Genetic Analysis And Gene Cloning Of Flow-,and Sink-related Traits In Rice

Vascular bundle plays an important role in plant growth,water transport,nutrient transport and mechanical strength of stem.The number,size and function of vascular bundle directly affect grain filling and yield.In this study,440 rice accessions from 3k were used to perform genome-wide association analysis（GWAS）for flow-and sink-related traits with 4.8Mb high quality SNP markers.We analyzed the genetic bases of flow-and sink-related traits in rice and cloned gene influencing flow-and sink-related traits.These results will have important guiding significance for breeding of super-high-yielding rice cultivars with flow fluency by molecular design breeding,and will also lay a foundation for the further functional studies of genes influencing flow-related traits.The main results were as follows:1.Genetic bases of flow-,and sink-related traits in rice revealed by genome-wide association analysis（GWAS）.A total of 140 QTL were identified by GWAS with 3,188,500 high-quality SNPs.Among these,16clusters simultaneously affected flow and sink traits were identified in this study.We selected nine candidate genes in two consistent QTL regions simultaneously affecting multiple vascular bundle and sink size traits by gene-based association analysis and haplotype analysis.Among them,D2（LOC＿Os01g10040）and Gn1a（LOC＿Os01g10110）for q PLVN1.1,q SLVN1.1,and q PRN1.2,Os PIN5b（LOC＿Os08g41720）for q PLVN8,q SLVN8,q SSVN8 and q STSN8.2 were considered as the most likely candidate genes based on functional annotations.2.Cloning of Os LVN6 affecting large vascular bundle number at panicle neck.In this study,a quantitative trait locus（QTL）q LVN6 affecting number of large vascular bundle in rice panicle neck was detected in the chromosome region of RM136–RM3 on chromosome 6 by genome-wide association analysis and linkage analysis.A large q LVN6 near-isogenic line was constructed by marker-assisted selection.The F₂population derived from heterozygous near-isogenic lines of q LVN6 was used for fine mapping,and the q LVN6 was finally delimited into a region of 55.3 kb on chromosome 6,including nine genes.Based on haplotype analysis and functional annotation of nine genes,the most likely candidate Os LVN6gene was predicted.gene was knocked out by CRISPR/Cas9,and three homozygous mutants with different mutation forms were obtained.The number of large vascular bundles in panicle neck was significantly reduced in all three mutants compared with the control.3.Genetic effect analysis of GNP1 on flow-and sink-related traits.One QTL cluster（q PTSN3 and q STSN3）simultaneously affecting grain number in primary and secondary rachis branches was detected in the region of 35.73?36.36 Mb on chromosome 3by GWAS.The chromosome region contained a cloned grain number GNP1 gene.In this study,GNP1 near-isogenic lines NIL-GNP1^TQ（Wu et al.,2016）and its recurrent parent Lemont were used as test materials to compare and analyze the genetic effects of GNP1 on flow and sink traits.Compared with Lemont,NIL-GNP1^TQsignificantly increased large and small vascular bundle number and area,and significantly increased grain number per panicle by 30.9?35.1%,but significantly decreased effective panicle number per panicle,seed setting rate and grain weight,finally increased actual yield by 6.8?7.5%.However,the increase in grain yield was not as significant as expected,mainly due to the capacities of stem vascular bundle transporting photosynthate could not fully meet the increased sink capacity.4.Cloning and functional identification of large vascular bundle phloem area gene LVPA4.In this study,we isolated and cloned a new gene,LVPA4,affecting large vascular bundle phloem area of panicle neck from 440 natural accessions.LVPA4^LT,a superior allele of LVPA4derived from Lemont,was introduced into Teqing,a high-yielding cultivar with poor quality,to construct a near-isogenic line NIL-LVPA4^LT.The results showed that NIL-LVPA4^LTexhibited significantly increased large vascular bundle phloem area,enlarged flag leaf size,and improved panicle type compared with Teqing,leading to 7.9–9.8%yield increase in NIL-LVPA4^LT.Due to significant improvement of grain filling in inferior spikelets in NIL-LVPA4^LT,NIL-LVPA4^LTsignificantly increased grain weight in inferior spikelets and obviously improved grain quality compared with Teqing.The one nucleotide variation in the third exon of LVPA4,which led to amino acid change is associated with large vascular bundle phloem area,spikelet number,and leaf size throughout sequencing analysis in 440 panels.LVPA4 can be used in rice molecular assisted selection breeding to improve rice yield and quality.