Positional Cloning, Function Analysis And Breeding Utilization Of Grain Length Gene QGL3in Rice(Oryza Sativa L.)

Grain number, panicle number and grain weight are three important components of grain yield of rice (Oryza saliva L.). When grain number per panicle and panicle number per plant reach an ideal level, improvement of grain weight plays a key role in further yield increase in rice breeding program. Grain weight is largely determined by grain size, which is specified by its three dimensions (grain length, width, and thickness) and the degree of filling. Meanwhile, grain shape is also a quality trait for rice so that study on grain size or weight in model plant rice is of great economic and academic meaning.For a better understanding of the genetic characteristics of rice grain size or weight, we screened an extra-large grain japonica accession, N411, with more than7.0grams of100-grain weight (HGW) and constructed five F2populations with five small-grain accessions (N643,93-11, RY, LLHN and SWWR). Genetic analysis of the grain shape and weight traits with those five F2populations indicates that there are many genetic differences between the extra-large grain N411and the other five small-grain accessions. The quantitative trait loci (QTL) for four grain traits and three panicle traits were analyzed in the N411×N643F2population. A total of30QTL for the seven traits were detected, including four QTLs for grain length (GL) on chromosome3,10and11; four for grain width (GW) on chromosome2,3and5; eight for grain thickness (GT) on chromosome2,3,5and6; seven for100-grain weight (HGW) on chromosome2,3,5,6,10and11; two for spikelete per panicle (SPP) on chromosome1and3; three for panicle length on chromosome2,3and6; two for grain rate on chromosome7and8. Most of the QTLs for grain traits were mapped to nearly the same intervals or adjacent regions. These pleiotropic performances provided satisfactory explanations for the results of high phenotypic correlations between grain traits in the F2population. Compared with previous studies, the mapping regions of the major QTL qGL3a, qGW2.2and qGW5, explaining25.54%,50.63%and15.38%of the phenotypic variation, were coincident with those of cloned genes GS3, GW2and qSW5/GW5, respectively. qSPP1, which positive alleles from line N643, was localized to the same interval of the cloned gene Gnla. A new grain length QTL, qGL3, which explained38.38%of the total variation of grain length, was mapped to the interval of RM5864-RM3199on the long arm of chromosome3, where the QTLs for GW, GT, HGW and PL were also detected. That most of the positive QTL for the grain traits came from N411provided a genetic explanation for the mechanism of the extra-large grain formation in N411.The novel major grain length QTL, qGL3, which explained variations for38.38%grain length,5.96%width,11.89%thickness and27.99%weight, was further disassembled to a single genetic factor in the background of indica accession93-11with marker-associated selection and successive backcrosses. Using a BC2F3population and BC2F4progeny testing, the qGL3QTL was mapped to a46.6-kb region with five predicted ORFs (ORF1, LOC_Os03g44460; ORF2, LOC_Os03g44470; ORF3, LOC_Os03g44484; ORF4, LOC_Os03g44500, and ORF5, LOC_Os03g44510)(Nipponbare genome sequence as a reference).Based on RT-PCR analysis and EST information, we found that only ORF3and ORF4were expressed, while ORF1and ORF2encoding retrotransposon proteins and ORF5encoding a transposon were not expressed in rice. RT-PCR analyses showed no expressional difference of ORF3and ORF4in young panicles between93-11and its near-isogenic line (NIL)93-11NIL-qgl3. Sequence comparison of these two expressed ORFs between93-11and N411revealed no difference in the ORF3sequence and four SNPs in the ORF4. SNP1, a single nucleotide transition from C (93-11) to A (N411)(c.+1092Câ†'A), is present in the10th exon of the ORF4, and SNP2, a single nucleotide transition from C (93-11) to T (N411)(c.+1495Câ†'T), in the11th exon. These two transitions cause amino acid residue changes from aspartate to glutamate (Asp364Glu) and histidine to tyrosine (His499Tyr), respectively. SNP3(c.+2643A->G, in the18th exon) and SNP4(c.+2838Tâ†'C, in the21th exon) do not cause amino acid residue changes. Therefore, ORF4was most likely the candidate gene for qGL3.The ORF4encodes a putative phosphatase with two Kelch repeat domains (OsPPKL1). There are two OsPPKL1homologues, OsPPKL2and OsPPKL3, in rice genome. Sequence alignment of plant homologues of OsPPKL1showed that the amino acid transition (Asp364Glu) caused by SNP1in OsPPKLlN411occurs in a conserved AVLDT motif of the second Kelch domain while the transition (His499Tyr) caused by SNP2occurs in a non-conserved region. Transgenic analysis found that over-expression of OsPPKL193-11and its closer homologue OsPPKL3forms shorter grains in Zhonghua11while over-expression of the other homologue OsPPKL2forms longer grains. Over-expression of the peptide cutoff PP2A domain forms shorter grains but over-expression the peptide without the two Kelch domains causes no effect on rice grain. Accordingly, the T-DNA insertion mutant of OsPPKLI and OsPPKL3exhibits longer grains and mutant of OsPPKL2exhibits shorter grains as compared with its wild type accession Dongjin. Those data indicated that OsPPKLI and its closer homologue OsPPKL3function as negative regulators of grain length, while OsPPKL2as a positive regulator. The Kelch domain is essential for OsPPKL193-11nagetive function.In the93-11genetic background,(NIL-GS3/qGL3), single loss qGL3increase grain length much more (～2.7mm) than single loss of GS3(～1.7mm), and loss of qGL3plus GS3together showed partially additive function (～3.7mm). And loss of qGL3in the functional GS3background could increase grain (2.7mm) more than in the null gs3background (～2.0mm). These data indicated that GS3and qGL3take in partially together in rice grain regulation. Some genes were overlapped and in different levels regulated by those two genes indicated by microarray.The near-isogenic line in the93-11background analysis indicated that the long glumes of93-11NIL-qgl3resulted from an increase in cell numbers longitudinally, which was consistent with the observation that the ovaries elongation of93-11NIL-qgl3was faster than93-11. Comparison of grain yield and other agronomic traits between93-11and N\L-qgl3showed that qgl3increased grain weight (+37.03%), length (+19.69%), width (+1.15%) and thickness (+8.26%) without changing other agronomic traits significantly. The heterozygote could increase grain weight about16.24%. The qgl3allele can increase16%of grain yield in inbred rice, and10%～13%in hybrid rice by regulating grain length, weight and filling rate.Ninety-four rice germplasms with abundant diversity in grain size were selected for sequencing a3.6-kb genomic DNA fragment that covers the four mutation sites of the qgl3. We found only one variety, DT108(grain length=12.08mm) have the same SNP1as N411, while polymorphisms of SNP2, SNP3and SNP4are widely distributed in either japonica or indica varieties. By association analysis of the SNPs and InDel markers in the3.6-kb region with grain length of the94germplasms, we found that SNP1had a high contribution to grain length while other polymorphic sites had no significant contributions to grain length. These results indicate that the qgl3is a rare allele in rice and SNP1is a functional mutation for long grain. Using this collection, the gene alleles of the reported grain shape genes/QTLs were analyzed, and the genetic architecture of rice grain shape was discussed.Those results indicated that qGL3may be used in either cross-selection breeding by MAS, or hybrid-rice breeding to increase rice yield and to improve appearance quality. Breeding application of this rare and desirable allele for extra-large grain may make a great improvement in the rice yield.