Source: MAYVILLE STATE UNIVERSITY submitted to
DISEASE RESISTANCE ORTHOLOGS AND THEIR LOCALIZATION IN THE COMMON BEAN GENOME
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0207432
Grant No.
2006-35300-17153
Project No.
NDR-2006-03590
Proposal No.
2006-03590
Multistate No.
(N/A)
Program Code
52.1
Project Start Date
Aug 1, 2006
Project End Date
Jan 31, 2009
Grant Year
2006
Project Director
Khwaja, H.
Recipient Organization
MAYVILLE STATE UNIVERSITY
330 THIRD STREET NE
MAYVILLE,ND 58257
Performing Department
(N/A)
Non Technical Summary
The genomic database resources of important plant and animal species and tools required to analyze these resources are now publicly available. The exploitation of these resources enables the rapid identification of genes and facilitates the examination of their functions in related species. The common bean is one of the most important species of leguminous crops but it lacks adequate genomic resources and the average global yield of this species is low because of its susceptibility to wide range of diseases. To maximize and sustain the production, it is essential to develop high yielding varieties of common bean with disease resistance traits. Classical breeding in common bean has given excellent results in the last two decades. To speed up the progress in this work and harness the valuable genes conditioning disease resistance in common bean, it is necessary to identify the portion of the genome responsible for the expression of these resistance traits along with their associated molecular markers. Using genomic database resources of related species, this project is aimed at delivering equivalent genomic portions of common bean conditioning disease resistance and associated markers to monitor their introgression into the breeding lines of common bean. This project will expand the research base of Mayville State University (MSU) and strengthen the PD`s position for future competitive grants. Hands-on experience with these problem based research will allow students to explore a rapidly changing area and the conceptual tools involved in these areas of biology.
Animal Health Component
(N/A)
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20114101080100%
Knowledge Area
201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1410 - Beans (dry);

Field Of Science
1080 - Genetics;
Goals / Objectives
Biological sciences have been revolutionized, not only in the way research is conducted but also in the way findings are communicated to professionals and to the public. The ever increasing number of publicly available genomic database resources of genomic sequences of different plant and animal species, full length genomic sequences and corresponding coding sequences, underlying protein sequences, gene and gene families, gene ontology, and comparative genomics have opened up the scope of utilizing gene information from one species for the benefit of other species lacking basic genomic tools. The common bean consisting of dry and snap beans, is one of the most important species of leguminous crops but there are many diseases that significantly reduce the persistence and productivity of common bean in the US and worldwide. Due to the lack of adequate genomic resources, bean breeders and growers across the world have been suffering from a shortage of functional markers for tagging and monitoring disease resistance traits in this species. Using the genomic database resources of related species, this project will identify equivalent genomic resources and develop molecular markers associated with disease resistance genes in common bean. It will take advantage of the high level of gene conservation in an inter-specific manner in the search for functional genes. In this project we plan to use this information with the following objectives: 1)Develop and validate orthologous gene sequences from Leguminosae in common bean focusing on candidate genes for different disease resistance 2)Develop functional markers segregating in different mapping population and localize them in the genome of common bean The development and validation of orthologous gene sequences and addition of gene-based markers in common bean focusing on candidate genes for disease resistance will augment the resistance gene tagging, mapping, and marker-assisted breeding.
Project Methods
1. Identify disease resistance gene orthologs in common beans Genomic databases will be searched for all possible disease resistance related genes in leguminous species. The tentative consensus and singleton EST sequences of common bean will be identified by blasting those gene sequences against the eukaryotic gene orthologs (EGO) and the P. coccineus EST databases. Corresponding common bean sequence of each query sequence obtained from these two databases will be compared and common bean ESTs with higher scores and probability will be considered. The queries with no hit from any of the databases will be blasted against Medicago truncatula and soybean ESTs databases. 2. Develop markers for disease resistance gene PCR-primers will be designed from the identified sequences. Based on the predicted average intron size of 161 bp in Medicago trancatula, primer pairs will be designed to amplify genomic DNA of maximum 350 bp in size using the web-based PCR primer designing program. For larger size gene sequences (over 350 bp), overlapping primer pairs will be designed, so that assembled amplified products corresponds to the equivalent size of the gene. All of the designed primers will be used to amplify genomic DNA of the parents of mapping populations segregating for different disease resistance traits. Primer pairs producing polymorphic products will be grouped according to the segregating populations and considered as EST-STS markers. The amplified genomic DNA will be sequenced and SSRs will be identified using Simple Sequence Repeat Identification Tool (SSRIT). The primers developed from the flanking SSR sequences will be considered as EST-SSRs and will be assessed against the populations segregating for disease resistance traits. In the cases, where no EST-STSs and EST-SSRs are found, amplified DNA sequences between the parents will be aligned to survey the parental alleles for polymorphic sites. Single-nucleotide polymorphisms (SNPs) will be converted to cleaved amplified polymorphic sequences (CAPS) by identifying SNPs that confer differential restriction enzyme sites between the two parental alleles. In cases in which a suitable restriction enzyme site is not identified, oligonucleotide primers with a single nucleotide mismatch will be designed to the polymorphic position and will be considered as EST-SNPs. To validate the resistance gene orthologs in common bean, the resulting gene sequences in common bean will be compared with the starting resistance gene sequences as well as with the sequences from which primer pairs were designed. 3. Localize resistance gene orthologs in the bean genome All the sequence and marker information will be made available through LIS (Legume Information Service) to assess and map disease resistance traits for the bean mapping populations as well as other related legume species. The resulting polymorphic markers developed in this study with strong associations with disease resistance will be integrated in the bean BJ core mapping population.

