Map-Based Cloning And Functional Analysis Of Photoperiod-Sensitive Genic Male Sterility Gene (pms1) In Rice

Rice is the staple food for more than half of the world population,and how to improve the rice yield has been the major strategic issues related to people's livelihood,national economic development and social stability,waiting to be solved by scientists.Fifty years ago,Longping Yuan proposed the theory and ideas of three-line hybrid rice,then applied it to practical production,mading enormous contribution to our country food security.The hybrid rice production in China developed from three-line hybrid to two-line hybrid,and planting proportion of the latter in hybrid rice has been growing.Comparing with CMS lines,the fertility of two-line sterility lines could be recovered by more restorer lines with more freedom,which greatly simplifying the hybrid process and having great potential for application.The two-line sterility lines are classified into photoperiod-sensitive genic male sterility(PSMS)lines and thermo-sensitive genic male sterility(TGMS)lines,whose fertility are influnced by temperature and day-length.PSMS lines are male-sterile under long-day conditions and convert to male-fertile under short-day conditions,while TGMS are male-sterile under high temperatures and male-fertile under low temperatures.Nongken 58S(japonica)is the first found and also the most extensive researched and widely used PSMS line,whose gene source is transferred to other cultivars to breed lots of excellent two-line sterility lines.It is necessary to isolate the PSMS and TGMS genes and elucidate their molecular mechanism for better utilization.pms1 is the major locus controlling PSMS in Nongken 58 S,and the advanced backcross population between Nongken 58S(58S)and Minghui 63(MH63)was constructed to map the pms1 gene and be further investigate the gene function.The major results are as follows:1.Combined analysis of Genotyping and the spikelet fertility of the advanced backcross F2 population indicated that pms1 was semi-dominant rather than completely recessive as previously assumed.Plants homozygous for the 58 S allele were highly sterile and plants homozygous for the MH63 allele were fertile under long-day conditions;spikelet fertility of the heterozygotes varied from completely sterile to fully fertile,most of them were less than 60%.The 1:2:1 ratio of homozygous for the 58 S allele to heterozygotes to homozygous for the MH63 allele was in accordance with Mendelian separation ratio of single gene.2.For fine mapping pms1,genotyping 6841 individuals from the BC5F2 mapping population derived from a cross between 58 S and NIL(MH)identified 88 recombinants between two flanking markers Fssr and pj23,covering a 138 kb genomic region in MH63.More molecular markers were developed surrounding Fssr and Rssr(now P5)based on the previous work.Because of extensive phenotypic overlap between heterozygotes and 58 S homozygotes,only the recombinants between MH63 homozygotes and heterozygotes were used for further mapping.Genotyping the recombinants resolved the pms1 locus to a 4.0 kb region flanked by markers P4 and P6 with one recombinant at each side,with the SSR marker P5 co-segregating with pms1.3.The genomic sequence covering this 4.0 kb from 58 S was complementary transformed into the near isogenic line NIL(MH)and could ruduce the spikelet fertility under long-day conditions,while no effect under short-day conditions.On the contrast,the corresponding fragment from MH63 could not change the spikelet fertilty of 58 S.These results confirmed that the transformed genomic fragment indeed contained the pms1 locus.4.A full length c DNA named PMS1 T by us was isolated within this mapping region using 5' RACE and 3' RACE.The suppression expression of PMS1 T in 58 S increased the spikelet fertility of transgenic plants under long-day conditions,while the spikelet fertility of transgenic plants with suppression expression of PMS1 T in NIL(MH)did not change regardless of the day length.Moreover,overexpression of full-length PMS1 T from 58 S driven by the maize ubiquitin promoter in NIL(MH)greatly reduced fertility under long days.The results above confirmed that the PMS1 T corresponding the pms1 locus confered the PSMS in rice.5.The expression pattern of PMS1 T showed that PMS1 T was expressed at low levels and in a tissue-specific manner.In general its expression levels in both 58 S and NIL(MH)were low in all tissues assayed and especially so in green tissues.It was expressed preferentially in young panicles and florets,with an expression peak at secondary branch primordium differentiation stage,pistil/stamen primordium differentiation stage and pollen mother cell formation stage.This expression pattern corresponded well with previous results that the most critical period for PSMS occurred.Moreover,the transcript abundance was lower in 58 S long-day conditions than 58 S under short-day conditions and also than NIL(MH)under both long-day and short-day conditions at these three stages.6.This PMS1 T transcript from 58 S was 1,453 nt that was 65 nt longer than the one from MH63(1,388 bp).The two SNPs S1 and S2 were both located at the 5' terminus of PMS1 T,while SNP S2 and marker P5 both cosegregated with pms1.We compared sequences of 15 different rice lines at these sites,the sequencing results and recombination event narrowed the potential causal polymorphism to SNP S2.7.There was no intron in PMS1 T transcript locating in the intergenic region without annotation.Three small ORFs were predicted from PMS1 T.The guanine residue was inserted immediately downstream the ATG start codons of putative 58 S ORFs to disrupt the reading frames.These three vectors were transformed into NIL(MH)independently,could function properly to reduce the spikelet fertility of NIL(MH)under long-day conditions,demonstrating that PMS1 T encoded a long non-coding RNA.8.PMS1 T was predicted to be targeted by mi R2118,and we validated the cleavage site in PMS1 T in both 58 S and NIL(MH)by modified 5' RLM-RACE.The signature sequence of the highest abundance of Parallel Analysis of RNA ends(PARE)reads also corresponded to the cleavage site.Sequencing of small RNA libraries constructed using young panicle tissues from pistil/stamen primordium differentiation stage and pollen mother cell formation stage and meiosis stages of 58 S and NIL(MH)grown under long-day and short-day conditions showed that 18 pairs of 21 nt phasi RNA could be generated from the cleavage site triggered by mi R2118.Comparative analysis revealed that the quantities of 21-nt PMS1T-phasi RNAs were higher in pollen mother cell formation stage and meiosis stages than in pistil/stamen primordium differentiation stage,and most phasi RNAs were generated from the sense strand corresponding to the 4s,6s phasi RNAs.The phasi RNA reads in 58 S under long days at pollen mother cell formation stage were the highest followed by 58 S under short days,while the reads in NIL(MH)were much lower.We noticed a trend of negative relation between the abundance of phasi RNAs and the PMS1 T transcript.We also quantified small RNAs from pollen mother cell formation stage in the transgenic plants,which showed that the 21-nt phasi RNA reads of PMS1 T were much higher in the 58 S PMS1T overexprssion transgenic-positive plants than in negative ones under long-day conditions.Conversely,their levels were lower in 58 S PMS1T suppression exprssion transgenic positive than in negative plants.These data suggesteed that PMS1T-phasi RNA associated with PSMS.Without the complete target sequence of mi R2118,the transformed 58 S PMS1T fragment did not reduce spikelet fertility of NIL(MH),indicating the important roles of phasi RNA for causing male sterility by pms1.PMS1 T was the first identified PHAS gene with biological function,implying the importance of this kind of small RNA for plant growth and development,and also deepening our understanding about the mechanism of PSMS in rice.