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United States Department of Agriculture

Agricultural Research Service

Research Project: ALLIUM, CUCUMIS, AND DAUCUS GERMPLASM ENHANCEMENT, GENETICS, AND BIOCHEMISTRY

Location: Vegetable Crops Research Unit

2012 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.


3.Progress Report:
New wild carrot germplasm was collected in California, USDA experimental carrot breeding entries were grown in California, & yield and quality components were evaluated. Populations were used to develop genetic maps & resistance to nematodes and Alternaria leaf blight was evaluated. New USDA hybrids performed very well. Flavor was evaluated for all entries. For carrot candidate gene evaluation, nine flavonoid and four carotenoid pathway genes and three additional nematode resistance genes were identified and genetically mapped. Families were developed to determine the genetic basis of carrot cytoplasmic male sterility restoration, alternaria resistance, and additional nematode resistance. The carrot transcriptome was analyzed and characterized and simple sequence repeat (SSR) markers were developed. Carrot transposable element variation was evaluated but not observed in a wild relative of carrot from North Africa. These results help breeders develop improved stock for growers and consumers.

Onion germplasm was screened for resistance to pink root and Fusarium basal rot. Evaluated crosses among different sources of semi-glossy foliage to determine if this phenotype is conditioned by multiple genes. Continued development of onion families segregating for health-enhancing fructans and anthocyanins, male-fertility restoration, leaf waxiness, & bulb colors. Sequencing identified large numbers of single nucleotide polymorphisms in onion. Gynogenic haploids were previously extracted from hybrids, asexually propagated, planted in trials and measured for health-enhancing fructans and flavonoids. Deoxyribonucleic acid (DNA) was isolated from over 180 haploids for genetic mapping. 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. Crosses were completed to validate a major quantitative trait locus on chromosome 3 of cucumber that controls mitochondrial sorting. A major grant from the USDA-Specialty Crops Research Initiative (SCRI) program will support cloning of the Zucchini Yellow Mosaic Virus resistance in cucumber.

Three new cucumber mapping populations were developed for genetic mapping of fruit quality & yield-related genes and powdery mildew resistance quantitative trait loci (QTLs). Two populations were observed & data collected for fruit quality and yield related traits including fruit number, size and flowering dates, as well as powdery mildew resistance. Molecular markers were added for construction of a high-resolution genetic map. An integrated genetic-physical map for cucumber was developed. Molecular and cytological investigations are revealing the evolutionary history of chromosomes in several cucurbit species. Machine trials were conducted in commercial fields and cucumber genomic resources are being developed which include whole genome sequencing & characterization. NB-LRR type resistance gene homologs were identified and characterized from cucumber genome and were genetic mapped. Molecular mapping of genes for resistances against the anthracnose and powdery mildew pathogens are underway.


4.Accomplishments
1. Carrot genetic markers. Genetic markers are valuable tools for crop improvement but had not been developed for carrot. In collaboration with researchers in California, Poland, and Argentina, many genetic markers were developed to help track carrot genes important to improve the crop for growers, such as disease resistance, and consumers, such as flavor and nutritional value. These biotechnological resources provide tools for breeding to improve carrot and its relatives, such as celery.

2. Development of an integrated cucumber genetic-physical map. A high-density, integrated genetic-physical map is indispensible for many basic and applied studies. A high-density linkage map (735 markers) and consensus map (1,681 markers) for cultivated cucumber have been developed. Whole genome scaffolds covering 96% of the assembled Gy14 genome were anchored to this genetic map. A number of assembly errors in draft genome scaffolds were identified and corrected. This research emphasized the importance of systematic and independent validation of the quality of draft genomes from de novo assembly of sequences by next generation sequencing technologies, which are showing exponential increase in many organisms. This integrated genetic-physical map provides a solid base for developing a reference map for cucumber and an important resource for the larger cucurbit research community in many uses such as map-based gene cloning and marker-assisted selection in cucumber breeding.

