Dissection Of The Genetic Basis Of Grain Quality Using A Multi-parental Population And Rapid Cloning Of A Grain Length QTL Based On Pseudo-near Isogenic Lines In Rice

Part Ⅰ:Rice grain quality is controlled by quantitative trait loci（QTLs）and is largely affected by environmental conditions.Identifying stable superior alleles across different environments such as high and low temperature is the prerequisite for improving quality traits,especially chalkiness and eating and cooking quality.The multi-parent advanced generation intercross（MAGIC）population takes both the advantages of robust QTL detection that a bi-parental population has and the associated population’s rich genetic diversity.Besides,the MAGIC population segregates with several alleles and enables the identification of the superior alleles.In this study,a bin-and SNP-based GWAS were conducted to scan QTL for nine grain quality traits including grain shape traits（grain length,grain width,and length-width ratio）,chalkiness traits（transparency,percentage of grain with chalkiness,and degree of endosperm chalkiness）,and eating and cooking quality traits（amylose content,gel consistency,and alkali spreading value）with an 8-parent MAGIC population.The main results were as follows:1-The 8-way MAGIC population consisted of 560 recombinant inbred lines（RILs）.Each parent genomic contribution in the 8-way MAGIC population was 11.4-12.7%except for MH63 accounting for 15.0%,which was in line with the expectation.The phylogenetic tree and principal component analysis revealed that the MAGIC population had no apparent population structure.The bin map was constructed with 12405 bins.The length of the bins varies from 10 kb to 490 kb,and 84.1%of the bins are about 10-50 kb in length,with an average length 29.2 kb.The average bins per line were 282.2-302 RILs heading within 15 days were selected to dissect the genetic basis of grain quality in the main crop and ratoon crop.The average temperature at filling stage was 29.2℃and 19.4℃in the main crop and ratoon crop,respectively.The high temperature negatively affected the grain quality,particularly the grain chalkiness and eating and cooking quality traits.Bin-based GWAS showed a more powerful QTL detection than SNP-based GWAS under both environments,where 17 and 20 QTLs were detected for the nine quality traits in the main crop by SNP-and bin-based GWAS,respectively.Meanwhile,14 and 16 QTLs were recorded in the ratoon.Ten and 12 QTLs were stably detected across the two environments by SNP-and bin-based GWAS,including the major genes affecting the grain quality（GS3,GW5,Wx,and ALK）.3-The novel minor QTL,qASV6.2,controlling the alkali spreading value,was fine mapped to bin B06＿271,which contains ten candidate genes.Five genes（LOC＿Os06g15380,LOC＿Os06g15370,LOC＿Os06g15410,LOC＿Os06g15430,or LOC＿Os06g15440）highly expressing in the embryo and the mature seed might be the candidate gene.4-According to the grain quality of the best five and the worst five RILs,we elucidated that RILs with slender long grain owned the GS3 superior allele from IR34,while RILs with short grains carried the inferior allele from ZS97 and GC2.Also,these RILs carried the superior allele of GW5 from GC2 and the inferior one from ZS97.For grain chalkiness,Cypress and GC2 offered the superior and inferior alleles of Chalk5,respectively.For amylose content and gel consistency,Wx superior allele was presented by Cypress while the inferior allele by MH63 and IR34.For the alkali spreading value,the ALK superior allele was introduced by GC2 and YJSM,while the inferior by Cypress,Pratao,and DA5.Part Ⅱ:The traditional QTL cloning method is time-consuming due to developing near-isogenic lines（NILs）.This study aimed to develop a pseudo-NIL population to clone QTL rapidly.Zhenshan 97 and Egy316（Egyptian mutant）were used to construct an F₂ population for QTL mapping.A pseudo-near isogenic F₂（Pseudo-NIF₂）was developed at F₃ generation for QTL cloning.The main results were as follows:1-Twenty-five QTLs controlling the grain yield-related traits were detected in the F₂ and F_2:3 populations.The q NEP5.1 is a stable minor novel QTL controlling the number of effective panicles per plant with-2.3 and-0.46 additive effects in the F₂ population and F_2:3 families.2-Forty-seven QTLs have been identified for grain shape traits across the F₂ and F_2:3populations.qGL3.1 and q GW5.1 are major QTLs for grain length and grain width,respectively.Moreover,qGL3.1 recorded additive effects of 0.36 mm and 0.4 mm,while q GW5.1 recorded-0.15 mm and-0.19 mm across the F₂ and F_2:3 populations,respectively,corresponding to the major genes GS3 and GW5.Three loci presented a pleiotropic effect on grain shape on chromosomes 1,2,and 10.Two novel minor QTLs,qGL1.2 and qGL10.1,were stable in both environments,with LOD of 9.9 and 7.9 in the F₂ population and of 3.1 and 3.6in the F_2:3 population,respectively.3-For the novel QTL,qGL10.1,we selected the plant F₂-120 according to the genome constitution of F₂ plants in which other grain length QTLs are fixed except qGL10.1.A pseudo-near isogenic F₂ population（Pseudo-NIF₂）was generated by selfing the plant.Then qGL10.1was fine mapped to a 19.3-kb region containing a gene Os MADS56,which was verified to regulate grain length.In a word,bin-based GWAS has a more vital QTL detection ability in the MAGIC population and the superior alleles identified in the MAGIC population is valuable for breeding application.High temperature has a harmful effect on the grin quality.In addition,the pseudo-NIF₂ approach is demonstrated for quick cloning of QTL,which is recommended to explore in the future.