Natural Variation, Evolutionary Pattern And Functional Analysis On Indica-japonica Hybrid Sterility Gene S5in Rice

Three allelic types, indica-like, japonica-like and WCG (wide compatibility gene)-like were found during the comparative sequencing of a hybrid sterility gene S5recently cloned in2008within Asian cultivated rice population. The indica-like alleles differ from japonica-like alleles by two nucleotides in the coding region (from5'to3', indica-like are C and C, japonica-like are A and T). The protein level can be distinguished by two AA replacements, and the WCG-like S5alleles are defined as "neutral" type due to the deletion in N-terminal and further loss of function. In our study, we analyzed the genomic sequences of S5alleles within138rice accessions including Asian cultivated rice O. sativa, annual wild rice O. nivara and perennial common wild rice O. rufipogon, covering upstream promoter, coding region and downstream flanking sequence totally4.7kb in length. Results from PCR-amplified comparative sequencing showed19haplotypes (natural variation in coding region) and could be further classified into three groups as indica, japonica and WCG-like alleles. There were8haplotypes in indica-like and WCG-like group separately, and only3haplotypes in japonica-like group.Analysis on evolutionary relationship among the three allelic groups indicated haplotype H19and H5were primitive to indica-like and japonica-like groups separately, with the highest frequency in each group and could only be distinguished by SNP C819A and C1412T. H17was primitive to WCG-like group resulting from a deletion mutation in H19. Calculation on evolutionary selective force suggested the WCG-like group was favored by positive selection due to the high compatibility in hybrids from individuals carrying correspondant alleles to inflict increased frequency. To conclude all, the formation of indica, japonica and WCG-like alleles were earlier than speciation of O. sativa, O. nivara and O. rufipogon, and the incompatible allelic types indica-like and japonica-like already existed in more ancestral species, suggesting that reproductive isolation exists not only within indica and japonica cultivated rice but also in earlier speciation events. The reproductive isolation might be established within the untamed wild rice populations. Also in this isolation system, the similar indica and japonica rice populations might had already differentiated out and the result also supported the independent origination hypothesis of indica and japonica subspecies.Further study found the two characteristic SNPs between indica and japonica in the sampled rice germplasm, C819A and C1412T were tightly associated, suggesting the coexistence of these two sites played an essential role of gene function. On basis of the published data from transformation of indica full length S5into japonica recipient, both female gametic and spikelet fertility were decreased, we constructed6donor-fragments from all recombinations of these two SNPs and driven by S5indica and japonica native promoters for genetic transformation to evaluate the spikelet fertility of single copy positive individuals in To and T1generations. Results indicated only the coexistence of C819and C1412could repeat the semi-sterile phenotype as indica-japonica hybrids, and the association between these two sites was necessary for S5to act functionally. In the same time we commenced expression-repressing transformation of S5into japonica rice recipient due to the lack of allelic mutant, and to observe possible hybrid fertility restoration after test-crossed with indica. The result showed no significant fertility difference between hybrid-positive and hybrid-negative individuals and no dosage effect of S5. We also used complementary and over-expression transformation into Arabidopsis to estimate the functional conservation, and results indicated no fertility-related traits were influenced but the dwarfing phenotype still matched the reported functions of aspartic protease gene family members in Arabidopsis, granting S5the potential to be further utilized as a new pathogen resistance genetic source in trait improvement.The intersubspecific hybrid sterility between indica and japonica rice is a typical reproductive isolation which plays an extremely important role in allelic evolution, differentiation and speciation. The hybrid sterility gene S5in rice demonstrated its specific complexity in evolution. The indica, japonica and WCG-like alleles of S5also showed very important functions on speciation and evolution, further study into natural variation and functional evolution of this gene will be helpful for better understanding of reproductive isolation and speciation procedures from formational and developmental level.