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作物学报 ›› 2021, Vol. 47 ›› Issue (2): 285-293.doi: 10.3724/SP.J.1006.2021.03015

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

玉米籽粒突变体smk7的表型分析和基因定位

蒋成功1,2, 石慧敏2, 王红武2, 李坤2, 黄长玲2, 刘志芳2, 吴宇锦2, 李树强2, 胡小娇2,*, 马庆1,*   

  1. 1作物抗逆育种与减灾国家地方联合工程实验室, 安徽合肥 230036
    2中国农业科学院作物科学研究所 / 作物分子育种国家工程实验室, 北京 100081
  • 收稿日期:2020-03-11 接受日期:2020-08-19 出版日期:2021-02-12 网络出版日期:2020-09-10
  • 通讯作者: 胡小娇,马庆
  • 基金资助:
    国家自然科学基金项目(31500984);中国农业科学院科技创新工程和中国农业科学院重大科研联合攻关任务“玉米藏粮于技”项目(CAAS-ZDRW202004)资助

Phenotype analysis and gene mapping of small kernel 7 (smk7) mutant in maize

JIANG Cheng-Gong1,2, SHI Hui-Min2, WANG Hong-Wu2, LI Kun2, HUANG Chang-Ling2, LIU Zhi-Fang2, WU Yu-Jin2, LI Shu-Qiang2, HU Xiao-Jiao2,*, MA Qing1,*   

  1. 1National Engineering Laboratory of Crop Stress Resistance Breeding, School of Life Sciences, Anhui Agricultural University, Hefei 230036, Anhui, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, National Engineer Laboratory of Crop Molecular Breeding, Beijing 100081, China
  • Received:2020-03-11 Accepted:2020-08-19 Published:2021-02-12 Published online:2020-09-10
  • Contact: HU Xiao-Jiao,MA Qing
  • Supported by:
    National Natural Science Foundation of China(31500984);Agricultural Science and Technology Innovation Program of CAAS, and the Storing Grain in Technology for Maize (CAAS-ZDRW202004).

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摘要：

利用甲基磺酸乙酯(EMS)对玉米自交系B73进行诱变, 获得一个可以稳定遗传的小籽粒突变体smk7 (small kernel 7)。smk7成熟籽粒表现为体积变小, 胚和胚乳发育缺陷, 百粒重显著降低。突变籽粒发芽率仅为10%, 且幼苗黄化不能生长成正常植株。成熟smk7胚乳中淀粉、蛋白、油分含量与野生型籽粒相比无显著差异, 但突变体胚乳淀粉粒体积明显变小且形状不规则。smk7突变籽粒在授粉后12 d即可观察到明显的小籽粒和空瘪表型, 石蜡切片显微观察显示突变籽粒的胚和胚乳发育迟缓, 胚乳基部转移层细胞(BETL)相对于野生型细胞壁向内生长减少, 发育受阻。用杂合植株(+/smk7)与多个自交系分别杂交, 构建不同背景的F2分离群体, 遗传分析结果表明该性状受单隐性核基因控制。利用靶向测序基因型分型(genotyping by target sequencing, GBTS)技术将基因初定位于2号染色体短臂, 进一步精细定位发现该基因位于RM1433917和RM1535316两个标记之间约120 kb的物理范围内, 共有8个蛋白编码基因。本研究为进一步克隆和解析SMK7基因调控玉米籽粒发育的分子机制奠定了基础。

关键词: 玉米, 小粒突变体, 遗传分析, 基因定位

Abstract:

In this study, a stable small kernel mutant, named small kernel 7 (smk7), was isolated from ethylmethane sulfonate (EMS) mutagenesis of maize inbred line B73. Compared with wild type, the smk7 mutants showed smaller kernel size, defective embryo and endosperm development and a significant decrease in 100-kernel weight. The smk7 kernels showed a low level of germination rate at 10% and cannot grow into normal plants. No significant changes were detected in protein, starch and oil content between mature wild type and smk7 kernels, but the starch grains became significantly smaller and irregular in smk7 kernels compared with wild type. The smk7 kernels could be clearly distinguished from the wild type as early as 12 days after pollination (DAP), on the basis of their smaller and emptier phenotype. Microscopic inspection of the paraffin sections revealed that the development of embryo and endosperm were delayed, and the cell wall in growth in basal endosperm transfer layers (BETL) were arrested in smk7 compared with wild type. The F2 populations with multiple backgrounds were constructed by crossing heterozygous plants (+/smk7) with several other inbred lines. Genetic analysis showed that the mutant phenotype was controlled by a single recessive gene. Based on genotyping by target sequencing (GBTS) strategy, the SMK7 was initially mapped on the short arm of chromosome 2. The fine mapping results suggested that SMK7 was located between markers RM1433917 and RM1535316, with a physical distance of 120 kb. There were eight protein-coding genes in this region. This study laid a foundation for further genes cloning and research of the SMK7 function in regulating maize kernel development.

