correlations between traits, and the assessment of genetic differences among adapted and exotic germplasms. The accessions identified and the accompanying information will result in the release of unique inbred lines and germplasm pools. Cucumber germplasm from Crete and Japan were evaluated using molecular markers to identify unique accessions in diverse genetic backgrounds. For onion, new low-pungency and red inbreds will be testcrossed and hybrid performance evaluated with release of new inbreds by year 5. Carrot germplasm from primary and secondary centers of diversity will be evaluated. Milestones for Objective 2 The identification of genes conditioning nutritional value and flavor in these vegetable crops will provide the information and germplasm for increasing the farm value and improving the nutritional value of the food supply for consumers. A project was initiated that will allow for the creation of unique cucumber germplasm with high carotene concentration in the processed product. For onion, evaluations for low pungency and high fructan content will occur in years 1 through 5 by selection of these traits in elite germplasms. Carrot carotenoid and sugar genes will be mapped and gene expression evaluated. Milestones for Objective 3 The identification of disease resistance in these vegetable crops will reduce the cost of production and pesticide applications. Because of our vegetable breeding programs, we are in excellent positions to incorporate these resistances from unadapted germplasms into elite genetic backgrounds for release to public and private vegetable breeders. Populations for the mapping of aphid melon resistance in melon were created and molecular marker screening of these populations has led to the development of strategies for the identification and mapping of genes for resistance. For onion, evaluations for smut resistance will occur in years 1 through 3. If resistance is identified, incorporation of resistance into elite germplasms will begin in years 4 and 5. Carrot nematode resistance genes will be mapped and located on a BAC library. Milestones for Objective 4 Populations of onions and carrots with improved yield will provide growers with higher economic returns. Replicated trialing of USDA inbreds, hybrids and populations have allowed for the identification of unique germplasm that will likely have value in cucumber machine harvest operations. Selection of yield components in melon has allow for the identification of unique germplasm from exotic sources that will increase yield potential in this species. For onion, inbreds and populations will be selected for low pungency and high quality, but because of the biennial generation time of onion progress will be slow over the 5 years. Carrot germplasm with uniformity, length, and smoothness of the storage root will be developed and released. Milestones for Objective 5 The identification of molecular markers closely linked to major economically important loci in these vegetable crops will reduce the cost to develop elite inbred populations and germplasms. Haploid development avoids the time-consuming and labor-intensive inbreeding cycles and allows for the relatively quick production of completely homozygous inbred lines. Unique chlorplast markers have been developed that allow for the rapid sequencing of the chloroplast genome of cucumber in lines resistant and susceptible to chilling injury that will allow for the development of strategies for the rapid incorporation of chilling resistance in commercial cucumber. For onion, molecular markers revealing nuclear genotypes at the fertility-restoration locus and cytoplasms of onion will be developed in years 1 through 3 and released for use by year 5. Carrot and garlic genetic markers for nuclear and mitochondrial genes will be developed and tested. 3. Milestones: A. List the milestones that were scheduled to be addressed in FY 2004. How many milestones did you fully or substantially meet in FY 2004, and indicate which ones were not fully or substantially met, briefly explain why not, and your plans to do so. Milestones: The genetic diversity of Spain, Japanese, and Greek melon accessions were evaluated for their genetic diversity using morphological and biochemical genetic markers. These evaluations lead to a better understanding of melon diversity for subsequent development of a core collection in this species. New low-pungency and red onion inbreds were testcrossed to male-sterile female tester lines in 2003 and hybrid performance was evaluated in 2004. We are on target for release of these inbreds by year 5. The development of a population of high carotene cucumber by random mating of exotic lines derived from Indian origin. This represents the initial phase of the development of U.S. processing cucumbers with high carotene content. Onion populations were evaluated for low pungency and high fructan content and selection of elite germplasms with