Study On Mapping Of Lentinula Edodes Vegetative Growth Rate Traits By BSA And Development Of InDel Markers

Xianggu(Lentinula edodes)is an edible fungi with the earliest cultivation origin and the most extensive cultivation area in China.Its output has been ranking first among edible fungi for many years.In 2019,the output of L.edodes reached 11.15 million tons,accounting for 28 % of the total annual output of edible fungi in China.It has an important position in the development of Chinese edible fungi industry.China has a vast territory,diverse climate types,and abundant germplasm resources for breeding and cultivation of L.edodes.Vegetative Growth Rate(VGR)is the most important agronomic traits in the cultivation of L.edodes,VGR determines the optimal time of log making and inoculation for its cultivation.With the continuous increase in the cultivation scale and the transformation of cultivation modes from traditional practice to intensive cultivation within agricultural facility the demand for different types of varieties of L.edodes is increasing.Among them,the demand for short VGR varieties that can shorten the cultivation cycleis a particularly urgent.At present,the breeding of L.edodes varieties is still dominated by traditional hybrid breeding,and it is not possible to achieve directional breeding for the VGR traits.For the lack of effective molecular markers of key traits of L.edodes,molecular marker-assisted selection(MAS)breeding technology is immature.Therefore,it has increased the difficulty and workload for breeding high-yield,high-quality and short VGR varieties.In this study,based on the genetic diversity analysis of the core germplasm resources of L.edodes in China,two distant relative strains,a long VGR strain "L135" and a short VGR strain "931",were selected as the hybrid parents to construct a VGR differentiated F1 population,in order to study the heredity law of VGR traits in F1 population.Firstly,extreme populations with different VGRs were utilized to carry out genomic mapping through Bulked Segregant Analysis(BSA)method,then InDel markers associated with the VGR traits were developed.Finally,candidate gene prediction around the associated locations.On this basis,molecular marker development and assisted selection can be carried out,in order to improve the breeding efficiency of VGR traits,reduce breeding costs.The main conclusions of this study are as follows:Genetic diversity analysis was carried out on the main germplasm resources of L.edodes in china to clarify their relationship.five strains with significant different characters were selected from major L.edodes germplasm resources containing 44 strains in total.Resecsequencing using L808-1 as the reference genome,107 pairs of InDel primers covering the whole genome have been developed Subsequently.These107 pairs of InDel primers were used to analyze the genetic diversity and population structure of major L.edodes germplasm resources,and divided 44 strains into 5subgroups.These results of cluster analysis and population structure analysis were consistent with that of principal component analysis.The construction of genetically isolated populations is the basis of trait mapping.According to the genetic diversity analysis,two strains of L135(long-VGR strain)and 931(short-VGR strain)with relatively distant genetic relationships were selected as the hybrid parents.The single spores were obtained by plate dilution separation method,and AS-PCR and InDel molecular markers were developed for mating type identification.A hybrid population of 1700 strains had been constructed by random hybridization and incomplete diallel hybridization,which were divided into three different VGR for fruiting.The VGR of each strain of F1 generation population was calculated according to the budding time and yield of the.The VGR differentiation of three batches strains showed partial separation that tended to short VGR distribution,indicating the exit of major genes controlling the VGR traits,suitable for using BSA analysis to locate the VGR traits.Based on BSA analysis,InDel molecular markers were developed to locate the VGR-related loci.According to the VGR differentiation of L.edodes in the cultivation experiment,30 strains of short VGR,intermediate VGR and long VGR were screened out to construct 3 gene pools for DNA sequencing.A total of 38 loci were mapped by BSA preliminary localization.Then 160 pairs of InDel primers were developed for fine positioning on 38 mutation loci.According to the amplification results of the primers on the mutation sites,20 loci were finally located,and the genetic contribution rate of different loci was analyzed as well.Finally the correctness of developed InDel primers for VGR traits were verified in natural population strains with different VGR traits.The InDel molecular markers developed in this study can be applied to the genetic diversity research,the construction of core genebank and the selection of breeding parents for L.edodes.The combination of BSA and InDel molecular markers was used for the first time in the quantitative trait localization related to VGR traits of L.edodes.The identification of VGR traits of L.edodes and the development of molecular markers provided materials and methods for molecular marker-assisted breeding of L.edodes,and laid a theoretical foundation and technical support for the breeding of ultra-short VGR strains.