Map-based Cloning Of Hybrid Pollen Sterility Locus QHMS7 Between Oryza Sativa And Oryza Meridionalis

Rice(Oryza sativa L.)is not only one of the most important staple food in the world,but also has been used as a model monocot plant for functional genomics research of Gramineae.Among the AA-genome Oryza species,there are two cultivated species(Oryza sativa L.and Oryza glaberrima Steud)and six relative wild species(O.nivara,O.rufipogon,O.barthii,O.longistaminata,O.glumaepatula and O.meridionalis).The Asian cultivated rice has been dived into indica and japonica subspecies.The possible methods for improving the yield potential are taking advantage of heterosis in rice.However,the hybrid sterility occurring among rice species has long been a puzzle and hampers progress in breeding crops with improved performance and yield characteristics.So we have to resolve the problem resulting from hybrid sterility,which can induce spikelet or pollen sterility.This explanation for how reproductive isolation is maintained among species of rice,and perhaps other organisms,also offers approaches for boosting yields by intersubspecific heterosis.So we performed QTL analysis using BC1F1 population and map-based cloning of qHMS7 between Dianjingyoul and O.meridionalis.The main results as follows:(1)The Asian cultivated rice DJY1(O.sativa L.ssp.japonica cv.Dianjingyoul)and the wild species(O.meridionalis NG)were a pair of distantly related species.Pollen from their hybrids F1 were almost sterile.The resultant F1 plants were backcrossed with DJY1 plants as the recurrent to produce BC1F1 population for hybrid pollen sterility loci detection.SSR or InDel markers were used to determine their genotypes of BC1F1 plants.Finally,four QTL loci located at the first,second,seventh and ninth chromosome were detected by IciMapping software.Among these loci,perhaps there were three new loci,including qHMS1,qHMS2 and qHMS9.The proportion of the phenotypic variance explained by them was about 90%.The value of Additive effect suggested that qHMS1 locus caused the frequency of the allele from DJY1 was low,but these other three loci caused the frequency of the alleles from O.meridionalis NG.(2)We constructed the NIL-qHMS7 by backcross and Marker-assisted selection.And we compared the morphological and developmental differences of anthers and microspores through meiosis to mature pollen grains stage between recurrent parent and NIL-qHMS7.In vitro germination test of pollen grains shown that sterile pollen failed to germinate.From pollen mother cell formation to mature pollen grains generation,although a number of shrunken pollen grains appeared at the stage 12,the anther wall of DJY1 and NIL normally took place and then degenerated.By Acetocarmine Staining,we found that there was no obvious morphological difference between-DJY1 and NIL during meiosis stage,unicellular stage,bicellular stage.But at the tricellular stage,about half of pollen grains from NIL plants were small and had only two nuclei.The results were confirmed by the transgenic plant with germ cell marker vector in NIL genetic background.Totally,hybrid sterile genes caused smaller pollen with little starch accumulation and only two similar nuclei.(3)We took advantage of the BC6F2 population with 478 plants to analyze the genetic effect of qHMS7.Pollen fertility of the BC6F2 population showed clear-cut bimodal distribution.And the genotypes of these semi-sterile plants were "DJY1/Mer" and fertile plants were "DJY1/DJY1" without any "Mer/Mer" type.To confirm these results,we again assayed patterns of gametophytic transmission using three populations,including one self-pollinated population of NIL and another two reciprocal crosses of DJY1 and NIL.It also showed that male gametes carried "M" allele couldn’t transmit to the progeny.We delimited the interval of qHMS7 between the markers YU-29 and YU-19.For map-based cloning of the qHMS7,we screened recombinants from 18,930 BC6F2 plants with the flanking markers YU-29 and YU-19,and finally delimited the qHMS7 into a 75-kb genomic interval.Through genomic and cDNA sequence analyses of target region,we found ten putative open reading frames without domain annotation.To fond out these target genes,we constructed genomic expression vector and CRISPR vector to perform transgenic experiments and awaited the results.