these characteristics is underway. We continued our evaluation of a unique cucumber interspecific hybrid and its derived cross progeny which have resistance to nematode and gummy stem blight. This is the first fertile hybrid which offers a bridge between wild and cultivated species in cucumber. Evaluations for onion smut resistance and virus resistance in cucumber are underway. We are presently testing progenies from resistant plants to determine if these resistances are heritable. If these progenies are resistant, we will cross to elite germplasms to transfer the resistance. The genetic basis of parthenocarpy (seedless fruit) in cucumber was determined. This knowledge will allow for the more efficient and effective incorporation of such genes for increasing the yield and quality of cucumber fruit. The development of chloroplast markers contributed to a better understanding of chloroplast genome of cucumber. The sequencing and construction of genetic markers will allow for the identification of genes associated with chilling tolerance in cucumber. Molecular markers are being evaluated for their linkage to the male-fertility-restoration locus of onion. All milestones were met. The milestones for carrot research listed below were scheduled to be completed this year. Identified carrot germplasm and genes with bioavailable lutein and beta-carotene. Identified correlation between garlic phenotypic diversity and molecular diversity. Identified new nematode resistance genes and markers in carrot Completed carrot yield trials and evaluated new nematode resistant germplasm. Developed molecular markers for carrot linkage groups, invertase, male sterile mitochondrial genomes and nematode resistance, and mapped specific AFLP amplicons. All milestones were completed. B. List the milestones that you expect to address over the next 3 years (FY 2005, 2006, and 2007). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Milestones for FY2005 For onion, new low-pungency and red inbreds will be testcrossed and hybrid performance evaluated. Evaluations for low pungency and high fructan content will occur in year this year. Evaluations for smut resistance will occur in this year. We will continue to evaluate for molecular markers revealing nuclear genotypes at the fertility- restoration locus of onion. The development of a core collection for melon. Evaluate newly collected garlic and carrot germplasm. Identify map location for 25 carrot carotenoid biosynthetic genes. Perform carrot yield trials. Develop garlic marker for flowering. Milestones for FY2006 For onion, new low-pungency and red inbreds will be evaluated for hybrid performance. Evaluations for low pungency and high fructan content will continue this year. Evaluations for smut resistance will also continue. Molecular markers revealing nuclear genotypes at the fertility- restoration locus of onion will be developed. The development of a population and lines in cucumber producing high carotene containing fruits. The development of lines derived from exotic germplasm which have increased nematode and gummy stem blight resistance. Evaluate carrot germplasm for transposons. Develop carrot carotenoid full length biosynthetic gene sequences. Develop carrot cytogenetic map. Perform carrot yield trials. Develop carrot transposon tagging system. Milestones for FY2007 For onion, new low-pungency and red inbreds will be released after assessment of hybrid performance. Evaluations for low pungency and high fructan content will be completed and beneficial germplasms identified. Evaluations for smut resistance will be completed. Resistant germplasms (if any) will be incorporated into the breeding program. The genetic mapping of yield and quality components (genes) in cucumber and melon for its use in the improvement of plants with unique vine and fruiting habits. The development of molecular marker technologies that increase the efficiency and effectiveness of the improvement of cucumber and melon. Evaluate garlic alliinase variation in germplasm. Clone carrot transposon. Perform carrot yield trials. Complete carrot SSR map. 4. What were the most significant accomplishments this past year? A. Single most significant accomplishment during FY 2004. The chlorplast genome of cucumber was sequenced. In order to accomplish this approximately 400 molecular markers were developed which will augment the previously developed 47 markers by our laboratory for genetic and evolutionary studies in cucurbit species (melon, cucumber, watermelon, squash). B. Other Significant Accomplishment(s), if any: The genes which control orange carrot root color have now been placed on the carrot genetic map. The USDA-ARS in Wisconsin grew plants and mapped 19 genes for orange, red, yellow and white color. Most genes were clustered in three groups along the carrot chromosomes. This research demonstrated that a few key genetic regions control root color and this is important for carrot