Fine-mapping Of SegD8 Gene For Rice Hybrid Segregation Distortion

Segregation distortion（SD）means that the observed genotype or phenotype does not conform to Mendelian’s laws of inheritance and cannot be analyzed using traditional methods.It is a very common phenomenon in genetic research and is considered one of the driving forces of biological evolution.In rice hybridization,SD is more likely to occur due to the different viability of homologous alleles between subspecies.In this study,we performed genetic analysis of an introgressive line population derived from an indica japonica rice cross combination Hua Zhan（HZ）/Koliya（KLY）,and identified a segment on chromosome 8 that displayed obvious deviated segregation ratio from Mendelian’s separation law.And it was verified that there was a SD loci.In this paper,we used multi-generational inbred populations to locate and analyze candidate genes,the specific results are as follows:1.In the BC₃F₂ population,there is a clustering of molecular markers on chromosome 8,And the SD direction does not conform to Mendelian’s law of inheritance The validation results of the BC₃F₃ population show that there is a loci on chromosome 8 to control this SD.Further analysis revealed that the SD was controlled by two loci on chromosome 8,which were named as Seg D8A and Seg D8B.Seg D8A was the segregation loci,but its segregation attribute was regulated by Seg D8B.When both Seg D8A and Seg D8B sites were heterozygous,the ratio of Seg D8A^HH and Seg D8A^KK in progeny was close to 1:4.When Seg D8B was homozygous HZ genotype,the ratio of Seg D8A^HH and Seg D8A^KK in progenies was close to1:1.When Seg D8B was homozygous KLY genotype,Seg D8A^HH almost did not appear in its progeny.Combined with the ratio of plant genotype composition and offspring separation,the Seg D8A gene was initially positioned between the markers CHR8-45 and CHR8-53 with a518.1 kb region,and the Seg D8B gene was positioned between the markers CHR8-53 and CHR8-86,with a 1160.2 kb region.2.We screened recombinant strains with overlapping overlaps and smaller heterozygous intervals in BC₃F₄ generations,identified genotype composition,and counted offspring segregation ratios.The results showed that there was a bias separation effect between marker CHR8-78 and marker SNP8-6.Combined with genotype composition analysis,Seg D8A was located between these two markers,a 46.5 kb region,and Seg D8B could not further narrow the interval due to the lack of recombinant plants.3.The Seg D8A candidate interval contains three open reading frames,and three prediction genes are obtained after sequence comparison,including ORF2（Loc＿Os08g0203300）and ORF3（Loc＿Os08g0203350）amplified successfully,ORF1（Loc＿Os08g0203201）amplification failed.The sequencing analysis results show that compared with KLY,there are a large number of base substitutions and deletions in the sequence of the coding region of ORF2 in HZ.ORF3 presents 14 base substitutions in the sequence of coding regions in HZ,and due to the G/C mutation of termination codon TGA causes translation to be delayed by 15 bp to terminate.Quantitative analysis results show that the expression of ORF2 in KLY is 50%lower than in HZ.The expression of ORF3 in KLY was 30.89%lower than in HZ,all of which were significantly different.