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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

2013 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.

BSL-1; Recertified through August 5, 2012. Certificate #SC09-161R.


3.Progress Report:
New wild carrot germplasm was collected in Tunisia, California, and Morocco. USDA experimental carrot breeding entries were evaluated for field productivity and consumer quality traits, as well as resistance to nematodes, and Alternaria leaf blight was evaluated in California and Wisconsin, respectively. New USDA hybrids performed very well in the trial, as did USDA germplasm. Flavor evaluation was also performed for all entries. Nine flavonoid and four carotenoid pathway genes and three additional nematode resistance genes were identified as candidate genes 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. Carrot breeding is more efficient with genetic information about important traits and molecular tools to facilitate the breeding progress.

Onion germplasm were screened for resistance to pink root and Fusarium, foliage variation evaluated, and traits genetically analyzed including fructans, male-fertility restoration, leaf waxiness, and bulb colors. Completed sequencing of onion complementary deoxyribonucleic acid (cDNAs) and 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 to map deoxyribonucleic acid (DNA) polymorphisms. This will shorten time for hybrid development to reduce development costs and increase grower competitiveness.

New cucumber mapping populations were developed for framework or fine genetic mapping of genes controlling fruit skin and spine color, little leaf, parthenocarpy fruit setting, anthracnose resistance, and powdery mildew resistance quantitative trait loci (QTL)S. Phenotyping was conducted in both the greenhouse and field for fruit number, size and flowering dates, parthenocarpy fruit setting, as well as disease resistances. Linkage maps are being developed for linkage analysis of these genes or QTL with molecular markers. Molecular and cytological tools were used to reveal the evolutionary history of chromosomes in Cucumis species. Machine trials were conducted in commercial fields. These results will accelerate genetic selection in cucumber breeding programs.

This research relates to Objective 1 by elucidating the genetic basis of carrot, onion, and cucumber disease resistance, consumer quality, and seed productions, and to Objective 2 to develop genetic and breeding stocks, by developing genetic maps, transposon mobility patterns, and marker-assisted selection populations of carrot, onion, and cucumber.


4.Accomplishments
1. Molecular evidence for a single origin of domesticated carrot. Wild carrot is thought to have its geographic origins in Central Asia, based on historical records, although it is widespread globally today. While carrot has been cultivated as a root crop about 1100 years, the genetic relationship between wild and cultivated carrot has not been understood, and the geographic origins of cultivated carrot are uncertain. Utilizing 3481 deoxyribonucleic acid (DNA) markers, ARS scientists at Madison, WI, in collaboration with researchers at five universities determined that carrot was first domesticated in Central Asia and deduced that wild carrot in North America originated in Europe, and did not likely arrive with the first American settlers that arrived from Asia, or as escapes of domesticated carrot. These studies help scientists understand the relationship between wild carrots and domesticated carrots, they help us understand where North American wild carrot originated, and they indicate that there is a wealth of genetic diversity available in both wild and cultivated carrot for future carrot breeders to utilize for crop improvement.

2. Plant deoxyribonucleic acid (DNA) moves from mitochondria to plastids. DNA is the genetic material in all organisms that makes up genes, and most DNA is located in chromosomes. But in plants there is a small amount of DNA in two sub-cellular compartments: the mitochondria and the plastids. There is some research evidence over the last several decades that in rare cases DNA has moved between the chromosomes, mitochondria, and plastids. In this research, ARS scientists at Madison, WI, in collaboration with researchers at five universities uncovered evidence that DNA in the carrot mitochondrion has moved to the carrot plastid. This is the first report of DNA transfer into plastid genomes of higher (flowering) plants. This research helps scientists understand fundamental mechanisms of DNA mobility. This research broadens the thinking about the origins of genetic variation which accounts for all biological diversity, both within a species, and between species on earth.

