Progress 07/01/02 to 06/30/07
Outputs
Experiments have been conducted on the potential of monoploid potato germplasm in both basic and applied studies. The germplasm was derived by anther culture or through the use of a haploid inducing pollinator on selected clones of the diploid potato, Solanum phureja, that were adapted to temperate climate and long photoperiod. Monoploids derived independently could be combined by protoplast fusion to recover fertile plants. Their chromosome number could also be doubled or quadrupled to derive homozygous diploids and tetraploids that were used to study the influence of ploidy on gene expression. This homozygous germplasm is unique in potato and its fertility offers the potential to study inheritance of traits in a simpler genetic background than has previously been available in potato. Populations have been constructed that segregate for high specific gravity, an important processing character, horizontal late blight resistance, and high carotenoids that are
responsible for yellow tuber flesh in popular cultivars such as Yukon Gold. The utilization of monoploids in potato breeding has been disseminated through academic publications.
Impacts
It has not been possible to study the effect of ploidy on gene expression in potato before the development of germplasm through this project because of the confounding influence of genetic differences in plants that differed in ploidy. The uniqueness of isogenic lines that differed by ploidy allowed us to determine those genes that were upregulated or downregulated in plants at the monoploid, diploid and tetraploid levels. Their allelic simplicity has been exploited to assist SNP (single nucleotide polymorphism) investigations for tetraploid potato. Monoploid potato is perforce sterile because of the absence of homologous chromosomes resulting in aberrant meiosis. Doubling the chromosome number of monoploids to develop homozygous diploids was accomplished, with no clear expectation that the resulting lines would be fertile. Test crosses revealed that nearly all of the homozygous diploids were female fertile and male sterile. Male fertility could be restored by genetic
complementation when monoploids of different origin were combined through protoplast fusion. Although the direct products of protoplast fusion between monoploids were tetraploid plants, their chromosome number could be reduced by anther culture resulting in dihaploids that were also fertile. Markers based on retrotransposons were used to characterize the germplasm. These markers did not reveal somaclonal variation. Much of the germplasm developed through this project is currently maintained in tissue culture. In addition, there is an abundance of seed available that was derived by crosses of homozygous lines with various diploid pollinators.
Publications
- Levy D, Veilleux RE (2007) Adaptation of potato to high temperatures and salinity -- a review. Am. J. Potato Res. 84: 437-456
- Lightbourn GJ, Veilleux RE (2007) Production and evaluation of somatic hybrids derived from monoploid potato. Am. J. Potato Res. 84: (in press)
- Palumbo RE, Veilleux RE (2007) GFP expression in potato. Am. J. Potato Res. 84: (in press)
- Lightbourn GJ, Jelesko JG, Veilleux RE (2007) Retrotransposon-based markers from potato monoploids used in somatic hybridization. Genome 50: 492-501
- Stupar RM, Bhaskar PB, Yandell BS, Rensink WA, Hart AL, Ouyang S, Veilleux RE, Busse JS, Erhardt RJ, Buell CR, Jiang JM (2007) Phenotypic and transcriptomic changes associated with potato autopolyploidization. Genetics 176: 2055-2067
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Progress 10/01/05 to 09/30/06
Outputs
In an effort to improve the vigor and fertility of anther derived inbred lines of Solanum phureja, we have taken two tactics. One is to reduce the ploidy of fertile tetraploid somatic hybrids between two monoploids. We have obtained several dihaploids after one round of anther culture and performed a second round of anther culture on the dihaploids. A few monoploids have been obtained that are now in the process of chromosome doubling by leaf disk regeneration to determine if genetic complementation between monoploids can restore male fertility to inbred lines. Studies on the response of anther-derived sibling doubled monoploids of a 2x heterozygous clone of Solanum phureja to in vitro bacterization with Burkholderia phytofirmans strain PsJN revealed genetic differences in their ability to establish symbiosis with the endophyte. The anther donor responded positively bacterization, i.e., greater in vitro shoot and root fresh wt. We continue to develop and share fertile
inbred lines of potato using adapted S. phureja. A study was conducted at the University of Wisconsin on one of our isogenic series (1x, 2x and 4x) to determine the effect of ploidy on gene expression. The diploid level was found to have the most robust phenotype. Microarray studies revealed few differences in gene expression levels among the ploidies. Diploid selections with high carotenoids, PVY resistance, and high specific gravity from the adapted Solanum phureja/S. stenotomum populations were obtained from USDA and are being used in crosses with homozygous lines in order to obtain half-sib segregating populations for these traits. Such populations will be useful in marker assisted selection.