breeders to know when they improve color. Determined that a carrot invertase mutant is caused by a DNA insert. This insert is suggestive of a transposable element which could shape the carrot genome. We characterized onion germplasms that are different for the accumulation of health-enhancing fructans to reveal that sucrose availability to the onion bulb is a major determinant of the amounts of fructans accumulated. We also developed the first genomic resources for onion by producing over 11,000 unique expressed sequence tags. DNA analyses on these ESTs demonstrated that expressed regions of the onion DNA is very different from other monocots, such as rice. This has important implications for DNA analyses of these important crops. Molecular markers were developed by sequence techniques that will be used for studies of synteny between cucumber and melon. The genetics of parthencarpy in cucumber was determined and the genes for parthenocarpic fruit development were mapped. To improve cucurbit crops it is important to establish the genetic characterization of a a unique intraspecific hybrid in Cucumis, and the determination of the genetic control of tolerance to chilling injury in cucumber. The USDA curcurbit genetic improvement project in collaboration with a scientist in China worked to hybridize wild and elite cucumber germplasm, and the USDA project has determined that chilling injury in cucumber is controlled by cytoplasmic factors, likely those found in the chloroplast. Embryos were rescued from the female parent to produce, after chromosome doubling, a fertile synthetic species which is cross-compatible with cucumber, and the fertile offspring cytogenetics and growth potential have been characterized, and the inheritance of chilling injury has been defined in two genetic backgrounds using exact reciprocal cross-progeny. This synthetic species could be used as a bridge between wild species and elite cucumber to transfer economically important disease resistant genes not now present in cucumber leading to the release of germplasm for improved yield and quality so that plant breeders will be able to more effectively introgress genes for chilling tolerance into elite germplasm. Germplasm collections are the source of germplasm for breeders interested in improving cultivars, phyisologists studying biotic and abiotic stresses, and pathologists characterizing the mechanisms of disease resistance and it is important to determine that different gene pools exist in melon and cucumber that can be used by plant breeders for crop improvement. The USDA cucurbit genetic improvement project in cooperation with Spanish researchers developed genetic marker (RAPD and SSR) systems to characterize the genetic variation in melons and cucumbers from Africa, Spain, Japan, and Greece. This required the analysis of hundreds of collections for public and private breeding programs and national plant introduction stations by using a set of reference accessions and a standard marker array from previous studies. Genetic diversity data will provide cucumber and melon germplasm curators and breeders with unique perspectives on the genetic differences among economically important germplasm pools to allow for more strategic use of exotic germplasm for the improvement of these crop species. C. Significant activities that support special target populations: None. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Objectives include continued multidisciplinary efforts to develop and evaluate multiple disease resistant cucumber hybrids and inbred lines, and identify appropriate cultural practices for genotypes with unique vining and fruiting habits. Evaluation of Cucumis and Cucurbita germplasm for diversity will continue to facilitate the construction of core collections, the enhancement of germplasm management methodologies, the identification of tolerance to suboptimal conditions (i.e., water and heat stress), and the development of efficient breeding methodologies. Continued progress will be made to identify linkage associations between molecular markers and economically important traits in order to identify technologies which will increase the efficiency of cucumber breeding. This will require the identification, inheritance and mapping of traits for disease resistance and fruit yield and quality using isozymes, restriction fragment length polymorphisms (RFLPs), DNA fragments obtained from random amplification polymorphism detection (RAPDs), amplified fragment length polymorphisms (AFLPs), single sequence repeats (SSRs), and single nucleotide polymorphisms (SNP) technologies. These markers will be used to more clearly understand the physiology and genetics of horticulturally important characters in cucumber and melon. Numerous germplasm releases have been made to carrot growers for development of new carrots with improved production, less pesticide application and with improved flavor and nutritional