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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETICS AND GENETIC IMPROVEMENT OF DISEASE RESISTANCE AND QUALITY TRAITS IN WATERMELON, BROCCOLI, AND LEAFY GREEN BRASSICAS

Location: Vegetable Research

2013 Annual Report


1a.Objectives (from AD-416):
1. Determine the genetic control of resistance to important diseases and pests of watermelon and release resistant breeding lines developed. 1.A. Determine genetic control of root-knot nematode (Meloidogyne incognita) resistance in watermelon; identify and map DNA-based markers closely linked to the resistance locus (loci). 1.B. Determine genetic control of Fusarium wilt (Fon race.
2)resistance in watermelon; identify and map DNA-based markers closely linked to the resistance locus (loci).

2. Utilize genomic tools to develop genetic linkage maps for watermelon and diagnostic DNA-based markers for host-plant resistance to viruses and key watermelon fruit traits. 2.A. Map and isolate DNA sequences associated with Zucchini Yellow Mosaic Virus (ZYMV) resistance in watermelon. 2.B. Identify and map DNA sequences associated with watermelon fruit quality traits (e.g., fruit size and shape, flesh color and texture, carotenoid levels, and soluble content).

3. Develop lines of broccoli improved for economically important traits. 3.A. Develop inbred broccoli lines with tolerance to high temperature stress, elucidate the underlying genetic control of the tolerance, and identify quantitative trait loci (QTL) and associated DNA-based markers for the tolerance. 3.B. Breed high yielding, self-compatible inbred broccoli lines with high productivity and high levels of health-promoting compounds (e.g., glucoraphanin) as compared to that of standard hybrid cultivars.

4. Elucidate the genetic control of bacterial leaf spot resistance in leafy green Brassicas (B. juncea and B. rapa), identify DNA markers closely linked to the resistance locus (loci), and release resistant breeding lines developed in this project.

5. Develop pinkeye-type southernpea (cowpea) lines that exhibit yield potential equivalent to leading blackeye-type cultivars.


1b.Approach (from AD-416):
Select parental lines of watermelon, broccoli, or leafy green brasscias based on phenotypic expression of resistance, tolerance, or quality traits under study. Use the selected parents to construct conventional (i.e., F2, BC1, recombinant inbred) and doubled haploid (for broccoli only) populations that segregate for the traits of interest, and then employ those populations in studies to determine mode of inheritance of each character. Utilize PCR-based markers and other genomic technologies to identify sequences linked to the studied characters and to locate controlling genes on linkage maps. Use particular markers (i.e., SSR, SRAP, SNPs, or SCARs) closely associated with traits of interest to develop tools for marker-assisted selection. Based on knowledge gained through above studies, devise breeding strategies, and applications of marker technologies to use in the further development of horticulturally enhanced lines or hybrids that express resistances and other traits of interest and that also produce high quality vegetables. Make enhanced lines available through public releases or commercial licensing. Continue ongoing searches for new resistances and tolerances among watermelon and vegetable Brassica accessions from the U.S. PI and other collections. In addition to the above, the southernpea pinkeye-type cultivar GreenPack-DG and the high-yielding blackeye bean cultivar California Blackeye No. 46 will be used as the parental lines to initiate a plant breeding project with the major goal of breaking the apparent yield barrier in pinkeye-type southernpeas; the two cultivars will be crossed and pedigree and single-seed-descent breeding procedures will be employed to quickly advance progeny populations three generations per year (spring field cycle, fall field cycle, and a winter greenhouse cycle).


3.Progress Report:
This project developed or cooperated in developing new watermelon breeding lines including: one resistant to root knot nematodes (RKN); two containing cytoplasm of the desert watermelon Citrullus colocynthis; and three exhibiting Fusarium wilt (FW) resistance. Inheritance studies showed that genetic populations derived from crosses between RKN or FW-resistant citroides lines and susceptible lanatus cultivars do not show Mendelian segregation. Instead, strong preferential segregation due to extensive differences in genome structure between the two subspecies has been documented. This is a problem when attempting to elucidate inheritance, but ongoing research will work to resolve this problem. This project also worked to complete genome sequencing and assembly of the watermelon cultivar ‘Charleston Gray’. In addition, cDNA libraries were conducted, a large number of EST-unigenes expressed in the watermelon fruit were developed, and numerous genes involved in fruit development were characterized. Genotype by sequencing of 95 watermelon accessions was also conducted and this revealed over 400,000 SNP markers representing most regions of the genome. In addition, a set of disease resistance gene analogs [e.g. nucleotide binding site-Leucine-rich-repeats (NBS-LRR)] likely associated with disease resistance in citroides accessions were identified. Studies clearly show that the NBS-LRR genes are not present in the genome of the cultivated watermelon, possibly having been lost during the history of cultivation and selection for desirable fruit quality traits. Separate research resulted in development of PCR-based markers in watermelon associated with resistance to zucchini yellow mosaic virus.

