2011 Annual Report
1a.Objectives (from AD-416)
Objective 1: Determine the genetic basis of and initiate selection for carrot, onion, cucumber, and melon quality attributes influencing human nutrition and health, disease resistances, and yield and quality components, and stress tolerance in cucurbits, and perform field performance and quality trials.
Objective 2: Utilize current biotechnology to discover and evaluate genetic variation and to map agriculturally important traits in Allium, Cucurbit, and Daucus germplasm, and to develop genetic and breeding stocks.
Sub-objective 2.A. Construct genetic maps of nuclear and organellar genomes using candidate genes, SCARs, SSRs, SNPs, transposon insertions, BACs, and cytogenetic stocks.
Sub-objective 2.B. Fine map pigment and carbohydrate genes in carrot and onion, resistance genes for nematode in carrot and viruses in cucurbits, and epistasis, yield and quality components in cucumber.
Sub-objective 2.C. Perform marker-assisted selection of carrot nematode resistance, onion male sterility, and cucurbit yield and quality.
Sub-objective 2.D. Evaluate transgene escape in cucurbits.
Sub-objective 2.E. Determine transposon mobility in carrot.
1b.Approach (from AD-416)
The long-term potential for improving a crop is only as great as the breadth of diversity that breeders utilize. Objective 1 targets evaluation and genetic characterization of carrot, onion, cucumber, and melon germplasm for traits important to growers and consumers.
Discovery Goal 1 - Identify unique phenotypic variation in germplasm collections and breeding stocks to improve nutritional and processing quality, disease resistance, stress tolerance, and yield of Allium, Cucurbit, and Daucus vegetables, genetically characterize observed variation and initiate genetic incorporation of these phenotypes into elite germplasms.
Many biotechnological tools have been developed to improve the efficiency of crop improvement. Objective 2 evaluates and develops these tools of carrot, onion, cucumber, and melon improvement. Identify adequate DNA polymorphisms in elite onion, cucumber, melon, and carrot germplasm to construct genetic maps for marker-facilitated selection of major horticultural traits.
Discovery Goal 2.A – Identify adequate DNA polymorphisms in elite onion, cucumber, melon, and carrot germplasm to construct genetic maps for marker-facilitated selection of major horticultural traits.
Discovery Goal 2.B – Evaluate variation at candidate genes in pigment and carbohydrate biochemical pathways for mapping in onion, cucurbit, and carrot.
Discovery Goal 2.C – Identify and utilize markers to accurately identify desirable genotypes for male sterility restoration in onion, cucurbit yield, and carrot nematode resistance.
Discovery Goal 2.D – Appraise the potential benefit(s) that transgenes might confer on transgenic populations using the ELISA test to estimate the degree of viral infection in wild populations and to determine the potential risk of virus gene introgression from commercial transgenic cultivation.
Discovery Goal 2.E - Determine if native transposable elements in the carrot genome, such as DcMaster, and introduced ones, such as maize elements Ac and Ds transpose to new chromosomal regions.
USDA experimental carrot breeding entries were grown in Holtville, CA. Yield components evaluated root length, shape, forking, smoothness, tip shape, length, uniformity, premature bolting, vigor, earliness & powdery mildew. Quality components include color, smoothness, flavor, texture & nutritional value. Resistance to nematodes & Alternaria leaf blight was evaluated in CA & WI, respectively. New USDA hybrids performed very well in the trial as did USDA germplasm. Flavor evaluation was also performed for all entries. For carrot candidate gene evaluation, four flavonoid pathway genes & two additional nematode resistance genes were identified & genetically mapped. Families were developed to determine the genetic basis of carrot cytoplasmic male sterility restoration, alternaria resistance, & additional nematode resistance. Carrot transposable element variation was evaluated but not observed in a wild relative of carrot from North Africa.
