Identification Of Tissue Culture Responsive Genetic Loci In Rice Based On Xian-geng Specific Markers

There are wide-ranges of genetic variation in agronomic,economic,and quality traits between xian and geng subspecies of cultivated rice（Oryza sativa L.）.Integration of elite characters from different subspecies in breeding program based on crossing between xian and geng genotypes is an important solution to broaden the genetic basis of rice varieties,and to obtain large breeding gain.Tissue culture response of xian varieties is generally much lower than geng varieties,being a key factor limiting the efficiency of anther culture and genetic transformation in xian subspecies.A multi-parental intercross double haploid（MIC-DH）population has been developed from 8xian and 8 geng parental lines.It is hypothesized that the geng alleles of tissue culture response loci are under selection during anther culture,causing segregation distortion of xian-geng specific markers.Using the genotypic data released by 3K rice genome program,typical xian and geng accessions are selected based on PCA to obtain xian-geng specific markers.Using xian-geng specific markers called from the re-sequencing data of the MIC-DH population,loci with enriched geng alleles are screened out,and used to select representative lines that carry favored alleles of target markers.Tissue culture experiments of mature embryo were conducted to identify the genetic loci determining the xian-geng differences in tissue culture reponse.The main results of this study are as follows:1.Using the SNP genotypic data published by the 3K Rice Genome Project（3KRG）,17,847 xian-geng specific SNP markers were identified.Among them,324xian-geng specific SNP markers had segregating distortion in the MIC-DH population.2.According to the genomic locations of above mentioned 324 SNP markers and previous GWAS results of callus inducing rate in a natural population,3 candidate genes were predicted for primary callus induction,namely CIR1,ROC1 and CIR8.Homologous protein and phylogenetic tree analysis of CIR1,ROC1 and CIR8demonstrated their conservativeness in the genus of Oryza.3.Tissue culture experiments were carried out using DH lines holding geng alleles(SNP^GJ)versus xian alleles(SNP^XI)on CIR1,ROC1 and CIR8.The callus induction rates of lines with SNP^GJ haplotype were significantly higher than lines with SNP^XI haplotype.There was no significant difference in the rates of seedling differentiation and tissue culture capacity.4.Significant higher expressions of CIR1,ROC1 and CIR8 were detected in callus in the SNP^GJlines than in the SNP^XI lines at induction stage.During the differentiation stage,there was no significant difference in the expression of all three genes between the SNP^GJlines and the SNP^XI lines.Comparing the expression of all three candidate genes in callus tissue,CIR1 has higher expressions during the induction and differentiation stages,and is more likely a candidate gene participating the regulatory process of callus induction.