3. Mapping genes for carrot flowering and pollen fertility. Most people growing carrots never see the plant flower, but exposure of carrots to cool temperatures stimulates carrot flowering. Carrots in warmer parts of the world flower without exposure to cold. In flowering carrots, some plants produce pollen (are male fertile) and some do not (are male sterile). This characteristic is controlled by several genes including one called Rf1 or “restorer of fertility.” In this research, ARS scientists at Madison, WI, in collaboration with researchers at Cuyo University discovered that this difference in initiation of flowering in carrots is controlled by one gene called Vrn1, and we determined the chromosomal locations of Vrn1 and Rf1 in the carrot genome. This research provides carrot breeders with information and tools to more efficiently breed the crop, and it provides plant scientists information to determine the biological foundations of these two interesting reproductive traits.

4. Genetics and mapping of important traits of onion. Resistance to diseases, pollen fertility, and nutritional quality are all traits controlled by onion genes. Using over 1000 single nucleotide polymorphisms (SNPs) these important characteristics of onion were located on the onion genetic map, and information about these SNPs was published and available to stakeholders in the public and private sectors and in 2014 will be added to website http://alliumgenetics.org/cmsmadesimple/index.php?page=maps. This research is valuable to onion breeders since it provides them with information for tracking these traits while they breed for improved onions for the U.S. market.

5. Development of an integrated cucumber genetic-physical map. Genetic maps are useful for crop improvement, and relatively little genetic map development has been undertaken for cucumber. ARS scientists, in collaboration with researchers in the Chinese Academy of Agricultural Sciences in China, developed a consensus genetic map with 1,681 deoxyribonucleic acid (DNA) markers for cultivated cucumber. Information of the molecular markers, and associated genetic and physical maps has been released to researchers and seed companies through relevant publications (available at http://www.biomedcentral.com/1471-2229/13/53/). These will be very useful tools for cucumber breeding by seed companies.

6. Map-based cloning of a candidate gene for spine and mature fruit color and powdery mildew resistance in cucumber. Cucumbers of different market classes differ in the color, number, and density of spines on the fruit skin. A single gene called B controls both the spine color and mature fruit color of cucumber, but genetic control of mildew resistance is more complicated. In a study conducted by ARS scientists in Madison, WI, the B gene was found to be located in a small deoxyribonucleic acid (DNA) region, and two major chromosome regions were identified to control mildew resistance. This research provides new insights into genetic mechanisms controlling fruit characters and powdery mildew resistance in cucumber, and will help cucumber breeders develop higher quality disease resistant cucumbers for consumers and growers.


Review Publications
Lu, J., Qi, J.J., Shi, Q.X., Shen, D., Zhang, S.P., Shao, G.I., Li, H., Sun, Z.Y., Weng, Y., Shang, Y., Van Treuren, R., Van Dooijeweert, W., Zhang, Z.H., Huang, S.W. 2012. Genetic diversity and population structure of cucumber (Cucumis sativus L.). PLoS One. Available: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0046919.

Alessandro, M.S., Galamarini, C.R., Iorizzo, M., Simon, P.W. 2013. Molecular mapping of vernalization requirement and fertility restoration genes in carrot. Theoretical and Applied Genetics. 126(2):415-423.

Iorizzo, M., Grzebelus, D., Senalik, D.A., Szklarczyk, M., Spooner, D.M., Simon, P.W. 2012. Against the traffic: The first evidence for mitochondrial DNA transfer into the plastid genome. Mobile Genetic Elements. 2(6):261-266.

Iorizzo, M., Senalik, D.A., Ellison, S., Grzebelus, D., Cavagnaro, P., Allender, C., Brunet, J., Spooner, D.M., Van Deynze, A., Simon, P.W. 2013. Genetic structure and domestication of carrot (Daucus carota subsp. sativus) (Apiaceae). American Journal of Botany. 100(5):930-938.