Impacts
Homozygous lines of diploid and tetraploid potato represent unique germplasm that can be used to study the inheritance of any trait of economic importance.
Publications
- Oosumi T, HA Gruszewski, LA Blischak, AJ Baxter, PA Wadl, JL Shuman, RE Veilleux and V Shulaev. 2006. High-efficiency transformation of the diploid strawberry (Fragaria vesca) for functional genomics. Planta 223:1219-1230.
- Veilleux RE, H De Jong, 2006. Potato. In: Genetic Resources, Chromosome Engineering, and Crop Improvement, Vol. 3, Ram J. Singh (ed.), CRC Press, Boca Raton (in press).
- Trader BW, HA Gruszewski, HL Scoggins and RE Veilleux. 2006. Somaclonal variation of Coreopsis regenerated from leaf explants. HortScience 41:749-752.
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Progress 10/01/04 to 09/30/05
Outputs
Studies on the response of anther-derived sibling monoploids of a 2x heterozygous clone of Solanum phureja to in vitro bacterization with Burkholderia phytofirmans strain PsJN revealed genetic differences in their ability to establish symbiosis with the endophyte. The anther donor responded positively bacterization, i.e., greater in vitro shoot and root fresh wt. Of 23 monoploids examined, most behaved similarly after bacterization; however, for five, in vitro growth parameters declined in the presence of PSJN, suggesting a genetic determinant in the host for ability to exploit beneficial interactions with the endophyte Leaf disc regeneration of the monoploids was done to double their chromosome number for use in sexual crosses and verification of bacterization results with isogenic lines. We now have 18 sibling doubled monoploids (DM) and four quadrupled monoploids. We continue to develop and share fertile inbred lines of potato using adapted S. phureja. We sent
several DM clones to the International Potato Center in Peru for their use in crossing with native diploid potato selected for high pigmentation and stress tolerance. Seed representing a half diallel cross was obtained. We also sent DM germplasm to a cooperator in Great Britain at his request. Initial efforts to select a monoploid for high throughput transformation have been made. Callusing of leaf segments from plants grown in a controlled environment was observed in nearly 100 percent of explants in one of three clones tested. The other clones were not responsive. In anticipation of a study on the genetic basis of heat tolerance in potato, we obtained seed from crosses among clones previously selected for ability to form tubers under heat stress in growth chambers (30 C day/20 C night) or in the field.
Impacts
Homozygous lines of diploid and tetraploid potato represent unique germplasm that can be used to study the inheritance of any trait of economic importance.
Publications
- Veilleux, RE, 2005. Cell and tissue culture of potato (Solanaceae). In: Genetic improvement of solnaceous crops. Vol. 1: Potato. MK Razdan and AK Mattoo (eds.), Science Publishers, Inc., Enfield NH, pp. 185-208.
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Progress 10/01/03 to 09/30/04
Outputs
Anther-derived sibling monoploids of a 2x heterozygous clone of Solanum phureja were bacterized in vitro with Burkholderia phytofirmans strain PsJN. The anther donor reacted positively to bacterization, (greater in vitro shoot and root fresh wt). Of 23 monoploids examined, most behaved similarly; however, for five, in vitro growth declined in the presence of PSJN, suggesting a genetic determinant in the host for ability to exploit beneficial interactions with the endophyte. Through anther culture of 2x clones of adapted S. phureja, we have developed several inbred lines of potato. Leaf disc regeneration of first and second cycle anther-derived monoploids has resulted in many homozygous doubled (DMs) and quadrupled monoploids (QMs). Cross pollination of inbreds with various non-inbred 2x and 4x clones resulted in fruit and seed set, revealing female fertility in the homozygotes. Male fertility, absent in the inbreds, has been restored by protoplast fusion of
independently derived monoploids. Tetraploid intermonoploid somatic hybrids (SH) have been self-fertile and reciprocally fertile with other 4x clones, indicating that the causes of male sterility in the DMs vary and fertility may be restored through complementation and re-extraction of monoploids and DMs from SH. Limited success with anther culture of SH has thwarted this effort so far. Test crosses with DMs and QMs have resulted in a substantial seed inventory of half-sib families. Monoploid and DM germplasm has been sent to research groups on request. The monoploids have been used to verify haplotypes based on single nucleotide polymorphisms (SNPs) identified in heterozygous germplasm of higher ploidy. Molecular markers were developed from monoploid sequences from inter-retrotransposon (IRAP) and sequence-specific amplified polymorphic (S-SAP) bands amplified from the retrotransposon element Tst1 and MseI adapter sites. The markers were polymorphic in a 1x population and also
