Dissection And Fine-Mapping Of Two Closely Linked QTL For Grain Size On Long Arm Of Chromosome 1 In Rice

Grain size is a key determinant of grain weight and a trait having critical influence on grain quality in rice.While increasing evidences are shown for the importance of minor-effect quantitative trait loci?QTL?in controlling complex traits,the attention has not been given to grain size until recently.In previous studies,five QTL having small effects for grain size were resolved on the long arm of chromosome 1 using near isogenic lines?NILs?derived from indica rice cross Zhenshan97?ZS97?³/Milyang 46?MY46?.One of them,qTGW1.2c,was was resovled using 18 NIL-F₂ populations and 15 sets of NILs in the present stuty.Consequently,two QTL for grain size were dissected in a 420kb region.1 Dissection of two QTL for grain sizeIn a previous study,qTGW1.2c was located within a 2.1-Mb region included the segregating region RM11807-RM265 and two flanking cross-over regions,i.e.,RM11800-RM11807 and RM265-RM11885.In the present study,the genotypes of the cross-over regions were determined using polymorphic markers developed based on sequence differences between ZS97 and MY46 detected by whole genome re-sequencing.The homozygous region newly identified was removed and qTGW1.2c was narrowed down to a 1.1-Mb region flanked by RM11800 and RM11844.Then,two BC₂F₁₀ plants were selected from progenies of a single BC₂F₉ plant,carrying heterozygous segments RM11807-RM11842 and RM265-RM11842,respectively.Homozygous non-recombinants in the BC₂F₁₁ populations were selected to develop two sets of NILs in BC₂F_11:12.Two-way analysis of variance was performed to test the phenotypic differences between the two genotypic groups in each NIL set.Consistent genotypic effects were detected for grain size traits in these populations.Thus,qTGW1.2c was located in same segregating region of these populations,which was a672.3 kb region flanked by Wn35060 and RM11844.Five BC₂F₁₂2 individuals were selected from progenies of another BC₂F₁₀ plant,carrying sequential heterozygous segments extending from RM265 to RM11842.In the five resultant NIL-F₂ populations in BC₂F₁₃,homozygous non-recombinants were identified and five sets of NILs in BC₂F_13:14 were developed.Using these NIL-F₂ and NIL populations,two QTL were resolved in the target region.The first QTL was located within the Wn35183-RM11828 interval that corresponds to a 132.4 kb region.This QTL primarily contributed to grain shape with the first segregating marker RM265 located at35.2 Mb on chromosome 1,thus we designated it qGS1-35.2.The other QTL was located in a 125.5 kb region flanked by Wn35518 and Wn35643.Because this QTL mainly contributed to grain width with the first segregating marker Wn35518 located at 35.5 Mb on chromosome 1,we designated it qGW1-35.5.2 Fine-mapping of qGW1-35.5Two plants were selected from the BC₂F₁₃ populations,which were heterozygous in qGW1-35.5 region but homozygous in another QTL region.In the resultant BC₂F₁₄populations,homozygous non-recombinants were identified and two sets of NILs in BC₂F_14:15 were developed.Using these four populations,the effect of qGW1-35.5 was validated.Two plants were selected from the BC₂F₁₄ populations,carrying sequential heterozygous segments covering qGW1-35.5 region.In the resultant BC₂F₁₅ populations,homozygous non-recombinants were identified and two sets of NILs in BC₂F_15:16 were developed.Using these four populations,the qGW1-35.5 region was updated to be interval Wn35518?Wn35561.Then three plants were identifed from the BC₂F₁₅populations,carrying sequential heterozygous segments covering the updated region.Using the three resultant populations in BC₂F₁₆,qGW1-35.5 was finally located within an86.3 kb region flanked by Wn35518 and Wn35603.In the two sets of NILs in BC₂F_15:16populations,the MY46 allele increased grain width by 0.008 mm and 0.013 mm,and increased 1000-grain weight by 0.09 g and 0.08 g,having R² of 11.4,22.0,8.3 and 4.4%,respectively.The qGW1-35.5 regulates grain width,simultaneously affecting grain weight,offering a new gene resource for enhancing grain yield.3 Fine-mapping of qGS1-35.2Three plants were selected from the BC₂F₁₃ populations,carrying sequential heterozygous segments covering qGS1-35.2.In the three resultant BC₂F₁₄ populations,homozygous non-recombinants were identified and three sets of NILs in BC₂F_14:15 were developed.Using the six populations,the qGS1-35.2 region was updated to be Wn35183?Wn35263.Two other plants were selected from the BC₂F₁₄ populations,carrying heterozygous segments RM265?Wn35263 and RM11824?Wn35393,respectively.In the two resultant BC₂F₁₅ populations,homozygous non-recombinants were identified and two sets of NILs in BC₂F_15:16 were developed.The qGS1-35.2 was finally mapped within a57.7 kb region flanked by Wn35183 and RM11824.In the NIL set in BC₂F_15:16segregating for qGS1-35.2,the ZS97 allele increased grain length by 0.030 mm,decreased grain width by 0.006 mm,and increased ratio of grain length to grain width by0.015,having R² of 29.0,5.2 and 36.5%,respectively.Nonsignificant on grain thickness and yield traits including 1000 grain weight,number of panicle per plant,number of grain per panicle and grain yield.This QTL affects grain shape but has little influence on grain weight and other yield traits,providing a potential gene resource for fine-tuning grain shape to modify grain appearance quality without yield penalty.The NIL^ZS97 and NIL^MY46 were used for observation of outer glume epidermal cell,transcription analysis of cell cycle genes and transcriptome analysis.Nonsignificant difference was detected on the cell length and width,but the cell number in the longitudinal direction was higher in NIL^ZS97 than NIL^MY46.As compared with NIL^ZS97,the expression levels of CYCB2.1 encoding a B-type cyclin and KPR4 encoding a cyclin-dependent kinase inhibitor were higher and lower in NIL^MY46,respectively.These results suggest that qGS1-35.2 affects grain length by controlling cell division.According to the Rice Annotation Project Database,there are six annotated genes in the 57.7 kb region for qGS1-35.2.Of which five showed nucleotide polymorphisms between the two parental lines.Quantitative real-time PCR detected expression differences between near isogenic lines for qGS1-35.2 at three of the six annotated genes.The mutants were constructed by CRISPR/Cas9 gene-editing technology for these annotated genes,laying foundation for cloning of qGS1-35.2.