Fine Mapping Of The Rice Photoperiod-sensitive Genic Male-sterile Gene Pms1 And Sequence Analysis Of Pms1 Candidate Region

Photoperiod-sensitive genic male-sterile rice (PSGMS), which under long-daycondition is sterile while under short-day condition is fertile, can be used as both sterileand normal line and thus be a very important germplasm to develop the "two-line" hybridin rice breeding program. Numerous studies on PSGMS from physiological, biochemicaland genetic aspects have showed that it concerned with very complicated phenomena,however, little has been known about its mechanism to date. Thus, this study tried toclone genes responsible for PSGMS and to elucidate the mechanism of male sterility andits reverse to fertility, which will have great significance on enriching mechanisms ofmale sterility in plants and will also give cues to finding eminent male-sterile line.Previous studies have shown that the sterility of Nongken 58S is controlled by one ortwo major loci, depending on the genetic backgrounds of the material used in the crosses,and the locus located on rice chromosome 7 designated pmsl can be detected in variouscrosses. Using an F₂ population from a cross between Nongken 58S and Minghui 63,pmsl was located within two RFLP markers RG477 and R1807, with the genetic distanceof 0.5 and 3.8 cM, respectively. Construction of physical map, sequencing of the oneBAC clone, and fine mapping of the gene helped to narrow it within two SSR markers,and mini-physical map overlapping the candidate region has been achieved. The mainresults of this study are summarized as following:1. Construction of shotgun library of BAC clone 18P4 from Nongken 58 BAC library,sequencing and thus assembling of subclones gave rise to an 81,029 bp-in-lengthinsert, which overlapped with 2109 by 55,046 bp.2. Editing the sequence of 2109 and detailed annotation resulted in thirteen predictedgenes, with the coding region and putative TEs accounted for 28.3% and 40.7% of thetotal sequence, respectively. Further comparison indicated five of the seven predictedgenes had the potential to be the candidate gene for pmsl, which should befunctionally tested in future studies.3. Comparison of the orthologous sequences from japonica cultiva (Nipponbare andNongken 58) and indica cultiva (93-11 and Minghui 63) in pmsl region showed that4 they shared microcolinearity at the gene level, meanwhile, striking differences existedwithin intergenic region, which were mainly caused by invasion and/or subsequentdeletions of LTR-retrotransposons. The highest level of polymorphisms was detectedbetween 93-11 and Nongken 58, while the lowest between Minghui 63 and 93-11.Inter-subspecific comparisons detected higher polymorphisms than intra-subspecificcomparisons, however, the comparison between Nipponbare and Nongken 58 hadhigher polymorphism than Nongken 58 and Minghui 63 and the DNA polymorphismsin this region were abnormally higher than the average genome. Further dating ofLTR-retrotransposons confirmed that this region has undergone a rapid evolutionsince the indica-japonica divergence.4. Investigation on all transgenic plants didn't yield lines of good correlation betweenphenotypes and molecular markers, with no expected result from furthertransformation of one gene concemed with photomorphogenesis, thus pmsl wasprobably located out of this region.5. Construction of a NIL using a cross between Nongken 58S and Minghui 63 gave riseto 500 highly sterile individuals from a BC₂F₃ population with about 2200 individuals,and further genotyping using SSR markers ch743 and ch745 identified 50recombinants. Abundant detection of parental lines using subclones as probesproduced several useful markers, which helped to narrow the gene between Rssr and5C1aG8, and most likely between Rssr and H1/N2 if combining previous mappingdata.6. Sequencing of two BAC clones produced a 119,695 bp-in-length insert of 24L2 andan 89,893 of 57N1, thus the three sequenced clones resulted in a contig of 290,641 bp.Comparison of Nipponbare and Minghui 63 genomic sequences identified deletions orinsertions of large fragments in this region, which induced the physical distancebetween Rssr and 5C1aG8 was 109 kb in Minghui 63 while 242 kb in Nipponbare andwere concerned with several LTR-retrotransposons and tandem repeat genes. The twogenomes overlapped only by approximately 85 kb with 8 predicted genes, 5 of whichwere located within the 21 kb-in-length region between Rssr and N6B5 indicating it agene-enriched and functional important region. Above analysis strongly supported the results of mapping, thus the gene was undoubtedly located between Rssr and N6B5considering with no predicted within H1/N2 and N6B5.7. Bioinformatic analysis showed that there were five predicted genes between Rssr andN6B5, three of which shared highly homologous proteins from other organisms. Byusing vector pCAMBIA 1301, five overlapping DNA fragments from Minghui 63were introduced into Nongken 58S, for which many transgenic plants were achieved.The copy numbers of fragment ulpl were detected in T₀ generation and furtheranalysis was needed for all transgenic plants.8. Homologous DNA fragments from parental lines with sterile genotypes Nongken 58S,Nongken 58 and other PSGMS lines, as well as parental lines with fertile genotypes1514, Lunhui 422, were amplified and sequenced, among which consistent mutations(five SNP and one bp insertion/deletion) between the two groups were found.In total, by combining mapping datas from two population and results of comparativesequence analysis, pmsl was located within a 21 kb-in-length fragment, where twoimportant candidate genes were offered. Thus this work has set a firm base for the finalcloning of pmsl.