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作物学报 ›› 2018, Vol. 44 ›› Issue (11): 1621-1630.doi: 10.3724/SP.J.1006.2018.01621

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

水稻矮化大粒突变体sdb1的鉴定与基因定位

陶怡然,熊毓贞,谢佳,田维江,张晓琼,张孝波,周倩,桑贤春,王晓雯()   

  1. 农学基础实验教学中心 / 西南大学农学与生物科技学院, 重庆400715
  • 收稿日期:2018-03-09 接受日期:2018-07-20 出版日期:2018-11-12 网络出版日期:2018-08-07
  • 通讯作者: 王晓雯
  • 基金资助:
    本研究由重庆市基础科学与前沿技术研究项目(cstc2015jcyjA80008);重庆市社会事业与民生保障科技创新专项(cstc2016shms- ztzx8007);重庆市社会事业与民生保障科技创新专项(cstc2017shms-xdny80057);国家级大学生创新创业训练计划项目(201710635043)

Identification and Gene Mapping of sdb1 Mutant with a Semi-dwarfism and Bigger Seed in Rice

Yi-Ran TAO,Yu-Zhen XIONG,Jia XIE,Wei-Jiang TIAN,Xiao-Qiong ZHANG,Xiao-Bo ZHANG,Qian ZHOU,Xian-Chun SANG,Xiao-Wen WANG()   

  1. Basic Experiment Teaching Center of Agronomy / College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China
  • Received:2018-03-09 Accepted:2018-07-20 Published:2018-11-12 Published online:2018-08-07
  • Contact: Xiao-Wen WANG
  • Supported by:
    This study was supported by the Chongqing Research Program of Basic Research and Frontier Technology(cstc2015jcyjA80008);the Project of Chongqing Science & Technology Commission Grants(cstc2016shms- ztzx8007);the Project of Chongqing Science & Technology Commission Grants(cstc2017shms-xdny80057);the College Students’ Innovation and Entrepreneurship Training Scientific Research Project(201710635043)

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摘要：

适度矮化有利于提高水稻的抗倒伏性, 进而影响产量和品质, 是水稻育种中重要的选择性状之一, 因此研究矮秆形成的分子机制具有重要的意义。为鉴定新的矮秆资源, 探讨株高形成的分子调控机制, 我们对籼型恢复系缙恢10号的EMS (甲基磺酸乙酯)诱变体库进行了鉴定, 从中筛选到1个植株半矮化且籽粒变大的突变体sdb1。本文对其进行了形态鉴定、细胞学观察、遗传分析和基因定位等研究。田间种植条件下, 全生育期sdb1的株高都明显矮于野生型, 成熟期仅76.66 cm, 与野生型的117.43 cm相比, 下降了34.72%, 差异达极显著水平, 进一步分析发现sdb1的穗和各节间长均显著变短。在茎秆石蜡切片中发现, 纵向细胞的长度与野生型相比无显著变化, 横向细胞面积极显著变小、数量则极显著增加, 纵向细胞变少是导致sdb1植株半矮化的主要原因。除植株变矮外, sdb1的另一典型特征是籽粒变大, 千粒重由野生型的24.83 g变为突变体的29.00 g, 差异达极显著水平; 颖壳中薄壁细胞数量增加了22.05%, 致使籽粒的长、宽、厚均极显著变大, 从而提高了sdb1的粒重。此外, sdb1叶肉细胞层数增多, 导致其光合色素含量极显著高于野生型, 叶片呈现深绿色。遗传分析发现, sdb1的突变表型受单隐性核基因调控, 利用中花11/sdb1杂交组合的F2隐性植株, 最终将调控基因定位在第4染色体SSR标记RM16632和Indel标记J50-7之间约406 kb的物理范围内。这为SDB1的克隆和功能研究奠定了基础, 也有助于水稻株高发育分子机制的进一步阐释。

关键词: 水稻(Oryza sativa L.), 矮化, 大粒, 基因定位, sdb1

Abstract:

Moderate dwarfing is conducive to improving the lodging resistance of rice and further affecting its yield and quality, which is one of the important alternatives in rice breeding. Therefore, it is of great significance to study the molecular mechanism of shortened stem formation. In order to identify new dwarf resources and to explore the molecular regulation mechanism of plant height formation, a semi-dwarf and bigger seed (sdb1) mutant was identified from the progeny of indica restorer line Jinhui 10 with seed treated by ethyl methane sulfonate (EMS). This paper performed systematic studies in phenotypic identification, cytological observation, genetic analysis and gene mapping. At maturity stage, the plant height of sdb1 was only 76.66 cm, significantly shorter than 117.43 cm of wild type, resulting in a 34.72% decrease. It was found from the paraffin sections of the stem that the mutant had no significant change in stem cell length compared with the wild-type cells, it reduced cell width caused significantly smaller cell size, and the number of lateral cells significantly increased. The decrease of longitudinal cells should be the major cause of sdb1 semi-dwarfism. In addition to reduced plant height of sdb1, another typical mutational trait was the larger grain size, the 1000-grain weight increased from wild-type’s 24.83 g to mutant’s 29.00 g, reaching an extremely significant difference. The number of parenchyma cells increased from 666.30 in WT to 813.21 in sdb1, with an increase of 22.05%. The increase of cell number resulted in a significant increase of grain length, width and thickness. The mutant also had increased number of sdb1 mesophyll cells, resulting in significantly higher photosynthetic pigment content than wild type, and dark green leaves. Genetic analysis showed that a recessive nuclear gene regulated the mutant phenotype. Based on the F2 recessive plants of Zhonghua 11/sdb1, the gene was finally mapped between markers RM16632 and J50-7 on chromosome 4, with a physical distance of 406 kb. This research lays a foundation for gene cloning and function research, and is conducive to further understanding the genetic model of rice plant height, which has the potential value for agricultural production research.

