Construction Of DNA Fingerprint And Variety Purity Identification Of Faba Bean Based On SSR Markers

Nowadays,the fundamental of molecular marker researches on faba bean developed very slowly in the world.In previous studies,SSR markers were sedomly used by employing small number of SSR markers for exploratory attempts,due to estremely short of SSR markers of faba bean.A high-quality genetic linkage map is an important foundation for excellent gene mapping,association analysis of major agronomic traits,gene cloning and marker assisted selection breeding.The problem of seed quality,especially authenticity and purity of seeds,has become an important factor restricting the redevelopment of the seed industry of faba bean.Therefore,establishing an efficient,rapid and high-throughput SSR identification system is of great significance for the authenticity and purity identification of faba bean varieties.The main research results are as follows:1.3744 pairs of genomic SSR primer and 1152 pairs of EST-SSR primer developed in our lab were screened by F₂ parents.A total of 329 pairs of polymorphic primer were selected,including 211 pairs of G-SSR primer and 118 pairs of EST-SSR primer.The 242 more SSR markers were anchored to the previously constructed genetic linkage map in 2014 in our lab by applying Map Manager QTX b20software,and a renewed SSR based genetic linkage map with 465 anchored SSR markers was constructed by removing SSR markers with genotype data lose rate over 20%.This density enhanced genetic linkage map includes 7 linkage groups with total length of 4516.75 cM.The length of each linkage group ranged from 129.35 to 1180.21 cM,with average distance between two SSR markers of9.71 cM to 1.69 cM that is much shorter than before.The number of SSR markers on each linkage group ranges from 12 to 136.With P less than 0.05,29.53%SSR markers showed distorted segregation,and 11 SDRs?segregation distortion regions?were identified.2.Considering the distribution uniformity,polymorphism,PCR amplification stability,among the anchored 510 SSR markerd in the renewed genetic linkage map,17 pairs of core primer were selected for the genetic diversity analysis of 48 faba bean varieties.A total of 106 alleles were detected with 6.24alleles per locus ranging from 3 to 13.The PIC index ranged from 0.53 to 0.85 with mean value of 0.63,and the gene diversity ranged from 0.60 to 0.85 with mean value of 0.69.Based on UPGMA clustering,population structure analysis and principal component analysis,48 faba bean varieties were clustered into spring sowing ecotype and autumn sowing ecotype.The results were highly consistent with their geographical origins or plant types of these faba bean varieties.3.Considering the effective number of alleles,heterozygosity,polymorphism information content,gene diversity and PCR product of molecular size and so on,8 pairs of SSR core primers?amplified 35alleles?were used for the construction of DNA fingerprint and database of 48 faba bean varieties.The information of agronomic traits and the data of DNA fingerprinting for these faba bean varieties were conversed into two-dimensional code for the first time,which provides a fast and convenient way for variety and seed quality control of faba bean.4.Two faba faba lines?GF47 and TF23?were identified by 6 pairs of core SSR primer,with purity of 93.75%and 95.83%respectively.SSR primers EST1142 and SSR11052 were found the strongest for detection of all heterotypic individuals among populations of the two faba bean lines.Comprehensive consideration of identification costs,high-throughput and effects of purity identification,the method was established for fast purity identification of faba bean lines based on combination of two core primers EST1142 and SSR11052.