value for consumers. The understanding of carrot pigment genetics, disease resistance, genetic map, and germplasm diversity has been provided. Garlic seed production is now routinely possible because of this research. Garlic tissue culture and transformation are now feasible and genetic variation of the crop is better understood. Most, if not essentially all, hybrid onion cultivars grown in the U.S. are produced using USDA germplasm from this program. We continue to breed and develop superior germplasm and develop new technologies to make this process quicker and less costly. We identified differences in the DNA flanking the Ms locus, a major gene that determines whether male sterile inbreds can be developed from a population or family. To make these DNA differences useful in applied breeding programs, we converted these DNA differences to genetic markers revealed by the polymerase chain reaction (PCR). It now takes only hours, as opposed to years, to establish the cytoplasm and estimate the nuclear genotype of individual onion plants. We continued a major field and laboratory study to establish the genetic bases of correlated flavor, production, and health enhancing attributes of onion. These correlated traits appear to be under the control of genes conditioning the synthesis of inulins, which are complex carbohydrates with well established health benefits in humans. When onion bulbs synthesis inulins, they take up less water at maturity causing the concentration of carbohydrates, as well as the compounds associated with higher pungencies and blood thinning attributes. Plants and bulbs were scored for genetic attributes or selected for commercially acceptable types. We are in the final seed increase of the first male sterile and male fertile maintainer of a red inbred onion population to be released from the public sector in the US. The development of genomic resources for onion will be a major accomplishment, allowing breeders and geneticists to find target onion genes. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The investigations being carried out that seek to increase the yield and quality of cucumber and melon will positively effect growers, processors and consumers. These investigations are part of a broad research program to develop multiple disease resistant, high fruit number cucumber germplasm that is basic to the need of private industry and the consuming public. The release of multiple resistant germplasm will decrease product wastage and the use of pesticides, thus directly improving profitability of growers and processors, the food safety of consumers, and harmony between agriculture and the environment. Data concerning cultural practices to optimize germplasm with unique fruiting and vining habits will accompany releases of germplasm to allow for optimized yield. Since many of the growers (60%) reside in small rural communities and manage relatively small production areas (1 to 25 acres), such information will enhance their economic opportunity and quality of life. The information obtained from diversity analyses will be used by the National Plant Germplasm System to increase its managerial efficiency and effectiveness. During the refinement of this germplasm, breeding methodologies will be developed to assist plant geneticists in the subsequent use of this or similar plant material, and provide information potentially useful to other cucurbit species. Properly identified and characterized biochemical markers will be used as tools to determine physiological processes of horticulturally important characters in cucumber and melon. Description and characterization of economically important physiological fruit disorders will lead to the establishment of better crop management practices, and thus decrease postharvest losses to increase profitability of growers and processors. The genetic bases of fructan accumulation in onion will be important for breeders to select health-enhancing onion cultivars.

Impacts
(N/A)

Publications

Robbins, M.D., Staub, J.E. Strategies for selection of multiple quantitatively yield components in cucumber. In Proceedings of 8th Eucarpia Conference, Cucurbitaceae 2004: Progress in cucurbit genetics and breeding research, July 12-17, 2004, Olomouc, The Czech Republic. p. 401-410.
Chung, S., Staub, J.E. Consensus chloroplast primer analysis: a molecular tool for evolutionary studies in Cucurbitaceae. In: Proceedings of the 8th Eucarpia Conference, Cucurbitaceae 2004: Progress in cucurbit genetics and breeding research, July 12-17, 2004, Olomouc, The Czech Republic. p. 477- 484.
Paris, M., Staub, J.E., Mccreight, J.D. 2003. Determination of fruit sampling location for quality measurements in melon (Cucumis melo L.). Cucurbit Genetics Cooperative Report. 26:12-17.
Zhuang, F.Y., Chen, J.F., Staub, J.E., Qian, C.T. 2003. Assessment of genetic relationships in cucumis species by SSR and RAPD marker analysis. Plant Breeding. 123:167-172.