3. Domestication of cucumber and chromosome evolution in Cucurbitaceae. Through multiple, interdisciplinary approaches, global differentiations including six inversions between chromosomes of wild and cultivated cucumbers were revealed. This work supports the notion that C. sativus var. hardwickii as the progenitor of modern cultivated cucumber. These new findings advanced our knowledge in understanding the domestication process and chromosome evolution in the genus Cucumis, and revealed closely related Cucumis species for germplasm enhancement.

4. 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 and identified 1000s of sequence differences in these expressed regions. 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.


Review Publications
Havey, M.J., Kielkowska, A.Z. 2011. In vitro flowering and pollen viability of cucumber. Plant Cell Tissue And Organ Culture. 109:73-82.

Howard, N., De Leon, N., Havey, M.J., Martin, W. 2012. Diallel analysis of floral longevity in Impatiens walleriana. Journal of the American Society for Horticultural Science. 137(1):47-50.

Zhang, H., Wang, H., Guo, S., Ren, Y., Gong, G., Weng, Y., Xu, Y. 2011. Identification and validation of a core set of microsatellite markers for genetic diversity analysis in watermelon, Citrullus lanatus Thunb. Matsum. & Nakai. Euphytica. 186:329-342.

Calderon, C.I., Yandell, B.S., Havey, M.J. 2012. Genetic mapping of paternal sorting of mitochondria in cucumber. Theoretical and Applied Genetics. 125:11-18.

Grzebelus, D., Baranski, R., Spalik, K., Allender, C., Simon, P.W. 2011. Daucus. In: Kole, C., editor, Wild Crop Relatives: Genomic and Breeding Resources. New York, NY:Springer. p. 91-113.

Li, D., Weng, Y., Cuevas, H., Yang, L., Li, Y., Garcia-Mas, J., Zalapa, J.E., Staub, J.E., Luan, F., Reddy, U., He, X., Gong, Z. 2011. Syntenic relationships between cucumber (Cucumis sativus L.) and melon (C. melo L.) chromosomes as revealed by comparative genetic mapping. Biomed Central (BMC) Genomics. 12:396-409.

Bo, K., Song, H., Shen, J., Qian, C., Staub, J.E., Simon, P.W., Lou, Q., Chen, J. 2011. Inheritance and mapping of the ore gene controlling the quantity of ß-carotene in cucumber (Cucumis sativus L.) endocarp. Molecular Breeding. 30(1):335-344.

Cavagnaro, P.F., Chung, S., Manin, S., Yildiz, M., Ali, A., Alessandro, M.S., Iorizzo, M., Senalik, D.A., Simon, P.W. 2011. Microsatellite isolation and marker development in carrot - genomic distribution, linkage mapping, genetic diversity analysis and marker transferability across Apiaceae. Biomed Central (BMC) Genomics. 12:386.

Staub, J.E., Simon, P.W., Cuevas, H.E. 2011. USDA, ARS EOM 402-10 high B-carotene cucumber. HortScience. 46:1426-1427.

Cavagnaro, P.F., Weng, Y., Senalik, D.A., Simon, P.W., Harkins, T.T., Huang, S. 2010. Genome-wide characterization of simple sequence repeats in cucumber (Cucumis sativus L.). Biomed Central (BMC) Genomics. 11:569-588.

Baransi, R., Maksylewicz-Kaul, A., Nothnagel, T., Cavagnaro, P., Simon, P.W., Grzebelus, D. 2011. Genetic diversity of carrot (Daucus carota L.) cultivars revealed by analysis of SSR loci. Genetic Resources and Crop Evolution. 59(2):163-170.

Iorizzo, M., Senalik, D.A., Grzebelus, D., Bowman, M., Cavagnaro, P.F., Matvienko, M., Ashrafi, H., Van Deynze, A., Simon, P.W. 2011. De novo assembly and characterization of the carrot transcriptome reveals novel genes, new markers, and genetic diversity. Biomed Central (BMC) Genomics. 12:389.

Last Modified: 8/22/2014
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