hybridized to potato and tomato cultivars. A hybrid between a regenerative diploid clone of S. phureja and a haploid inducer was transformed with a plasmid containing genes for green florescent protein (GFP) and kanamycin resistance. GFP expression segregated in the selfed progeny of primary transformants in a 15:1 ratio (GFP:non-GFP), suggesting that primary transformants were double hemizygotes. GFP expression was observed in guard cells, trichomes, along vascular traces, flower petals, sepals and tuber flesh. The selfed progeny of plants that expressed the highest levels of GFP segregated for dwarfism, leaf malformation, and pigmentation. Some plants selected for highest GFP expression as seedlings underwent transgene silencing, such that their clonal propagules expressed little or no GFP. In anticipation of a study on the genetic basis of heat tolerance in potato, we propagated 200 seedlings from crosses among clones previously selected for ability to form tubers under heat stress
in growth chambers (30 C day/20 C night) or in the field. Tuberlings were sent to the Eastern Shore AREC for evaluation. After a hot, rainy season, we selected 30 clones that tuberized well under stressful field conditions.
Impacts
Much of the work examines the potential for developing homozygous lines that may be instrumental in genetic studies and breeding new cultivars of potato.
Publications
- Lightbourn, G, 2004. Development of intermonoploid somatic hybrids of potato and their molecular analysis based on polymorphism of retroelement Tst1. Ph.D. dissertation,Virginia Polytechnic Institute & State University, Blacksburg. 176 pp.
- Skoneczka, JA, 2004. Inheritance and expression of Cry3Aa and PVY-O coat protein transgenes in diploid and tetraploid potato. M.S. Thesis, Virginia Polytechnic Institute & State University, Blacksburg. 87 pp.
- Veilleux, R.E., 2004. Cell and tissue culture of potato (Solanaceae). In: Genetic Improvement of Solanaceous Crops, Vol. 2: Potato, M.K. Razdan (ed.), Science Publishers, Inc., Enfield, NH. (in press)
- Lightbourn, G, R Veilleux, 2003. Retrotransposon based markers to characterize somatic hybrids and assess variation induced by protoplast fusion of monoploid potato. Acta Hort 619:35-43
- Palumbo, R, 2004. The potential for green fluorescent protein as a screening tool in the production of haploid potato plants. M.S. Thesis, Virginia Polytechnic Institute & State University, Blacksburg. 88 pp.
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Progress 10/01/02 to 09/30/03
Outputs
Studies were conducted to determine the response of sibling monoploids derived by anther culture of a heterozygous diploid selection of Solanum phureja to in vitro bacterization with Pseudomonas syringae (PSJN). The anther donor exhibited the typical positive response to bacterization with this endophyte, i.e., greater in vitro shoot and root fresh wt. Of 12 monoploids examined, most behaved like the anther donor. However, for three, in vitro growth parameters actually declined in the presence of PSJN, suggesting a genetic determinant in the host for the ability to enter a symbiotic relationship with the endophyte. Fertile tetraploid somatic hybrids (SH) were developed by fusion of differentially derived monoploid potato lines. Different intermonoploid SH were intercrossed to recombine the genomes of four monoploids in tetraploid seedlings. A field study was done to compare these seedlings with related diploid seedlings that had not been through anther culture, and
others derived from crosses between SH and cvs. Atlantic or Katahdin. A cool wet growing season and late planting reduced yield in general. The only seedlings that approached the yield of the cvs. were derived from crosses between cvs. and SH. The seedlings from crosses between intermonoploid SH yielded poorly, perhaps due to their lack of adaptation. The intermonoploid SH were analyzed using S-SAP (sequence specific amplified polymorphism. Primer pairs were designed to target MseI adapter sites and the potato retrotransposon element Tst1. Little polymorphism (15 of 120 bands scored) was observed between monoploids derived from genetically distinct source plants. Cloned fragments were selected and sequenced. Preliminary sequence analysis revealed that Tst1 had inserted into distinct sites that had no match in the GenBank. The paucity of polymorphism among different genotypes suggests that Tst1 is an ancient retrotransposon not influence by tissue culture manipulation. Using ELISA to
determine transgene expression in reciprocal crosses of tetraploid potato lines, one of which carried the Cry3Aa gene as a single insert, we found that overall expression levels of progeny bearing the transgene did not dramatically differ. However, in crosses between transgenic Atlantic and wild type Katahdin, the ratio of expressors to nonexpressors varied significantly between reciprocals and differed significantly from the 1:1 segregation expected. Furthermore, an overabundance of transgenic progeny when the Cry3Aa gene was maternally inherited suggests differential gametic selection or an epigenic effect on transgene expression during megagametogenesis and/or pollination and fertilization. A hybrid between a highly regenerative diploid clone of Solanum phureja and haploid inducer was transformed with a plasmid containing genes for green florescent protein (GFP). Hemizygous primary transformants had variable GFP expression that was greatest in guard cells, trichomes and along
vascular traces. The selfed progeny of plants that expressed the highest levels of GFP segregated for dwarfism, anthocyanin pigmentation and GFP expression.