Key words: maize, small kernel mutant, genetic analysis, gene mapping

图1

smk7突变体表型 A: M4代果穗; B: smk7与野生型籽粒表型比较; C: smk7和野生型幼苗表型比较。"

图2

突变体籽粒和野生型籽粒的百粒重 **表示野生型与突变体在0.01水平差异显著。"

图3

smk7及野生型成熟籽粒扫描电镜照片 A: 野生型胚乳; B: smk7胚乳; SG为淀粉粒; PB为蛋白体; 标尺为20 μm。"

表1

基因定位引物序列"

引物
Primer		 正向序列
Forward sequence (5°-3′)		 反向序列
Reverse sequence (5′-3′)
In4.3 AACGCATCATCCTATGTCCAAC GGGTGAAGCCAGCCATTATTT
In1.9 ATGGTACGATCAACATAAAGGGAA GGCGTCACCGAAGAAATACAC
In0.83 GCAAGAAGCACCAGCCCT CGAGCGAAAGAAAGGAATGT
In1.16 ACATGACCCACGATCCAGACA TGCAGCCACTCTCCTTATGGT
SNP1 TTAGGGTGGAGTTGCTTCGC TTATGAATGAAGCACGGAAATGA
SNP2 TTAGGGTGGAGTTGCTTCGC TTATGAATGAAGCACGGAAATGA
SNP3 AAGGAATGGAGGCTTGGGTT ACAGCCGCCTTCGGATTC
SNP4 AGCGGTCCTTGACTTTATTTGA CATTACACGACCAATACAGCCA
SNP5 TGGCTTTTCATACCCTCCTCC CCTTCGCTGTGACTTGGATGT

图4

野生型籽粒和smk7籽粒的成分测定 **表示野生型与突变体在0.01水平差异显著。"

图5

授粉后不同时期胚和胚乳观察分析 A: 野生型籽粒在12、15、18、21、25和30 DAP胚和胚乳及完整种子的观察分析; 标尺为3 mm。B: 突变型籽粒(smk7)在12、15、18、21、25和30 DAP胚和胚乳及完整种子的观察分析; DAP: 授粉后天数; 标尺为3 mm。"

图6

授粉后不同天数野生型和突变体籽粒石蜡切片观察 A: 野生型授粉后12 d籽粒; 标尺为 1000 μm。B: smk7授粉后12 d籽粒; 标尺为1000 μm。C: WT授粉后12 d籽粒胚乳基部转移; 标尺为150 μm; D: smk7授粉后12 d籽粒胚乳基部转移层; 标尺为150 μm; BETL: 胚乳基部转移层细胞。"

表2

不同群体的遗传分离比检验"

世代
Populations		 总粒数
Total kernels		 正常籽粒数
Normal kernels		 突变籽粒数
Mutant kernels		 实际比例
Actual ratio		 理论比例
Theoretical ratio		 卡方值
χ2
M2 6372 4742 1630 2.91:1 3:1 1.1151
M3 9821 7412 2409 3.08:1 3:1 1.1367
F2 (Mo17×smk7) 5123 3870 1253 3.09:1 3:1 0.7730
F2 (Z58×smk7) 2016 1512 504 3:1 3:1 0.1120

图7

多态性SNP标记的数目"

图8

SNP index全基因组频率分布图"

图9

SMK7基因的精细定位 群体大小; Recombinants: 重组单株数。"

表3

候选区间内基因信息"

基因名称
Locus name		 基因注释
Gene annotation
ZM00001D001818 Probable polyol transporter 4
ZM00001D001819 N-acetylglucosaminyl-phosphatidylinositol de-N-acetylase family protein
ZM00001D001820 Protochlorophyllide reductase1
ZM00001D001821 Unknown
ZM00001D001822 Unknown
ZM00001D001823 Anthranilate synthase beta subunit 1 chloroplastic
ZM00001D001824 Dof zinc finger protein DOF1.6
ZM00001D001825 Transducin/WD40 repeat-like superfamily protein
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