Genetic Analysis Of Several Grain Type Genes In Rice And Fine Mapping Of QTL QLFP9 For Flag Leaf And Panicle Length

Rice is one of the most important food crops.Increasing rice output plays an important role in ensuring food security.With the increase of population and the improvement of people's living standards,the yield and quality of rice need to be further improved.The current progress in rice yield potential and quality requires constructing a more efficient breeding technology system.Molecular breeding based on the analysis of related genes for yield,quality,and regulatory networks has become an effective way to break through the bottleneck of yield breeding in rice.This study is based on the genetic effect analysis of multiple grain size genes using InDel-markers in rice(Oryza sativa L.)and the precise location of quantitative trait loci QTL qLFP9 that control the flag leaf and panicle length of rice.Part 1:InDel marker-based estimation of Multi-Gene Allele Genetic Variations for Grain Size in Rice(Oryza sativa L.)The market success of any rice cultivar is exceedingly dependent on its grain appearance and grain yield,which eventually describe its demand by consumers and growers.The present study was undertaken to explore the contribution of nine major genes viz.qPE9-1,GW2,SLG7,GW5,GS3,GS7,GW8,GS5,and GS2 in regulating grain size and weight related traits i.e.,grain length(GL),grain width(GW),grain thickness(GT)and thousand grain weight(TGW)in diverse 204 rice germplasm using Insertion/Deletion(InDel)markers.This provided a basis for the effective utilization and protection of rice.The major conclusions were described as follows:1.The 204 rice germplasm were classified into three distinct groups(?,?,?)based on phenotypic variation.Major groups,i.e.,Group I:182 genotypes,Group ?:8 genotypes,whereas Group III had 14 genotypes.Group I was further subdivided into cluster IA and cluster IB for simplification.Group IA consisted of 78 entries,and cluster IB contained 104 entries of germplasm.The average grain length of cluster ? was 8.01mm,whereas average grain width(GW)and grain thickness(GT)were 2.96mm and 2.16mm respectively.Thousand-grain weight(TGW)of this group was 25.63g.Group ?showed the highest values of average grain length(GL)(8.83mm),indicating that entries of this group had maximum grain length(GL).This group also had a maximum grain thickness(GT)(2.27mm),grain width(GW)(3.0mm)and TGW(30.43g).Cluster? contained genotypes with medium grain length(GL)(8.25mm),but their grain width(GW)(2.71mm),grain thickness(GT)(2.03mm)and Thousand-grain weight(TGW)(21.70g)were the lowest among groups.2.The correlation coefficient values suggest that the other studied grain size traits positively influenced the thousand grain weight(TGW)but with a different aptitude.The thousand grain weight(TGW)can be improved by using all three studied grain traits,most importantly grain thickness(GT),which contributes 49%,followed by grain width(GW)and grain length(GL),which contribute 37.4%and 24.9%of the final grain weight,respectively.Hence,assuming the trend of trait selection,i.e.,GT>GW>GL,for improving TGW in the rice yield enhancement programs.3.Grain length(GL)was associated with four genes(GS3,GS7,GW8,and GS2).Grain thickness(GT)was also found to be regulated by four different genes(GS3,GS7,GW8,and GS2)out of the nine studied genes.Grain width(GW)was found to be under the control of three studied genes(GW5,GW8,and GS2),whereas,thousand grain weight(TGW)was found to be under the influence of four genes(SLG7,GW5,GW8,and GS5)in the 204 germplasm under study.From these nine major genes viz qPE9-1,GW2,SLG7,GW5,GS3,GS7,GW8,GS5 and GS2,Fourteen Insertion/Deletion(InDel)markers were designed.The results showed that 28 alleles(A1,B1,A2,B2,A3,B3,A4,B4,A5,B5,A6,B6,A7,B7,A8,B8,A9,39,A10,B10,A11,B11,A12,B12,A13,B13,A14,and B14)were identified,with an average of 7.285 for each locus.4.The average polymorphism information content(PIC)and gene polymorphism(H)were 0.3310(0.1653?0.4359),indicating that each point had high polymorphism.The average expected heterozygosity(He=0.0193)was higher than the observed heterozygosity(Ho=0.2451),showing that a deficit of heterozygotes in rice.The studied markers also revealed higher gene diversity(D)ranging from 0.1730?0.5246.The study showed that InDel markers(i.e.,GW8-InDel,GW8-InDe12B,GS5-InDel1A,and GS2-InDell A)of the GW8,GS5,and GS2 genes are highly informative regarding these traits and can be used to understand the genetic variations in germplasm,whereas the rest of the markers are moderate to slightly informative for these traits.5.The AMOVA analysis was done which clearly emphasized that there was maximum(?PT=0.449)and significant(p?0.001)genetic variance between short grain and extra-long grain germplasm/sub-populations,while the least(?PT=0.035)genetic difference was observed between long grain and medium grain sub-populations.Part 2:Fine mapping of the QTL qLFP9 for controlling flag leaf and panicle length in rice(Oryza sativa L):Increasing rice