Staub, J.E., Fanourakis, N., Lopez-Sese, A.I. 2003. Genetic diversity in melon (Cucumis melo L.) landraces from the island of Crete as assessed by random amplified polymorphic DNA and simple sequence markers. Euphytica. 136:151-166.
Sun, Z., Lower, R.L., Staub, J.E. Generation means analysis of parthenocarpic characters in a processing cucumber (Cucumis sativus L.) population. In: Proceedings of the 8th Eucarpia Conference, Cucurbitaceae 2004: Progress in cucurbit genetics and breeding research, July 12-17, 2004, Olomouc, The Czech Republic. p. 365-372.
Staub, J.E., Zalapa, J.E., Paris, M., McCreight, J.D. Selection for lateral branch number in melon (Cucumis melo L.). In: Proceedings of the 8th Eucarpia Conference, Cucurbitaceae 2004: Progress in cucurbit genetics and breeding research, July 12-17, 2004, Olomouc, The Czech Republic. p. 381-388.
Bartoszewski, G., Malepszy, S., Havey, M.J. 2004. Mosaic (MSC) cucumbers regenerated from independent cell cultures possess different mitochondrial rearrangements. Current Genetics. 45:45053.
Santos, C.A., Simon, P.W. 2004. Merging carrot linkage groups based on conserved dominant AFLP markers in F2 populations. Journal of the American Society for Horticultural Science. 129:211-217.
Santos, C.A., Simon, P.W. 2003. Sequencing of specific AFLP amplicons reveals highly conserved DNA sequences mapping to the same linkage groups in two unrelated F2 populations of carrot. Genetics and Molecular Biology. 25:195-201.
Yau, Y., Simon, P.W. 2003. A 2.5-kb insert eliminates acid soluble invertase isozyme II transcript in carrot (Daucus carota L.) roots, causing high sucrose accumulation. The Plant Cell. 53:151-162.
Bach, I.C., Olesen, A., Simon, P.W. 2002. PCR-based markers to differentiate the mitochondrial genomes of petaloid and male fertile carrot (Daucus carota L.). Euphytica. 127:353-365.
Ipek, M., Ipek, A., Simon, P.W. 2003. Comparison of AFLPs, RAPD markers, and isozymes for diversity assessment of garlic and detection of putative duplicates in germplasm collections. Journal of the American Society for Horticultural Science. 128:246-252.
Boiteux, L.S., Hyman, J.R., Bach, I.C., Fonseca, M.N., Matthews, W.C., Roberts, P.A., Simon, P.W. 2004. Employment of flanking codominant STS markers to estimate allelic substitution effects of a nematode resistance locus in carrot. Euphytica. 136:37-44.
Barttoszewski, G., Katzir, N., Havey, M.J. 2004. Organization of ribosomal RNA coding and repetitive DNAs in the cucurbit mitochondrial genomes. Theoretical and Applied Genetics. 108(6):982-992.
Havey, M.J., Galmarini, C.R., Gokce, A., Henson, C.A. 2004. QTL affecting soluble carbohydrate concentrations in stored onion bulbs and their association with flavor and health-enhancing attributes. Genome. 47:463- 468.
Kuhl, J.C., Cheung, F., Yuan, Q., Martin, W.J., Zewdie, Y., Mccallum, J., Catanach, A., Rutherford, P., Sink, K., Jenderek, M.M., Prince, J.P., Town, C.D., Havey, M.J. 2004. Unique set of 11,008 onion (Allium cepa) ESTs reveals expressed sequence and genomic differences between monocot orders Asparagales and Poales. The Plant Cell. 16:114-125.
Zalapa, J.E., Staub, J.E., Mccreight, J.D. Genetic analysis of branching in melon (Cucumis melo L.). In: Proceedings of the 8th Eucarpia Conference, Cucurbitaceae 2004: Progess in cucurbit genetics and breeding research, July 12-17, 2004, Olomouc, The Czech Republic. p. 373-380.