Simon, P.W., Iorizzo, M., Senalik, D.A., Szklarczyk, M., Grzebelus, D., Spooner, D.M. 2012. De novo assembly and characterization of the carrot mitochondrial genome using next generation sequencing data from whole genomic DNA provides first evidence of DNA transfer into an angiosperm plastid genome. Biomed Central (BMC) Plant Biology. 12:1-17.

McManus, M.T., Joshi, S., Leung, S., Albert, N., Pither-Joyce, M., Shaw, M., Mccallum, J., Searle, B., Shigyo, M., Jakse, J., Havey, M.J. 2012. Genotypic variation in sulfur assimilation and metabolism of onion (Allium cepa L.) III. Characterization of sulfite reductase. Phytochemistry. 83:34-42.

Yang, L., Li, D., Li, Y., Gu, X., Huang, S., Garcia-Mas, J., Weng, Y. 2013. A 1,681-locus consensus genetic map of cultivated cucumber including 67 NB-LRR resistance gene homolog and ten gene loci. Biomed Central (BMC) Plant Biology. 13(53):1-14.

Azhaguvel, P., Rudd, J.C., Ma, Y., Luo, M., Weng, Y. 2011. Fine genetic mapping of greenbug aphid resistance gene Gb3 in Aegilops tauschii. Theoretical and Applied Genetics. 124(3):555-564.

Weng, Y., Yang, L., Koo, D., Li, Y., Xuejiao, Z., Luan, F., Havey, M.J., Jiang, J. 2012. Chromosome rearrangements during domestication of cucumber as revealed from high-density genetic mapping and draft genome assembly . Plant Journal. 71:895-906.

Garcia-Mas, J., Benjak, A., Sanserverino, W., Bourgeois, M., Mir, G., Gonzalez, V.M., Henaff, E., Camara, L., Cozzuto, L., Lowy, E., Alioto, T., Capella-Gutierrez, S., Blanca, J., Canizares, J., Ziarsolo, P., Gonzalez-Ibeas, D., Rodriguez-Moreno, L., Droege, M., Du, L., Alvarez-Tejado, M., Lorente-Galos, B., Mele, M., Yang, L., Weng, Y., Navarro, A., Marques-Bonet, T., Aranda, M.A., Nuez, F., Pico, B., Gabaldon, T., Roma, G., Guigo, R., Casacuberta, J.M., Arus, P., Puigdomenech, P. 2012. The genome of melon (Cucumis melo L.). Genome amplification in the absence of recent duplication in an old widely cultivated species. Proceedings of the National Academy of Sciences. 109(29):11872-11877.

Duangjit, J., Bohanec, B., Chan, A.P., Town, C.T., Havey, M.J. 2013. Transcriptome sequencing to produce SNP-based genetic maps of onion. Theoretical and Applied Genetics. 126(8)2093:2101.

He, X., Li, Y., Pandey, S., Yandell, B.S., Pathak, M., Weng, Y. 2013. QTL mapping of powdery mildew resistance in WI 2757 cucumber (Cucumis sativus L). Theoretical and Applied Genetics. 126(8):2149-2161.

Li, Y., Wen, C., Weng, Y. 2013. Fine mapping of the pleiotropic locus B for black spine and orange mature fruit color in cucumber identifies a 50 kb region containing a R2R3-MYB transcription factor. Theoretical and Applied Genetics. 126(8):2187-2196.

Bowman, M.J., Simon, P.W. 2013. Quantification of the relative abundance of plastome to nuclear genome in leaf and root tissues of carrot (Daucus carota L.) using quantitative PCR. Plant Molecular Biology Reporter. 31(4):1040-1047.

Yildiz, M., Willis, D.K., Cavagnaro, P.F., Iorizzo, M., Abak, K., Simon, P.W. 2013. Expression and mapping of anthocyanin biosynthesis genes in carrot. Theoretical and Applied Genetics. 126(7):1689-1702.

Last Modified: 8/27/2014
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