yield is the main task of rice breeders.Flag leaf length and panicle length(LFP)affect grain yield and have become the main concern of more scientists.Flag leaf length and panicle length(LFP)directly affect rice yield.Therefore,studying the genetic and molecular basis of flag leaf length and panicle length(LFP)will help us to improve high yielding and high quality rice varieties.Flag leaf length and panicle length(LFP)are important factors determining grain yield potential in rice.To understand the molecular mechanism of the flag leaf length and panicle length,a multi-generation backcross from flag leaf length and panicle length was used derived from the previous construction of the research group with 9311 as the recurrent Wuyunjing 8 as the donor parent.The rice line L6238 with a significantly shorter length(SFP)was crossed with the long flag leaf and panicle length(LFP)of the indica rice variety Bing4114 and then backcrossed with Bing4114,and the influence of flag leaf length and panicle length(LFP).The main conclusions of the quantitative trait locus(QTL)are described as follows.1.In this study,genetic analysis for primary mapping and fine mapping of the flag leaf and panicle length gene qLFP9 was performed on BC1F2 and BC1F5 population,derived from a cross of two rice,L6238 short flag leaf and panicle length(SFP)and Bing4114 long flag leaf and panicle length(LFP)and the difference of flag leaf and panicle length was also significant.In order to identify QTL qLFP9(long flag leaf and panicle length)target gene,a linkage map was constructed using 450 SSR/InDel polymorphism markers data.All the markers positions were compared with the previous map(www.gramene.org)there is good consistency.The mapping markers covered the genetic distance of 1397.5cM in 12 rice chromosomes,and the average distance between the two markers was 12.3cM.2.The flag leaf and panicle length of BC1F2 backcross between parents L6238 and Bing4114 were investigated flag leaf and panicle length.The variation range of flag leaf length was 13.3?61.5 cm.There are 91 lines with long flag leaf length and 283 lines with short flag leaf length.The ratio of short flag leaf length plants to long flag leaf length plants of the BC1F2 segregation population is 1:3(?2=0.089<?0.05 2=3.84).At the same time,the variation range of panicle length was 16.1?36.2 cm.There are 107 lines with long panicle length and 267 lines with short panicle length.The ratio of short panicle length plants to long panicle length plants of the BC1F2 segregation population is 1:3(?2=2.599<?0.05 2=3.84),the segregation of flag leaf and panicle length is controlled by a single Mendelian factor,and short flag leaf and panicle length is dominant to long flag leaf and panicle length.3.For primary mapping BC1F2 segregation population of the QTL qLFL9 locus was initially located within the 0.24Mb intervals between InDel9-2(16.46Mb)and InDel9-10(16.70Mb)by using the single recessive plant in the isolated population of 374 individuals.The results showed that the QTL qLFL9 locus was located within the 0.24Mb intervals between InDel9-2 and InDel9-10 on chromosome 9.4.BC1F5 generation was obtained by screening BC1F2 recombinant plants from InDe19-2 and InDel-10.A total of 35 lines were used to expand the population for fine mapping.More intensive InDel markers were developed to detect 1950 recessive individuals,and finally,qLFL9 was located in the 18kb intervals between TS9-4 and TS9-18.5.According to the data published on the RGAP website,the 18kb region between the two markers TS9-4 and TS9-18 on chromosome 9 contains four candidate genes:LOC_Os09g27360,LOC_Os09g27370,LOC_Os09g27380,and LOC_Os09g27390.6.The four candidate genes were annotated through the sequence annotation database(http://www.gramene.org/).LOC_Os09g27360 encodes a protein with an unknown function.The LOC_Os09g27370 encodes a member of the spc97/spc98 family proteins and is a hypothetical protein.LOC_Os09g27380 encodes a zinc finger C3HC4 domain protein,and LOC_Os09g2 7390 encodes a transposable protein with a hypothetical function.7.The qRT-PCR results of candidate genes showed that LOC_Os09g27380 had the highest expression level than the other three genes LOC_Os09g273 70,LOC_Os09g27360,and LOC_Os09g27390.Also,the expression level of LOC_Os09g27380 in Bing4114 was significantly higher(P<0.05)than L6238,indicating that the qLFP9 allele in Bing4114 inhibited the flag leaf and panicle length development of rice.8.Sequence analysis also showed that there were mutations in LOC_Os09g27380 coding region between Bing4114 and L6238,which resulted in differences in amino acid sequences.Therefore,this study suggests that LOC_Os09g27380 is the candidate gene.LOC_Os09g27380 was identified as a significant candidate gene associated with qLFP9.9.This research provides new genetic resources for the molecular mechanism of phenotypic variation of important agricultural traits in rice and the genetic improvement of rice varieties.It lays the foundation for further gene function verification of qLFP9.