水稻粒厚主效位点qGT8精细定位和候选基因分析

doi:10.3864/j.issn.0578-1752.2015.24.001

摘要/Abstract

摘要： 【目的】在已鉴定的稻谷粒长、粒宽和粒厚QTL的基础上，对控制粒厚的主效QTL进行精细定位和候选基因分析，以解析川106B（C-106B）细长粒形的遗传基础，为进一步通过分子技术改良其产量水平提供科学依据。【方法】以细长粒形的优质籼稻保持系川106B与籽粒较宽厚的籼稻保持系川345B（C-345B）杂交，构建包含182个单株的F2群体，采用QTL Catographer v2.5软件基于复合区间作图法发掘与稻谷粒形性状相关的QTL；进一步从BC₃F₂群体筛选隐性单株（稻谷厚度较薄）对粒厚主效QTL（qGT8）进行精细定位，并对候选基因进行测序和荧光定量PCR分析。分别构建qGT8位点携带川106B等位基因的近等基因系（NIL-gt8^C-106B）和携带川345B等位基因的近等基因系（NIL-GT8^C-345B）并调查其稻米外观品质及产量性状。【结果】川106B和川345B的粒长、粒宽和粒厚表型存在显著差异。利用F₂群体检测到2个粒长QTL、3个粒宽QTL和3个粒厚QTL，其中，位于第7染色体区间RM21892—RM3589的粒长主效QTL（qGL7）可解释粒长变异的68.23%，川106B等位基因在该位点可增加粒长0.47 mm。控制稻谷粒宽和粒厚的主效QTL（qGW8和qGT8）位于第8染色体上相同区间RM6070—RM447，分别解释相应表型变异的26.48%和34.89%，增加粒宽或粒厚的等位基因均来自于川345B。利用1 732个BC₃F₂隐性单株，将粒厚主效位点qGT8精细定位在标记SG930和SG950间的11.2 kb区段，该区段仅包含1个注释基因LOC_os08g41940（OsSPL16）。对该基因测序分析发现，川106B和川345B在起始密码子ATG上游2 kb区段存在7个差异位点，在编码区有5个多态性位点，其中，川106B在第3外显子插入2 bp（c.1006_1007 插入CT）引起移码突变，且位于qGT8的OsmiR156结合位点，推测为川106B籽粒厚度变薄、宽度变细的关键位点。实时荧光定量PCR分析发现，qGT8在幼穗中表达量较高，且在川106B和川345B中的表达方式相似，表达量在1—8 cm长幼穗发育时期随幼穗发育逐渐增加，8 cm时达到最高，之后随幼穗发育逐渐降低，但2个亲本在各时期的表达水平存在差异。近等基因系NIL-GT8^C-345B的粒厚、粒宽、千粒重、单株产量和垩白粒率显著高于NIL-gt8^C-106B，而粒长、透明度、株高、单株有效穗数、穗长、每穗实粒数、结实率和播抽期与NIL-gt8^C-106B相当。【结论】控制粒长的主效QTL（qGL7）位于第7染色体区间RM21892—RM3589，控制粒宽和粒厚的主效QTL位于第8染色体的相同区间RM6070—RM447。粒厚主效QTL（qGT8）被精细定位在仅包含GW8的片段上，是控制粒形和产量的关键基因，但在近等基因系中高粒重与高垩白紧密连锁，表明该位点存在高产与外观品质改良的矛盾。

