Identification Of Grain Shape Genes And Their Interaction Effect In Rice

Grain length,width and thickness are important traits for rice yield and quality.Most of these properties belonged to quantitative traits controlled by a number of genes.At present,many QTLs or genes controlling grain shape were mapped,but only qGL3,GW8,GS5,GS3,GW2,GW5 and qSW5 genes were cloned,which were involved in different varieties of Zhenshan 97,Minghui 63,Nipponbare,9311 and WY3.The function and effect of these genes have been preliminary definited,but it is difficult to estimate the effect of the role of these genes in the same genetic background because of the influence of different parental genetic background.The ultimate goal of researches for grain shape genes is to improve grain traits for high yield by the effective utilization of these genes in breeding.Therefore,it is very important to further exploit new rice grain genes/QTLs,analyze and compare the effect and interaction of cloned genes in the same genetic background.In this study,240 RIL generated from the cross of a japonica rice TD70 with extra large grains and an indica variety Kasalath was employed to investigate the genetic characteristics of extra grain size of TD70,to map QTL associated with grain shape in order to discovery new loci.Based on the analysis of the potential genes donated by TD70,the differences of gene sequence of different varieties were studied via sequencing.The effects of grain shape genes were estimated.The results here eventually benefits the marker assisted selection in rice high yield breeding.The main results were as follows:1.Genetics and QTL mapping of grain shape traits on an extra large grain size in rice240 RIL generated from the cross of TD70 and Kasalath was employed to map QTL associated with grain shape in 2010 to 2013.The distribution of gain length and grain width was continuous with multi-peaks,while a single peak continuous distribution was observed in grain thickness.The grain weight measured in 2011 was normally distributed,with a wide distribution range of 18.8-63.83g and a peak between 30-35g.And most individuals were located in the range of 25-40g.Regarding the thousand seed weight,14 individuals weighed over 50g,and none weighed over that of the parental line TD70.It suggests that the extra large grain trait of TD70 is heritable,and random recombination of several genes/QTL enhancing grain weight was observed in the offspring.QTL analysis based on the RIL population data collected from four years revealed that there were totally 22 fragments carrying the loci associated with grain shape.The fragments between RM1347-RM5699,RM109-RM3264,RM6080-RM6832,RM3777 and RM169 were detected for four years,referring to the genes/QTL GW2,qGW2-1,GS3 or qGL3,GS5 and GW5,respectively.GS7,GL7 and qGW9 located between RM1328-RM7038 were detected in two years,while GW8 between RM5545-RM447 was detected only in 2013.Except for qGWT8-1(13)locus,10 loci were detected only in one year,including six loci associated with grain thickness(GT),one grain length(GL)loci,and three grain width(GT)loci.Among all genes/QTL,qGW2-1 and qGW9 were potential valuable sites.2.Sequence analysis of GS3,qGL3、GW2,GS5 and GW8 complete genome on TD70 and KasalathMany differences on sequences of GW2,qGW2-1,GS3 or qGL3,GS5 and GW5,between TD70 and Kasalath.GW2-TD70 has the same function to GW2-WY3 via homology-based cloning method.A single nucleotide substitution(C to A)was found in the position of +165 in the encoding region of the second exon,which is essential to the function change of GW2-TD70.As to qGL3 allele of TD70,a single nucleotide substitution(C to A)was found in the position of +1092 in the encoding region of the 10th exon,while as to GS5,the deletion of two codons(GCGGCG)was found at the position of 2059bp,causing the del-type of slender grain.There are several key differences on GW8 between TD70 and Kasalath.These results indicated that there were at 5 synergistic alleles from TD70,while the normal alleles were found in Kasalath.3.Sequence difference of grain shape gene GW2,GS3,GS5,GW8,qGL3 in different rice varieties59 rice varieties,together with TD70 and Kasalath were selected to perform haplotyping analysis on GW2,GS3,qGL3,GW8 and GS5.As to GS3,only two SNP and 1 InDel exit in the coding region among these varieties.Based on this difference,61 varieties are grouped into five haplotypes.A total of 16 haplotypes of GW2 is based on 27 SNP sites and 5 insertion/deletion sites in all exons and intron,in which GW2-9 group was the most represented allele.The phylogenetic tree indicates that GW2-7,8,11 and 12 are close to GW2-9,and GW2-10 is the closest to GW2-9.There are 5 SNPs and 6 Indels detected in all introns and exons of GS5,and based on these polymorphisms,the 61 varieties are grouped into 13 haplotypes.As to GW8,21 SNPs and 9 Indels were screened among the whole genome sequence of about 4,100bp,in which there are 7 SNPs and 1 Indel are located in the exons.And based on the differences,the 61 accessions can be categorized into 20 types.Only 8 SNPs and 1 Indel were detected in the genome sequence of qGL3,and 54 in 61 varieties share the haplotype of qGL3-2,while the other 7 varieties belong to other 5 haplotypes.4.Vector construction and cDNA clone of GW2,GS3,GS5 gene on extreme grain shape riceFragment length of GW2 gene cDNA sequence from TD70 and Kasalath were 1277bp and 1278bp,respectively.For absence of 316th base A,GW2 protein of TD70 only express 115 amino acids,while protein of Kasalath express 425 amino acids completely;cDNA fragment length of GS3 sequence gene from TD70 and Kasalath were 713bp and 696bp,encoding 54amino acids and 231 amino acids,respectively,cDNA fragment length of GS5 sequence gene from TD70 and Kasalath were 1449bp and 1443bp,encoding 482 amino acids and 480 amino acids,respectively.The GS5 sequence of Kasalath in the position of 106bp exit GGCGGC six base deletion.The sense and antisense gene over-expression vectors were constructed to indentify the gene function of GS3,GW2 and GS5 from both TD70 and Kasalath.The selective gene of Hygromycin was employed in the primary identification of positive transgenic plants.5.Grain effect analysis on different function genotype combinationAs to the genes of qGLS,GW2,GS3,GS5 and GW8 genes from TD70,random recombination was observed in the offspring lines with synergetic gene effect and the effects are accumulative,which is consistent with the previous reports.There are three major genes,qGL3、G W2 and GS3,with a single gene effect order as qGL3-TD70>GW2-TD70>GS3-TD70>GS5-TD70>GW8-7D70,which is also observed in multi-loci combinations.GS5 and GW8 have relatively weak effect without any major genes,indicating that it’s possible to adjust the loci number and the cluster of loci to reach different grain weight.