This project has developed broccoli germplasm adapted to hot summer conditions. Field studies have shown that unlike most commercial hybrids, many of our experimental hybrids produce similar good quality heads in both cool falls and hot summers. This breeding effort is recognized as a model program in breeding for tolerance to an abiotic stress. It has also developed unique doubled haploid broccoli populations that can be used to elucidate genetic control of the heat tolerance attribute, a trait that evidence indicates is inherited quantitatively. This project has documented the effects that genotype and environment have on the expressed levels of several phytonutrients (e.g., carotenoids, minerals) in broccoli heads. Research has shown that the levels of minerals in heads have remained stable as new hybrids have been released over time. Recent releases by this project include a broccoli inbred (‘USVL048’) shown to exhibit a high level of glucoraphanin which is believed to help protect humans against carcinogens. This project also found that a serious bacterial leaf spot disease of leafy green Brassicas is incited by two different bacteria. To date, a primary focus has been to identify host plant resistance to the bacteria that causes disease during the cool seasons. One mustard green accession have verified to be highly resistant, and inheritance studies have shown the resistance to be quantitative and likely controlled by 2 or 3 genes.


4.Accomplishments
1. Development of a tetraploid citron watermelon (Citrullus lanatus var. citroides) rootstock for grafting seedless watermelon. Following the international treaty “Montreal Protocol” to phase out the fumigant methyl bromide, there is an urgent need to develop effective techniques to reduce soil-borne diseases and pests in watermelon. Scientists at the U.S. Vegetable Laboratory (Charleston, South Carolina) have shown that Citrullus lanatus var. citroides watermelon accessions collected in southern Africa are resistant to soil-borne diseases including Fusarium wilt and root-knot nematodes. The scientists converted one of these citroides accessions into a tetraploid line (e.g., has four sets of chromosomes) and they designated it ‘USVL 360’. In field experiments during the summers of 2012 and 2013 in South Carolina, seedless watermelon plants grafted onto ‘USVL 360’ rootstocks produced higher yields than those that were grafted onto commercial rootstocks typically used for watermelon grafted productions. ‘USVL 360’ has excellent potential as a rootstock for grafted watermelon in the southeastern United States, and seed companies and producers are interested in testing this resistant rootstock.


Review Publications
Reddy, U.K., Aryal, N., Islam-Faridi, N., Tomason, Y., Levi, A., Nimmakayala, P. 2013. Cytomolecular characterization of rRNA gene sequences among Citrullus species and subspecies using fluorescent in situ hybridization (FISH) technology. Genetica. 60:427-440.

Manohar, S., Jagadeeswaran, J., Nimmakayala, P., Almeida, A., Tomasson, Y., Sunkar, R., Levi, A., Reddy, U. 2013. Dynamic regulation of novel and conserved miRNAs across various tissues of diverse Cucurbit spp. Plant Molecular Biology Reporter. 2:335-343.

Farnham, M.W., Lester, G.E., Hassell, R. 2012. Collard, mustard and turnip greens: Effects of varieties and leaf position on concentrations of ascorbic acid, folate, B-carotene, lutein and phylloquinone. Journal of Food Composition and Analysis. 27:1-7.

Guo, S., Zhang, J., Salse, J., Ren, Y., Wang, Z., Sun, H., Min, J., Zhang, H., Lucas, W., Murat, F., Zhao, H., Levi, A., Zhang, X., Wang, J., Li, Y., Fei, Z., Xu, Y., Giovanoni, J.L. 2012. The Genome of Watermelon (Citrullus lanatus). Nature Genetics. 45:51-58.

Levi, A., Thies, J.A., Wechter, W.P., Harrison Jr, H.F., Simmons, A.M., Reddy, U., Nimmakayala, P., Fei, Z. 2012. High frequency oligonucleotides – targeting active gene (HFO-TAG) markers reveal wide genetic diversity among Citrullus spp. accessions useful for enhancing disease or pest resistance in watermelon cultivars. Genetic Resources and Crop Evolution. 60:427-440.

Last Modified: 9/10/2014
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