Key words: rice (Oryza sativa L.), dwarfism, bigger seeds, gene mapping, sdb1

表1

基因定位引物序列"

引物名称
Primer name		 上游引物序列
Upstream primer sequence (5'-3')		 下游引物序列
Downstream primer sequence (5'-3')
J50-3 TGCCTCCTTGTGTGTGGAGT TCCAAGCACTGACTTCATAGC
J50-2 CCACCAGTGATCCCTCATACA TCCGAGCGATTCTACTGGTC
J50-7 CCTCGTGTGGAATCTCGTTC GTAACAGTAGGTGCGAAGATGG
J50-53 GCGTTGTGTGCTCACTGG CCACTTGTCAGGTCTCCATC
J50-65 CCGGACGAGCCAATCAG CGGTGGCTTCTCCCAAGG

图1

突变体sdb1和野生型WT的表型鉴定 A: 苗期野生型WT和突变体sdb1, Bar=5 cm; B: 分蘖期野生型WT和突变体sdb1, Bar=10 cm; C: 灌浆期野生型WT和突变体sdb1, Bar=15 cm; D: 灌浆期野生型WT和突变体sdb1的茎秆, Bar=5 cm; E: 灌浆期野生型WT和突变体sdb1的穗, Bar=5 cm; F: 野生型WT和突变体sdb1穗长及各节间长的统计分析。**在0.01水平上差异显著。"

图2

野生型与突变体sdb1茎石蜡切片A, B: 野生型WT和突变体sdb1茎秆纵切, Bar=100 μm; C: 图A红色小框的放大图, Bar=25 μm; D: 图B红色小框的放大图, Bar=25 μm; E:野生型WT和突变体sdb1茎秆纵切同一面积的细胞数目; F: 野生型WT和突变体sdb1茎秆纵切的细胞大小比较; G, H: 野生型WT和突变体sdb1茎秆纵切的细胞长度和宽度统计; I, J: 野生型WT和突变体sdb1茎秆横切, Bar=100 μm; K: 图I中红色小框放大图, Bar=25 μm; L: 图J的红色小框放大图, Bar=25 μm; M: 图I的黑色小框放大图, Bar=50 μm; N: 图J的黑色小框放大图, Bar=50 μm; O: 野生型WT和突变体sdb1茎秆横切同一面积的细胞数目统计; P: 野生型WT和突变体sdb1茎秆横切的细胞大小比较。**在0.01水平上差异显著。"

图3

野生型WT与突变体sdb1籽粒的比较 A: 野生型WT与突变体sdb1的籽粒, Bar=0.5 cm; B, C: 野生型WT和突变体sdb1横切籽粒的石蜡切片, Bar=500 μm; D: 图B红色小框的放大, Bar=50 μm; E: 图C红色小框的放大, Bar=50 μm; F: 野生型WT和突变体sdb1千粒重统计; G, H: 野生型WT和突变体sdb1细胞长度 (G)、宽度(H)、厚度(H)数据统计; I: 野生型WT(B)和突变体sdb1(C)颖壳薄壁细胞数目统计。**在0.01水平上差异显著。"

图4

冷冻切片和开花期光合色素含量分析 A: 成熟期野生型和突变体sdb1倒一叶中部, Bar=2 cm; B, C: 野生型和突变体sdb1叶片中部冷冻切片横切, Bar=50 μm ; D: 野生型与突变体sdb1倒一叶光合色素含量; E: 野生型与突变体sdb1倒二叶光合色素含量; F: 野生型与突变体sdb1倒三叶光合色素含量。Chl a: 叶绿素a; Chl b: 叶绿素b; Car: 类胡萝卜素。**在0.01水平上差异显著; *在0.05水平上差异显著。"

图5

SDB1在水稻第4 染色体上的分子定位"

表2

定位区间 RGAP 注释的表达蛋白和功能基因"

基因名称
Locus name		 基因注释
Gene annotation
LOC_Os04g23170 Expressed protein
LOC_Os04g23180 Cation efflux family protein, putative, expressed
LOC_Os04g23200 Expressed protein
LOC_Os04g23210 Expressed protein
LOC_Os04g23220 Expressed protein
LOC_Os04g23230 Expressed protein
LOC_Os04g23319 Expressed protein
LOC_Os04g23330 Expressed protein
LOC_Os04g23420 Expressed protein
LOC_Os04g23440 Helix-loop-helix DNA-binding domain containing protein, expressed
LOC_Os04g23460 Expressed protein
LOC_Os04g23550 Basic helix-loop-helix family protein
LOC_Os04g23580 Xylosyltransferase, putative, expressed
LOC_Os04g23600 D-mannose binding lectin family protein, expressed
LOC_Os04g23610 Expressed protein
LOC_Os04g23620 D-mannose binding lectin family protein, expressed
LOC_Os04g23630 Expressed protein
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