Impacts
Much of the work examines the potential for developing homozygous lines that may be instrumental in genetic studies and breeding new cultivars of potato.
Publications
- Johnson, AAT, BA Nault, RE Veilleux, 2003. Transmission of a Bacillus thuringiensis cry3Aa transgene from diploid to tetraploid potato using 4x-2x hybridization: effect of ploidy increase on transgene expression and implications for TPS hybrid production. Plant Breed 122:223-228
- Chen, P, GR buss, SA Tolin, RE Veilleux, 2003. A tissue culture method for the preservation of Soybean mosaic virus strains. Plant Cell Tiss Organ Cult 74:185-192
- Skoneczka, J, R Veilleux. 2003 Effects of parent-of-origin on transgenic Cry3Aa expression in potato hybrids. HortScience 38: 803 (Abstr.)
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Progress 10/01/01 to 09/30/02
Outputs
Intermonoploid somatic hybrids (ISH) of potato have been developed by protoplast fusion of genetically distinct monoploids derived from diploid clones of the adapted Solanum phureja population. Three differently constructed ISH have been established in the greenhouse and crosses are underway among them. Both male and female fertility have been observed in the 4x ISH, even though male fertility was absent in the monoploids used to construct ISH and their doubled monoploids derivatives. Therefore male fertility has been restored throught genetic complementation. Seeds from crosses between 4x ISH represent unique potato germplasm comprised of four reshuffled genomes after elimination of lethal alleles through monoploidization. We are currently using retrotransposon display to observe somaclonal variation acquired through the process of protoplast fusion. Anther culture studies are underway on the 4x ISH to reduce the genome to the dihaploid level and also on hybrids
between 4x ISH and potato cultivars to try to introgress S. tuberosum germplasm and relevant horticultural characters into the experimental population. A set of 58 anther-derived potato monoploid siblings has been used to construct an amplified length polymorphism (AFLP) map that highlights, through distorted segregation, regions of the genome that are preferentially excluded during the process of haploidization.
Impacts
This is the only cultivated potato germplasm available that is free of lethal genes that are present in all cross breeding population of organisms. This germplasm will serve as the basis for inbred line development. Such inbred lines can serve as parents of uniform hybrids or test plants to observe simplified segregation of horticultural traits.
Publications
- Johnson, AAT and RE Veilleux, 2002. Integration of transgenes into sexual polyploidization schemes for potato (Solanum tuberosum L.) Euphytica (in press)
- Boluarte-Medina, T, E Fogelman, E Chani, AR Miller, I Levin, D Levy, RE Veilleux, 2002. Identification of molecular markers associated with leptine in reciprocal backcross families of diploid potato. Theor Appl Genet 105:1010-1018
- Chani, E, V Ashkenazi, J Hillel and RE Veilleux, 2002. Microsatellite marker analysis of an anther-derived potato family: Skewed segregation and gene-centromere mapping. Genome 45:236-242
- Varrieur, JM, 2002. AFLP marker analysis of monoploid potato. M.S. Thesis, Virginia Polytechnic Institute & State University, Blacksburg, Va.
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