A large collection of onion germplasms were screened for resistance to pink root & Fusarium. Evaluated crosses among different sources of semi-glossy foliages to determine if conditioned by same genes. Continued development of new onion families segregating for health-enhancing fructans, male-fertility restoration, leaf waxiness, & bulb colors. Completed sequencing of onion complementary deoxyribonucleic acid (cDNAs) & presently identifying large numbers of single nucleotide polymorphisms in onion. Gynogenic haploids were previously extracted from hybrids from a cross of a doubled-haploid line with an inbred. These haploids were asexually propagated off of the basal plate & bulbils produced. These bulbils were planted in replicated trials & measured for health-enhancing fructans & flavonoids. DNA was isolated from over 200 haploids for genetic mapping of SNPs without heterozygosity. These markers will be used to more efficiently develop lines for commercial production. This will shorten time for hybrid development to reduce development costs & increase grower competitiveness. A series of crosses were completed to validate a major quantitative trait locus on chromosome 3 of cucumber that controls mitochondrial sorting. The source of this unique trait was sequenced & we are assembling the DNA sequence across the region on chromosome 3 to identify potential candidate gene(s) controlling mitochondrial sorting. A major grant from the USDA-SCRI program will support cloning the gene conditioning resistance to Zucchini Yellow Mosaic Virus in cucumber.
Three cucumber mapping populations were developed for genetic mapping of fruit quality & yield-related genes. Two populations were observed & data collected for fruit quality & yield related traits including fruit number, size & flowering dates. Molecular markers were added for construction of a high-resolution genetic map. Machine trials were conducted in commercial fields & cucumber genomic resources are being developed which include whole genome sequencing & characterization. Resistance gene analogs in the cucumber genome were identified & mapped in the cucumber genome. Molecular mapping of genes for resistances against two cucumber pathogens are underway.
Fluorescent labeled deoxyribonucleic acid (DNA). Carrot chromosomes were associated with linkage groups. This associates DNA variation with important agricultural traits. Researchers have developed genetic maps but the relationship between these carrot genetic maps and carrot chromosomes has not been established. In this research, 17 genes of carrot were located on the chromosomes. This information will help breeders track these genes more easily in breeding programs.
Cucumber whole genome and transcriptome sequencing. In collaboration with the Roche/454 Company, ARS in Madison, Wisconsin sequenced the genome and transcriptome of the pickling cucumber inbred line Gy14. The Gy14 genome was sequenced at 36× genome coverage covering 55% of the cucumber genome. Over 2.3 million ESTs from Gy14 have also been obtained. This work is providing an important new resource for the cucurbit research community for mining of molecular markers, comparative genetic mapping, gene tagging and cloning, as well as other basic and applied researches. The Gy14 draft genome and the EST assembly are now publicly available through the incumbent’s lab database (http://cucumber.vcru.wisc.edu). Microsatellite sequences from the Gy14 draft genome were identified and characterized. Primer sequences for more than 83,000 newly discovered microsatellites were made publicly available. The large amount of microsatellite markers presents a very valuable tool in many basic and applied researches such as molecular mapping, comparative genomics, gene cloning and marker-assisted selection in cucumber breeding.
Molecular mapping of economically important genes in cucumber. Fine genetic mapping (mapping at high resolution) was conducted for genes controlling compact (gene cp) growth habit and resistance against the scab pathogen (gene Ccu), resulting in candidate genes being identified. The compact plant architecture in cucumber has a potential to increase fruit yield in once-over machine harvest production of pickling cucumbers through high-density planting. This work provides molecular markers that are required for implementing marker-assisted selection in cucumber breeding to expedite the classical breeding process. It also provides a foundation for map-based cloning of those genes to understand the gene structure and functions, as well as molecular mechanisms underlying those traits, which may help to address some problems associated with compact cucumber.