关键词: 水稻, 粒形, 粒厚, 数量性状基因座位, 产量, 品质

Abstract: 【Objective】 The paper is order to identify quantitative trait loci (QTLs) for grain length (GL), width (GW) and thickness(GT), and further to narrow down the genomic region containing the major QTL for GT and analyze the possible candidate genes, which would help us to understand the genetic basis underlying the formation of slender grains and facilitate the efforts to the marker-assisted improvement of yield in a cytoplasmic male-sterile (CMS) maintainer line Chuan106B (C-106B). 【Method】 In this study, C-106B, which has a long slender grain type and good grain quality, was crossed with Chuan345B (C-345B), another CMS maintainer line with thicker and wider grain, to produce F₂ population for a preliminary QTL analysis. The chromosome interval of QTLs for grain shape was identified by composite interval mapping (CIM) using QTL Catographer v2.5. Then, a BC₃F₂ population derived from the same cross was developed and those plants with thinner grain were further picked out for the fine mapping of the major QTL (qGT8) for GT. Sequencing analysis and quantitative real-time PCR for the candidate gene were also conducted. In addition, a pair of nearisogenic lines (NIL) carrying the C-106B (NIL-gt8^C^-106B) and C-345B (NIL-GT8^C^-345B) alleles at qGT8 locus were bred and applied to survey for yield and quality traits, respectively.【Result】Significant differences were observed for GL, GW and GT between C-106B and C-345B. Using the phenotype and genotype data of the F₂ population, two QTLs for GL, three for GW and three for GT were identified. Among these, the major QTL for GL, accounting for 68.23% of phenotypic variance was mapped to the region between RM21892 and RM3589 on chromosome 7. The allele from C-106B was responsible for increasing GL by 0.47 mm at this locus. Two major QTLs (qGW8 and qGT8) controlled GW and GT, each explaining up to 26.48% and 34.89% of the variation, respectively, were found in the same interval of RM6070-RM447 on chromosome 8. The C-345B alleles contributed to the increase in GW and GT at the two loci. Furthermore, using 1732 plants with recessive phenotype (thinner grain) from the BC₃F₂ population and 11 markers including in the region RM6070-RM447, qGT8 was delimitated to a region bounded by insertion/deletion (Indel) markers SG930 and SG950, with a physical interval of 11.2 kb, which contains only one predicted gene (LOC_os08g41940, OsSPL16). Comparison of the nucleotide sequencesof the C-106B and C-345B alleles of qGT8 uncovered seven nucleotide changes within the promoter region upstream of the ATG start codon and five polymorphisms in the coding region. One of these, a 2-bp insertion（c.1006_1007 ins CT）in exon 3 at miR156 target site in qGT8 of C-106B, result in a frameshift mutation, which is probably associated with the formation of a more slender grain. Expression analysis indicated that qGT8 was preferentially accumulated in developing panicles. The expression patterns of qGT8 at eight panicle developmental stages were similar between C-106B and C-345B, which gradually increased at early stages (panicles length ranging 1-8 cm), peaked at the 8 cm young panicle and then declined in panicles of 12 cm to 23 cm in length. However, the levels of transcript for qGT8 were different at every developing stage between two parents. Comparison of the two NILs showed that NIL-GT8^C^-345B increased significantly in GW, GT, 1000-grain weight, grain yield per plant and the percentage of grain with chalkiness, while no significant differences were observed in other measured traits such as GL, transparency, plant height, number of panicles per plant, panicle length, number of filled grains per panicle, seed setting ratio and heading date. 【Conclusion】 A major QTL affecting grain length in C-106B was mapped to a region flanked by markers RM21892 and RM3589 on chromosome 7, while two major QTLs (qGW8 and qGT8) governing grain width and thickness, respectively, were found in the same interval of RM6070-RM447 on chromosome 8. Furthermore, qGT8 was fine mapped to a DNA region only covering GW8 gene and this result suggested that qGT8 may play a key regulator role in grain shape and yield traits. However, a coupling link of the increasing for grain weight and high chalkiness in NILs implied its counteracting effects on grain yield and quality at same locus.

Key words: rice, grain shape, grain thickness, QTL, yield, quality

高方远，罗正良，任鄄胜，吴贤婷，陆贤军，苏相文，吕建群，任光俊. 水稻粒厚主效位点qGT8精细定位和候选基因分析[J]. 中国农业科学, 2015, 48(24): 4859-4871.

GAO Fang-yuan, LUO Zheng-liang, REN Juan-sheng, WU Xian-ting, LU Xian-jun, SU Xiang-wen, Lü Jian-qun, REN Guang-jun. Fine Mapping and Candidate Gene Analysis of qGT8, A Major QTL for Grain Thickness in Rice[J]. Scientia Agricultura Sinica, 2015, 48(24): 4859-4871.

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