Development And Application Of An Efficient Identification System For S-RNase Gene Of Lycium Barbarum Germplasm Resources

Lycium barbarum is a typical gamete type self-incompatibility forest crop.The proper selection of pollinated trees and good combination of hybrid parents are limited by self-compatibility index and S-RNase gene.In order to establish a rapid genotyping method for S-RNase gene identification of Lycium barbarum,96 germplasm resources of Lycium barbarum and 210 F1 populations with Z44 and 14-401 as parents were used as experimental materials.The S-RNase genotyping database of Lycium barbarum germplasm resources was constructed by collating the S-RNase gene sequences of Lycium barbarum logged in the NCBI database.Combined with genomic DNA sequencing technology,an efficient S-RNase gene typing assay system for germplasm resources of Lycium barbarum was established by targeting sequencing technology.The S-RNase gene of Lycium barbarum F1 population was identified by efficient S-RNase genotyping system of Lycium barbarum germplasm resources,and the self-incompatibility of 210 Lycium barbarum F1 population was detected by cross-pollination technique in the field.The main results are as follows:1.Twenty-six S-RNase gene sequences of Lycium barbarum registered in NCBI database were searched and collated,and the S-RNase genotyping database of Lycium barbarum germplasm resources was constructed by combining with three S-RNase gene sequences of Lycium barbarum obtained by cloning sequencing.Among them,26 S-RNase gene sequence entry numbers registered in NCBI database were HM195023.1--HM357298.1(20 entries)and EU074797.1--EU074802.1(6 entries).The three S-RNase gene sequences not registered in NCBI database were temporarily named as S-BARB6n,S-BARB9n and S-CHIN6n.2.According to 29 base sequences in the S-RNase genotyping database of Lycium barbarum germplasm resources,96 specific primers with length of 120bp were designed,and an efficient detection system for S-RNase gene of Lycium barbarum germplasm resources was established.The accuracy of the S-RNase gene detection system based on targeted sequencing was verified by genomic DNA sequencing technology.The sequence of 96 S-RNase gene bases was used as reference to the transcription sequence of S-RNase gene of Lycium barbarum.DNA fragmentation,terminal repair,joint connection,fragment selection,library amplification,product purification,library quality inspection,library mixing,freeze drying,library hybridization,target capture,library amplification,product purification,library quality inspection,S-RNase gene sequence detection and identification,the results showed that the genes with coverage value of 0.6-1 and reads number between 205-2023 were S-RNase genes of the test materials.3.210 F1 populations with Z44 and 14-401 as parents were genotyped using the established efficient S-RNase gene detection system from Lycium barbarum germplasm resources.Meanwhile,cross-pollination experiments were carried out in the field and the self-compatibility index of each material was obtained.The self-compatibility of F1 population materials was analyzed by combining S-RNase gene and self-compatibility index.The results showed that:(1)F1 population can be divided into four S-RNase genotypes:S-BARB2/S-BARB8;S-BARB8/S-CHIN6;S-BARB1/S-BARB2;S-BARB1/S-CHIN6.(2)The self-compatibility index of self-flowering and cross-flowering of the materials containing S-BARB8 gene was greater than 5.44;The self-compatibility index of the materials without S-BARB8 gene was less than 1.92 for the self-flowering and cross-flowering of the same plant.4.Through the comprehensive analysis of three methods based on targeted sequencing of S-RNase gene detection system of germplasm resources of Lycium barbarum,self-pollination of the same plant and cross-pollination of the same plant,and the comprehensive evaluation of the self-compatibility of each material in F1 population,it was found that the ratio of self-compatibility and self-incompatibility in F1 population of the hybrid combination was 1:1 separation ratio.The S-BARB8 gene was the key gene for self-compatibility identification of Lycium barbarum.Therefore,the establishment of efficient detection system of S-RNase gene for germplasm resources of Lycium barbarum is an economical and efficient method for identification of self-incompatibility of Lycium barbarum,which accelerates the identification of S-RNase gene and mining of S-RNase gene resources of Lycium barbarum to a certain extent,and lays a scientific foundation for cultivation of high yield and breeding of new varieties of Lycium barbarum.