Sequencing of expressed regions of the onion genome. Onion has one of the largest amounts of deoxyribonucleic acid (DNA) among all plants. This enormous amount of DNA means that sequencing of random DNA fragments will reveal very few regions that are actually expressed. We completed deep sequencing of the expressed regions of the onion DNA by isolating first RNA from expressed genes, converting this RNA to DNA, and sequenced the DNA. This information will be useful to onion breeders and geneticists in the public and private sectors towards the development of molecular markers for genetic mapping and use in selecting traits of high economic value to develop superior populations.
Root and bulb crop data base (RoBuST) was developed. Information for genomics, genetics, and diversity assessment of important vegetable crops was not previously available, but is now available at this database. This database provides a convenient source of information for scientists.
Kang, H., Yang, Y., Zhang, Z., Zhang, S., Mao, Z., Cheng, G., Gu, X., Huang, S., Weng, Y., Xie, B. 2010. Fine Genetic Mapping Localizes Cucumber Scab Resistance Gene Ccu into an R Gene Cluster. Journal of Theoretical and Applied Genetics. Available: http://www.springerlink.com/content/lqu520225154x357/.
Garcia Lampasona, S., Burba, J.L., Simon, P.W. 2010. Molecular markers: are they really useful to detect genetic variability in local garlic collections? The Americas Journal of Plant Science and Biotechnology. 4(SI1):104-112.
Simkova, H., Safar, J., Kubalakova, M., Suchankova, P., Cihalikova, J., Robert-Quatre, H., Azhaguvel, P., Weng, Y., Peng, J.H., Lapitan, N.L., Ma, Y., You, F.M., Luo, M., Bartos, J., Dolezel, J. 2010. BAC Libraries from Wheat Chromosome 7D – Efficient Tool for Positional Cloning of Aphid Resistance Genes. Journal of Biomedicine and Biotechnology. Available: http://www.hindawi.com/journals/jbb/2011/302543.html.
Cavagnari, P.F., Weng, Y., Senalik, D.A., Yang, L., Simon, P.W., Harkins, T.T., Kodira, C.D., Huang, S. 2010. Genome-wide Characterization of Simple Sequence Repeats in Cucumber (Cucumis Sativus L.). Biomed Central (BMC) Genomics. Available: http://www.biomedcentral.com/1471-2164/11/569.
Miao, H., Zhang, S., Wang, X., Zhang, Z., Li, M., Mu, S., Cheng, Z., Zhang, R., Huang, S., Xie, B., Fang, Z., Zhang, Z., Weng, Y., Gu, X. 2011. A linkage map of cultivated cucumber (cucumis sativus l.) with 248 microsatellite marker loci and seven genes for horticulturally important traits. Euphytica. Available: http://www.springerlink.com/content/l8u2603qg4657g53/fulltext.html.
Li, Y., Pathak, M., Yang, L., Li, D., He, X., Weng, Y. 2011. Fine genetic mapping of Cp, a recessive gene for compact (dwarf) plant architecture in cucumber, cucumis sativus L. Journal of Theoretical and Applied Genetics. Available: http://www.springerlink.com/content/b3062187w253v464/.
Bhasi, A., Simon, P.W., Senalik, D.A., Kumar, B., Manikandan, V., Philip, P., Senapathy, P. 2010. RoBuST: An Integrated Resource of Genomics Information for Plants in the Root and Bulb Crop Families Apiaceae and Alliaceae. Biomed Central (BMC) Plant Biology. 10:161.
Behera, T.K., Behera, S., Bharathi, L.K., John, J., Simon, P.W., Staub, J.E. 2010. Bitter Gourd: Botany, Horticulture, Breeding. Plant Breeding Reviews (J. Janick ed.) 37:101-141. Wiley-VCH, Koln, Germany.
Iovene, M., Cavagnaro, P.F., Simon, P.W., Senalik, D.A., Buell, C.R., Jiang, J. 2011. Comparative FISH mapping of Daucus species (Apiaceae family). Chromosome Research. 19:493-506.