| Faba bean(Vicia faba L,2n=12)is an important legume crop with a long history of cultivation.China is one of the main producing areas of broad beans in the world,and broad beans are grown in most parts of China.Faba bean is mainly sown in spring in the areas of north Yangtze River,while it is mainly sown in autumn in the areas of south Yangtze River.Bean weevil has seriously restricted the production and development of faba bean as an important pest affecting the production and storage of faba bean.The screening of bean weevil resistant materials is of great importance for the breeding and genetic improvement of faba bean.At present,the molecular research on faba bean at home and abroad lags behind other crops,and the genetic studies on bean weevil resistance in faba bean are seriously insufficient.The number of available SSR molecular markers in faba bean is limited,so it is of great significance to develop new SSR markers and research on faba bean.In this study,a total of 80 faba bean germplasm resources were collected,and the resistance to Callosobruchus chinensis L.was identified by artificial inoculation test,and the genetic diversity of these germplasm resources was analyzed by SSR molecular markers developed based on faba bean transcriptome sequencing(RNASeq)data.The main results are as follows:1.Identification of resistance to Callosobruchus chinensis L.in faba bean germplasm resources: the resistance to Callosobruchus chinensis L.of 80 faba bean germplasms was identified by indoor artificial inoculation.According to the number of damaged seeds per material,the seed damage rate was calculated to evaluate the resistance level of each material.Among them,26 were immune(I)material,41 were highly resistant(HR)material,1 was moderately susceptible(MS)material,9 were highly susceptible(HS)material,and 3 were susceptible(S)material.Among the 9 highly susceptible accessions,7 accessions had 100% seed damage rate,and 67 accessions were resistant materials with seed damage rate less than 10%.2.The transcriptome sequencing data of two broad bean varieties Y134 and Y078 seed samples contained 207506 transcripts with a total sequence length of about 241 Mb.The SSR loci in all Unigene sequences were searched using the MISA program.It was found that 18868 SSR sites existed in 16170 Unigene sequences,among which 2203 Unigene sequences contained multiple SSR sites and 1077 carried compound SSR sites.SSR nucleotide motif repeat types were abundant,two to six nucleotide repeat types were present,with dinucleotide and trinucleotide repeats predominant.SSR primers were designed using Primer 3.0,and 400 pairs of SSR primers were randomly selected from them for validation.3.The developed 400 pairs of SSR primers were used to perform PCR amplification in 80 faba bean germplasm resources DNA,and 328 pairs of primers were successfully amplified,the effective amplification rate was 82%,among which 89 pairs of SSR primers showed polymorphic amplified bands,accounting for 27.13% of the effective amplified primers and 22.25% of the total designed primers.The genetic diversity of 80 faba bean resources was analyzed by using the screened 89 pairs of SSR markers,and it was found that six of the 89 pairs of SSR primers had a number of alleles of 1 and the PIC value of 0.This may be due to their low number of alleles and frequency of occurrence,which are not of practical use.The remaining 83 SSR primer pairs detected a total of 428 allelic locus variants in 80 bean weevil resistant materials,with the number of alleles per locus ranging from 2 to 21,with an average of 5.16 alleles per locus and an average effective number of alleles per locus was 2.37.The variation in polymorphic information content(PIC)ranged from 0.02 to 0.82,with an average value of 0.46.There were 16 low polymorphic primers with PIC values less than 0.25,accounting for 19% of the polymorphic primers.29 moderately polymorphic primers with PIC values between0.25 and 0.50,accounting for 35% of the polymorphic primers.38 high polymorphic primers with PIC values greater than 0.5,accounting for 46% of the polymorphic primers.the Shannon information index(I)ranged from 0.04 to 2.31,with a mean value of 0.89.4.Based on the unweighted pair-group method with arithmetic means(UPGMA)method,the clustering analysis of 80 faba bean germplasms was carried out,they were divided into two major categories at a similarity coefficient of 0.80.The first category contained 67 faba bean germplasm,including 25 I materials,39 HR materials,2 HS materials and 1 S material,accounting for 83.75% of the tested broad bean germplasm resources;Category II contained 13 faba bean germplasm,accounting for 16.25% of the tested faba bean resources.Among them,one I variety(line),two HR varieties(lines),one MS variety(line),seven HS varieties(lines)and two S varieties(lines).. |
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