Development Of Transcriptome SSR Markers For Bitter Gourd (Momordica Charantia) And Genetic Diversity Of Commercial Varieties

Bitter gourd(Momordica charantia L.)is an annual herb vegetable of the family Cucurbitaeae,which is mainly originated from tropical regions of Africa and Asia.It is cultivated all over China at present,especially in province of Guangdong,Guangxi,Hainan,Fujian,Taiwan,Hunan,Sichuan.As the medicinal and health functions of bitter gourd were gradually discovered,the consumption and cultivation area of bitter gourd were also increasing.In recent years,the varieties of bitter gourd on the market had been increasing,and the variety resources were also getting mixed.Therefore,research on genetic diversity of bitter gourd variety resources at the molecular level can help understand the genetic classification and genetic relationship of different bitter gourd varieties.There were a large number of simple sequence repeats(SSR)in the genome.SSR molecular markers had the characteristics of abundance,high polymorphism and co-dominant inheritance.SSR marker development in the whole genome of bitter gourd had been completed,but comprehensive transcriptome SSR marker development has not been reported.In this study,a set of specific transcriptome SSR primers based on transcriptome sequencing data of bitter gourd ‘Dali-11’ were developed.Used restriction site associated DNA(RAD)sequenced technology,133 commercial varieties of bitter gourd were sequenced.The genetic diversity and genome wide association study(GWAS)for seed coat color of bitter gourd were conducted based on SNP genotyping data.The main findings are as follows:1.SSR distribution in bitter gourd transcriptomeA total of 31,066 SSR loci were identified from 59,740 Unigene(about81,511,284 bp)of bitter gourd,with a frequency of 52.00%.There were 19,335 sequences containing SSR loci,with a frequency of 32.37%.2.Development of SSR molecular markers based on bitter gourd transcriptomeThrough the primer design of the identified SSR motif sites and the specific alignment of the primer sequences,a total SSR 9,135 pairs of specific primers were obtained.232 pairs of specific primers on MC00(genome sequences that have not been assembled to chromosome)were selected and validated their amplification in two bitter gourd lines ‘Tanbian dading’ and ‘Huayi 320’.The results showed that94.40% of the designed primers had amplification effectiveness;the polymorphic rate of 232 pairs SSR specific primers between the two bitter gourd lines was13.36%.3.Genetic diversity analysis of bitter gourd commercial populationThe SNP genotyping was performed on 133 bitter gourd commercial varieties using RAD sequencing technology,and a total of 59,527 SNPs were obtained after data filtering.Based on the genotype data,a population structure structure and phylogenetic NJ tree of 133 bitter gourd varieties were constructed.Both results indicated that the ‘Dading’ bitter gourd materials were divided into a relatively independent clade.‘Pearl’ bitter gourd and ‘Smooth’ bitter gourd materials were mixed and showed no obvious population structure.Among the bitter gourd commercial materials,‘Pearl’ bitter gourd showed the highest degree of genetic diversity,and ‘Dading’ bitter gourd shiowed the lowest;the degree of genetic differentiation between ‘Dading’ bitter gourd and ‘Smooth’ bitter gourd was the highest,and that between ‘Smooth’ bitter gourd and ‘Pearl’ bitter gourd was the lowest.4.Genome-wide association analysis of seed coat color of bitter gourdUsing the genotypic data obtained by RAD sequencing and the seed color phenotype of the bitter gourd commercial varieties,the GWAS for seed coat color was carried.The results showed that a significant correlation site was located on chromosome MC03,with a candidate range of 14.8-15.0 Mb(about 200 kb)on chromosome MC03,containing 25 candidate genes.