Development Of SSR Molecular Markers And Evaluation Of Germplasm Resources In Aesculus Wilsonii Rehd

Aesculus wilsonii Rehd,belongs to the Hippocastanaceae family,also called as“Hou Ban Li”,it’s one of the major sources of the traditional Chinese medicine“Suo Luo Zi”.Its dry seeds have been used to treat chest and abdominal pain,dysentery and ague.In recent years,with the more products are being developed,market demand is increasing.However,due to the continuous deterioration of the growing environment,the wild resources of A.wilsonii have been seriously damaged,so carring out the collection and evaluation of A.wilsonii germplasm resources is becoming more and more importance.In order to expand the germplasm resources and provide theoretical basis for genetic breeding of A.wilsonii,RNA-Seq technigical was used to develop EST-SSR molecular marker and reveal its genetic diversity and population structure.The morphological,physiological and medicinal quality of A.wilsonii were comprehensively evaluated,the relevance between SSR markers and the quantitative traits was further analyzed.This study laid a foundation for the germplasm resources development and genetic breeding of A.wilsonii,and provided germplasm materials for fostering A.wilsonii with superior quality.The main results are as follows:1 A total of 21562 potential EST-SSRs were identified from 287506 unigenes.The EST-SSR frequency was 7.49%and the distribution density was 9.12 per kb.Among these potential SSRs,dinucleotides and trinucleotides repeat units account for 22.0%and32.9%respectively;The most dominant dinucleotides and trinucleotide repeat motif was AG/CT（2544,11.8%）and AAG/CTT（464,6.79%）respectively.2 Based on the identifified EST-SSRs,220 pairs EST-SSR primers were randomly selected for the development of potential molecular markers.Among these SSR markers,90 pairs of primers（41%）can successfully amplify clear and bright bands with expected sizes.Eventually,25 pairs of primers were screened with high polymorphic and chosen to analysis the population structure and genetic diversity of A.wilsonii.A total of 122 alleles were identified by the 25 primer pairs,the average number of alleles detected at each primer was 4.88.Nei’s Genetic diversity index was ranged from 0.41–0.81,with an average of 0.64,The PIC was ranged from 0.35-0.79,with an average of 0.59,which shown a high level of polymorphism.3 The genetic differentiation（Fst）averaged at a value of 0.124,the gene flow（Nm）averaged at a value of 1.27,suggesting the low genetic differentiation among the different A.wilsonii populations.The AMOVA analysis results revealed that 95%of the total genetic variance appeared within populations,while only 5%genetic variance occurred among the populations.The NJ tree results shown that the all 29 A.wilsonii germplasm resources could be divided into 4 groups with a similarity coefficient of 0.724 and the four groups were divided into several subgroups.POP 1 includes two germplasm resources from Fang County and Zhuxi County.In POP2,including the germplasm resources from Fang County and Zhuxi county and other seven germplasm resources from all eight germplasms which collected from Shennongjia,two germplasm resources from Enshi were also grouped into this subgroup,POP 3 includes eight germplasm resources,four from Zhuxi County,three from Fang County,and one from Shennongjia.POP 4 includes two germplasm resources that both from Shennongjia.4 There are 29 A.wilsonii germplasm resources that their morphological characteristics,physiological characteristics and medicinal quality were investigated and evaluated.The results showed that the light saturation point of A.wilsonii was 800μmol.m^-2.s^-1,the light compensation point was 23.53-33.9μmol.m^-2.s^-1,the diurnal variation of the net photosynthetic rate of A.wilsonii showed bimodal curve and there was an obvious phenomenon of midday depression of photosynthesis,which indicating the A.wilsonii fear the strong light,so in the process of cultivation,take appropriate shading measures can provide a more suitable environmental conditions for A.wilsonii.Through comprehensive evaluation of the characteristics,we identified ZXLY2、FXJN2 and FXSZ as the germplasm resources with better quality and higher resistance,which provided new materials for the breeding of A.wilsonii.5 In order to determine the best Q value,the software Structure 2.3.4 was used to analyze the population structure of A.wilsonii.The GLM model was employed and the determined Q value was set as a covariate,SSR locies were correlated with the quantitative traits and quality traits of A.wilsonii.Finally 9 markers that significantly associated with 10 traits（P<0.05）were detected.The results provided a basis for the molecular assisted breeding of A.wilsonii.