| Pea(Pisum sativum L.) is a major winter crop in south China, as well as early spring crop and potential winter crop in north China. Pea plays an important role in human food and animal feed as fresh vegetable and dry grain, due to its high protein content and easy to be digested into various kinds of nutrient elements, along with its significant positive ecological effects and high economic benefits as intercropping and rotation crop in the sustainable and traditional integrated cropping systems of agriculture in China. China is the largest pea producer in the world and plays a vital role in the global pea production. The theoretical and practical value of studies on winter hardy ability of pea has been gradually raised by globally abnormal winter climatic flutuation during past years. In this study, pea genomic SSRs were developed by next generation sequencing(NGS) technology, and 24 genotypes of pea were selected to detect and verify the quality of developed markers. The developed SSR markers were then used to enhance the density of genetic linkage map of field pea based on F2 population. Another effort was putting them into association analysis of winter hardy genes through SSR markers with agronomic traits for discovery of freezing-resistant relative genes in pea. It is believed that the results of this study will lay important foundation for winter-hardy breeding projects of pea.The main research results were summarized as following:1. Combined with next generation sequencing technology, a total of 3275 pairs of non-redundant genomic SSR primers were developed, of which 1644 SSRs were selected randomly for primer validation using 24 accessions of cultivated pea(P.sativum L.) and its wild relatives(P. fulvum Sibth & Sm). Quality assesment obtained 1355 primer pairs with successful amplification and a subset of 841 primer pairs revealed polymorphism. Among them, 514 primer pairs showed single band in amplification products. In addition, a total of 2710 allelic variations were detected and the variation number ranged from 2 to 10 for each locus with an average Polymorphism Information Content(PIC) of 0.43, which was significantly correlated with the number of alleles. Furthermore, in combination with structure and cluster analyses using 841 SSR markers, the 24 pea accessions fell into three subgroups which were basically consistent with the geographic origin of the materials.2. Based on the first SSR genetic map of pea in 2013 constructed by our lab, the density enhancement of pea genetic linkage map was performed in this study. Combined with NGS technology, a total of 12491 pairs of SSR marker were newly developed and further screened for polymorphisms using the parents(G0003973 and G0005527) of hybrid population, among which 954 primer pairs revealed polymorphism and were applicable for further study. Based on these polymorphic SSR primers, genetic linkage analysis was conducted to the hybrid F2 population and a density-enhanced genetic linkage map containing 634 SSR markers was constructed. It was found that these SSR markers were distributed in 28 linkage groups covering the genetic distance of 7195.081 cM with an average genetic distance of 11.19 cM between neighboring loci, and the maximum number of markers were present in LG I. Three hundred and ninty-one new markers were anchored in the present genetic map with an extension of 4800 cM genetic distance comparing with the previous one, which will play an important role in further breeding studies of pea.3. Using 400 SSR markers on the new genetic map, a genome-wide scanning was conducted based on the winter-hardy screening data set of 145 core collections from China, 255 core collections from USDA-AR and 272 accessions in three locations(Qingdao, Binzhou and Yantai in Shandong province) for two years during winter in the open fields. The results showed that a subset of 198 SSR markers detected polymorphisms among the tested populations. In combination of genotype and phenotype data collected from three locations experiments in two years, winter-hardy related agronomic traits association analysis in pea germplasms were preliminarily explored with the Q value obtained from STUCTURE analyses as covariate correction. 85 SSR markers with a grand total of 148 loci were discovered to be associated with leaf age and number of branches before winter and survival rate after winter. Sixty-six loci distributing in 14 linkage groups were related to leaf age accounting for the largest portion; 47 and 35 loci were associated with survival rate after winter and number of branches before winter, respectively. In addition, loci 24398 and 23521 were persistently detected in three experiment environment, and 26333, 23358, 24588, CAASES-P878 and 23468 were identified to be related to all three traits. These aforementioned loci are most likely to associate with winter-hardy genes. |
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