Development Of Genome-wide SSR Markers And Genetic Diversity In Cucurbita Pepo L.

Cucurbita is an important plant population in Cucurbitaceae with long history.It contains many species,the most common of which are C.moschata,C.maxima and C.pepo.Pumpkin is not only nutritious,but also has a good preventive effect on various diseases.In China,the planting area has already reached 4.2×105 hm2 and the output is as high as 7.78 X 106 ton until 2016.Compared with other species in Cucurbitaceae,our country has fewer germplasm resources for pumpkins,and there is a lack of high-quality varieties on the market.Marker-assisted selection(MAS)can be unaffected by the environment and shorted the breeding period,has already become the main stream breeding method.This study has developed the genome-wide SSR markers and primers from C.moschata,C.maxima and C.pepo which are already been sequenced and counted different SSR type frequency and distribution.Then,in silico PCR we developed the cross-species SSR from C.moschata,C.maxima,C.lanatus,C.sativus and C.melo.Then,these SSRs were used to compare with C.pepo in order to analyze the chromosome synteny.In addition,based on the developed markers,96 pairs of SSR primers were selected for versatility analysis among 7 species of Cucurbitaceae.Finally,66 pairs of high polymorphism primers were selected to analyze the genetic diversity of 61 C.pepo germplasm resources.The results are as follows:1.A total of 34375,30577,38104 SSR markers were identified from the C.moschata,C.maxima and C.pepo genome and the average distance of markers are 127Mb,113Mb and 145Mb,of which 29133,28905 and 36234 markers were designed to be SSR primers,respectively.The result showed that C.pepo genome has the highest density of markers.2.The SSRs from the dinucleotide repeat to the octanucleotide repeat were statistically analyzed.The results showed that among the three genomes,the most frequent was the dinucleotide repeat SSR types,and the others were:trinucleotide,tetranucleotide,pentanucleotide,heptanucleotide,hexanucleotide and octanucleotide.Different types of SSR were randomly distributed on the chromosomes.Meanwhile,the number of each SSR type will gradually decrease as the number of repetitions increases.3.The results indicated that C.moschata has the largest number of cross-species,reaching 15274 and C.sativus has the lowest number,with only 391.The synteny between chromosomes was found that the chromosome homology is high among the three species of Cucurbita,and there were seven chromosomes with very high homology.4.A versatility of seven Cucurbitaceae crops was analyzed by using 99 pairs of primers,of which 87 pairs had good polymorphism.The versatility ratio of the 87 pairs of primers between crops ranged from 90.0%to 96.6%,the polymorphism ratio ranged from 5.7%to 48.2%,and the single polymorphism ratio ranged from 0 to 12.6%;the clustering results were consistent with those of traditional botany classification.It proved that the markers selected in this study have high versatility and polymorphism,and also verify the relationship between different crops of Cucurbitaceae.5.The average diversity index of 15 morphological traits of 61 C.pepo germplasm resources was 1.04,and the average coefficient of variation was 0.35.Principal component analysis could combine 15 traits into 5 principal components with a maximum contribution rate of 18.794%and cumulative rate of 67.741%;cluster analysis can be divided into 6 groups according to different characteristics of phenotype.6.A total of 276 loci were amplified from 61 germplasm resources using 66 pairs of SSR primers,and an average of 4.18 loci were amplified per pair of primers.The variation range of Na(observed alleles)was 2-9,variation range of Ne(effective alleles)was 1.03-1.67,the average of Shannon-index and polymorphism index was 0.83 and 0.43,respectively.The results indicated that the C.pepo germplasm resources in this study are rich in genetic diversity and can be screened and utilized during breeding;PIC values reflect the good polymorphism of SSR primers.7.According to the SSR markers,all germplasm can be clustered into 2 categories,among which category 1 includes 5 wild species;category 2 includes 56 cultivars;population structure analysis and principal coordinate analysis show that all germplasm can also be divided into two groups based on wild and cultivated.