| Rice yield is a complex agronomic trait,which is mainly composed of three key factors: effective panicle number per plant,grain number per panicle and thousand-grain weight.Among them,the relationship between grain number per panicle and the yield is the most direct.In this study,a substitution line X133 with a significantly increased grain number per panicle was identified in the population of chromosome segment substitution lines(CSSL)constructed by parents of indica rice variety Changhui 121 and japonica rice variety Koshihikari,Only three segments of X133 were replaced by the donor parent Koshihikari,and the rest of the genetic background of X133 was consistent with the recurrent parent Changhui 121.Compared with Changhui 121,X133 showed that the number of grains per panicle,the length of panicle,and the plant height increased.The number of grains per panicle of X133 was 365,which was 148% of Changhui 121.In order to identify the gene that lead to the significant increase of grain number per panicle of X133,a F2 segregation population was constructed by crossing X133 with Changhui 121.SSR markers and bulked segregant analysis(BSA)method were used to locate the genes,but no markers showing polymorphism between the two mixing pools were identified.Then,the BSA-seq and composite interval mapping method(CIM)and complete interval mapping method(ICIM)were further used to analyze the genes controlling the target traits.The results are as follows:(1)Among 556 individual plants of F2 population,the maximum number of grains per panicle was 448,the minimum number of grains per panicle was 78,the variation range of grain number per panicle was wide,and the kurtosis was > 0,which shows the continuous normal distribution law,indicates that grain number per panicle was controlled by multiple genes.The correlation analysis showed that there was a very significant positive correlation between the number of grains per panicle and the length of panicle and the height of plant,respectively,and it was speculated that the gene for grain nember per panicle had pleiotropism.(2)According to the BSA method,two DNA pools with extremely high and low large grain nember per panicle were mplified by SSR markers in substituted segments of X133 by Koshihikari,and no polymorphic markers were found.Then,the DNA of two extreme mixing pools and two parents were sequenced in high-throughput way.A total of 60.142 Gbp of original sequencing data was obtained.The filtered clean data was59.982 Gbp,Q30 was 89.97%,GC content was 40.83%-44.96%.The average sequencing depth of each sample is 59.88 ×.The average comparison efficiency between the sample and the reference genome was 92.38%,the average coverage depth was 34.38 ×,and the genome coverage was 91.17%.Based on the algorithms of SNP-index and InDel-index,the polymorphic SNP and InDel loci were found on chromosomes 2,4 and 5,including 15 candidate genes.(3)A total of 16 SSR markers which showed polymorphism between the two parents were screened to construct the genetic linkage map of substituted segments by 556 individual plants of F2 population.A total length of 198.8 c M was obtained,covering 3chromosomes in rice,and the average distance between the markers was 15.29 c M.By using composite interval mapping method(CIM)of Win QTLCart 2.5 software and556 plants of F2 population,two QTLs controlling grain number per panicle were detected,which were distributed on chromosome 2 and 4,and the contribution rates were 4.73% and 6.83%,respectively.By using the complete interval mapping method(ICIM)of QTL IciMapping V4.0 software,one QTL controlling tgrain number per panicle were detected,which was distributed on chromosome 4.qGN-4 could been detected by two mapping methods,the contribution rates were 6.83% and 7.22%,respectively,and the synergistic allele comes from female parent X133.(4)According to the results of BSA-seq and QTL detection,15 candidate genes were compared to the qGN-4 interval,and two candidate genes within this interval were selected,they were verified by Sanger sequencing and realtime fluorescent quantitative PCR.It was found that the 276 th base in the coding region of the LOC_Os04g42540 gene was replaced(A-G).It was found that two bases(TA)in the 3’UTR of the noncoding region of the LOC_Os04g47520 gene were deleted.It was found that the expression levels of the two genes was significant difference between two parents when the young panicle developed at 1-3 cm.It was preliminarily predicted that these two genes have the greatest correlation with the number of grains per panicle in rice. |
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