Results and Discussion
The total genomic DNA of B810S, which was collected from a deserted paddy field, and that of 810S were extracted using a Plant DNA isolation Reagent kit (Tiangen, Beijing, China) following the manual specifications. The entire sequences of the atp H genes of B810S and 810S were amplified by PCR, and were then subjected to detection in 1.5% agarose gel electrophoresis, which appeared two single bands, and both are approximately 250 bp in length.
The entire sequences of the atp H genes of B810S and 810S were 245 bp in size. The atp H gene sequence of B810S has been deposited in GenBank under accession number: MN879320. A comparison of B810S with 810S, indicates that the similarity of both sequences is 51.22%, and there are 99 variation sites in their atp H gene sequences. The results of haplotype (Hd) and nucleotide diversity (Pi) analysis of theatp H gene sequences of B810S and 810S showed that there are 2 haplotypes in their atp H gene sequences and that the haplotype diversity and nucleotide diversity were 1.0000 and 0.41250, respectively. Comparisons with other members of the Oryzaisgenus, indicated that the sequence differences were significantly higher, namely, 48.78-49.19% for their atp H gene sequences.
To test whether B810S with a color marker is a new male sterile rice line, the variation frequencies of allele genes between B810S and 810S were evaluated using Tajima’s D statistical analysis.The result of Tajima’s D statistical analysis showed that the Tajima’s Dis was 2.32478. These results indicate that there is genetic differentiation between B810S and 810S. Also, the mismatch distribution analysis of the atp H gene sequence of B810S and 810S, showed that there were multi-peaks in their atp H gene sequences, which suggests that B810S is a new male sterile rice line.
In addition, to infer the phylogenetic relationship of B810S and other members of the Oryzais genus, phylogenetic analysis was performed using the maximum likelihood (ML) method with the GTR+I model in the study. The results of phylogenetic analysis showed that B810S evidently separated from 810S and other rice strains, and formed an independent branch in the phylogenetic tree (Fig. 1). This result is in accordance with the mismatch distribution analysis, which showed that there was obvious genetic differentiation among B810S and other members of Oryzaisgenus(including 810S and other rice sequences which were downloaded from Genebank ), which suggest that B810S is a new male sterile rice line.
In conclusion, the genetic diversity and genetic structure of B810S were revealed by the atp H gene sequence of the cp genome from rice leaves, and this study also confirmed that B810S with a color marker is a new male sterile rice line. In addition, the molecular approach employed in this study provides a foundation for studying the genetic variations and structures of rice in different geographical regions of China and other regions of the world.