Progress 08/01/06 to 01/31/09

Outputs
OUTPUTS: The objectives of this project were to a) develop and validate orthologous gene sequences from Leguminosae in common bean focusing on candidate genes for different disease resistance and b) develop functional markers segregating in different mapping population and localize them in the genome of common bean. One hundred forty-two resistance gene sequences consist of complete and partial coding sequences of NBS-LRR, RGAs, microsatellite DNA clones, resistance protein clones, SCAR-RAPD fragments, disease resistance like protein, etc. downloaded from Phaseolus resistance gene database and analyzed. Resistance gene sequences were anlyzed using different bioinformatic tools and PCR primers were developed from overlapping sequences to cover complete sequences. The developed primers were screened against parents of seven ampping population segregating for difefrent disease resistance traits. Because of multiple copies of resistance gene sequences in the genome, we have also worked on cloning and sequencing of overlapping PCR products identified by complete coding sequences of NBS-LRR types. NBS-LRR sequences specific PCR amplified products of 13 parental lines were cloned and five random clones were sequenced. This project helped developing research attitude among the undergraduates at Mayville State University. The most notable achievement of this project is that one of the Native American students was pursuing her MS in plant sciences at North Dakota State University and later on obtained job in a biotechnology company. The undergraduate students participated in research works of this projects have their authorships in all publications and attended different in-state/out of state research meetings. PARTICIPANTS: 1. Khwaja Hossain, PD- Overall supervision and directions 2. Zahirul Takukder, Research faculty, involved in all laboratory and green house related activities 3. Six undergraduate students- Trained and involved in difefrent period of times- DNA extraction, PCR analysis, and gel electrophoresis TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Initially it was plan to work on disease resistance gene orthologs of legume and identify, and validate equivalent sequences in common bean by direct sequencing of PCR products. However, because of multiple copies of resistance gene sequences in the genome, we are worked on cloning and sequencing of overlapping PCR products identified by complete coding sequences of NBS-LRR types. Amplified products of 13 parental lines of seven mapping populations segregating for different disease resistant traits were excised, cloned and sequenced to identify NBS-LRR sequences in common bean.

Impacts
A total of 90 functional markers were developed and analyzed among parental combinations of the mapping population. Thirty-eight percent of these markers have been polymorphic and identified population specific resistance gene markers. In addition to the parental lines of the mapping population, these markers also have been analyzed among 16 common bean genotypes consist of six North Dakota common bean cultivars and 10 CIAT common bean lines selected for breeding for high mineral content. During this project period, we have also analyzed 34 SSRs markers and 21 markers developed from tentative consensus (TC) sequences of common bean in all of the 29 genotypes. Selected markers in this study have been used to estimate genetic diversity among the common bean genotypes. Over 150 NBS-LRR type incomplete sequences were obtained from different common bean genotypes. Genotype level NBS-LRR type sequence variations were evident and potentials of using these sequences for developing SNP to CAPS markers that could increase polymorphism by six folds were observed.

Publications

  • Z. I. Talukder, P. N. Miklas, K. Schimt, E. A. Anderson, J. Osorno, M. Dilwari, and K. G. Hossain. 2008. Genetic diversity and selection of common bean genotypes to enhance mineral content. Submitted to Plant Breeding. Erika Anderson, Kayla Schmidt, Zahirul Talukder, Phillip Miklas, Khwaja Hossain 2008. Genetic diversity and selection of common bean genotypes for mapping genes conditioning mineral contents. Proceedings of the North Dakota Academy of Science, April, 2008. Erika Anderson, Kayla Schmidt, Zahirul Talukder, Phillip Miklas, Khwaja Hossain. 2007. Functional Markers of Common Bean from Disease Resistance Gene Orthologs. The Annual Report of the Bean Improvement Cooperative. Vol. 50: 75-76. Zahirul I. Talukder, Kayla Schmidt, Erika L. Anderson, Phillip N. Miklas, Thomas P. Gonnella, and Khwaja G. Hossain. 2008. Selection of Markers for Mapping and Cloning Disease Resistance in Common Bean. The Annual Report of the Bean Improvement Cooperative. Vol. 51: 95-96. Poster Genetic Diversity Among Common Bean Lines Assessed With Molecular Markers Developed From R-Gene, TC, And Simple Sequence Repeat Sequences, 2008. Plant & Animal Genome XV, January 11-16, 2008, Town and Country Hotel, San Diego, California. Selection of Markers for Mapping and Cloning Disease Resistance in Common Bean. 2008. Plant & Animal Genome XV, January 11-16, 2008, Town and Country Hotel, San Diego, California. Selection of Markers for Mapping and Cloning Disease Resistance in Common Bean. 2007. Bean Improvement Cooperative 19th Biennial Meeting, Madison, Wisconsisn. Disease Resistance Gene Orthologs and their Localization in Common Bean. Plant & Animal Genome XV, January 13-18, 2007, Town and Country Hotel, San Diego, CA.