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作物学报 ›› 2024, Vol. 50 ›› Issue (12): 3046-3054.doi: 10.3724/SP.J.1006.2024.41014

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大麦稃壳白化突变性状的遗传解析

孙曼1,2(), 安朝丹2, 高广奇2, 郭杰1, 杨平2, 蒋枞璁2,*()   

  1. 1山西农业大学农学院, 山西晋中 030801
    2农业农村部粮食作物基因资源评价利用重点实验室 / 中国农业科学院作物科学研究所, 北京 100081
  • 收稿日期:2024-03-01 接受日期:2024-06-20 出版日期:2024-12-12 网络出版日期:2024-07-15
  • 通讯作者: *蒋枞璁, E-mail: jiangcongcong@caas.cn
  • 作者简介:E-mail: sunm1223@163.com
  • 基金资助:
    中国农业科学院科技创新工程项目(农科英才计划-杨平);Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences (Young Talent Project granted to Ping Yang)

Genetic dissection of the albino hull mutations in barley (Hordeum vulgare L.)

SUN Man1,2(), AN Chao-Dan2, GAO Guang-Qi2, GUO Jie1, YANG Ping2, JIANG Cong-Cong2,*()   

  1. 1College of Agriculture, Shanxi Agricultural University, Jinzhong 030801, Shanxi, China
    2Key Laboratory of Grain Crop Genetic Resources Evaluation and Utilization, Ministry of Agriculture and Rural Affairs / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2024-03-01 Accepted:2024-06-20 Published:2024-12-12 Published online:2024-07-15
  • Contact: *E-mail: jiangcongcong@caas.cn

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摘要：

大麦(Hordeum vulgare L.)稃壳是在小穗上包裹种子的花器官, 包括内稃和外稃。大麦稃壳具有光合作用能力, 为籽粒发育提供部分营养物质。通过EMS诱变获得的大麦稃壳白化突变体, 其稃壳明显呈白色, 叶枕、茎节和茎基部均呈白化变异, 但叶片和芒均为绿色。本研究对稃壳白化突变性状进行鉴定和遗传分析, 发现该突变性状由单个隐性基因控制; 通过候选基因测序及竞争性等位基因特异性PCR (KASP)共分离检测, 证实该白化突变是由基因HvGLK2的功能缺失所致; 并在HvGLK2基因上鉴定到3个独立突变事件, 均与已发表的大麦alm1和ebu-a等白化变异不同。HvGLK2基因编码一个Golden 2-like (GLK)转录因子, 为MYB基因家族GARP亚家族成员, 在多数的单、双子叶植物中均存在另一个同源基因GLK1。通过公共数据库检索HvGLK2基因的表达谱数据, 发现HvGLK2在大麦衰老叶片、外稃及穗轴中高丰度表达。本研究明确了HvGLK2基因对大麦稃壳和茎节等组织叶绿素合成的关键作用, 相关的稃壳白化突变体为稃壳光合作用对产量形成的机制研究提供了理想材料。

关键词: 大麦, 稃壳, 白化突变体, HvGLK2, 功能缺失

Abstract:

Lemma and palea are the outermost organs on each floret of barley (Hordeum vulgare L.), where the majority of spike photosynthesis occurs, supplying carbohydrates to the developing grains. The barley albino hull (alh) mutants, obtained from EMS mutagenesis, showed albinistic lemma and palea, as well as albinistic pulvini, stem nodes, and stem bases, while the leaves and awns remain green. In this study, multiple allelic alh mutants were identified, and segregating populations were generated accordingly. Genetic analysis indicated that a single recessive gene is responsible for the alh phenotype. Through re-sequencing of the candidate gene in multiple alh mutants, and co-segregation tests using competitive allele-specific PCR (KASP) markers, loss-of-function mutations in the gene HvGLK2 were shown to account for the alh phenotype. Each of the three independent mutations identified in this study is distinct from previously reported albino lemma variants such as alm1 or ebu-a. HvGLK2 encodes a Golden 2-like (GLK2) transcription factor, belonging to the GARP subfamily of MYB transcription factors, and has a paralog designated GLK1 in most monocot and dicot species. The temporal and spatial expression patterns showed that HvGLK2 is abundantly transcribed in senescent leaves, lemmas, and rachises. This study highlights the importance of HvGLK2 in chlorophyll synthesis in various organs, including lemma, palea, and stem nodes of barley plants. Moreover, it provides valuable materials for further studies aimed at evaluating the contribution of spike photosynthesis to the eventual grain yield.

Key words: barley, hull, albino mutant, HvGLK2, loss-of-function

图1

野生型HTX与稃壳白化突变体alh.a的形态对比 A: 野生型HTX (左)和T-M3-4073家系中稃壳白化纯合突变体alh.a (右)的整穗; B: 野生型HTX (左)和alh.a突变体(右)的穗子局部; C: 野生型HTX (左)和alh.a突变体(右)的茎基部; D: 野生型HTX (左)和alh.a突变体(右)的茎节; E: 野生型HTX (左)和alh.a突变体(右)的叶枕; F: 灌浆期野生型HTX (左)和alh.a突变体(右)中央小穗的结构解剖; G: 完全成熟期野生型HTX (左)和alh.a突变体(右)的种子(下排去除稃壳)。图中标尺均为1 cm。"

图2

野生型HTX与稃壳白化突变体alh.a的千粒重对比 **, 显著性水平P < 0.01。"

表1

10个T-M4-4073家系表型分离情况统计"

M4家系
M4 family		 野生型单株数目
Number of wild-type plants		 稃壳白化单株数目
Number of alh plants		 M3单株基因型
Deduced M3 genotype		 卡方测验
χ2-test (χ20.05=3.841)
T-M4-4073-1 0 12 M Na
T-M4-4073-2 12 0 WT Na
T-M4-4073-3 0 12 M Na
T-M4-4073-4 51 17 Het χ2(1:3)=0
T-M4-4073-5 52 19 Het χ2(1:3)=0.082
T-M4-4073-6 12 0 WT Na
T-M4-4073-7 7 5 Het Na
T-M4-4073-8 12 0 WT Na
T-M4-4073-9 12 0 WT Na
T-M4-4073-10 6 6 Het Na

图3

HvGLK2基因结构示意图(A), 及其在野生型HTX与多个稃壳白化突变体中的编码氨基酸序列比对(B)"

表2

稃壳白化突变基因HvGLK2的片段扩增引物"

引物名称
Primer name		 引物序列
Primer sequence (5'-3')		 退火温度
Melting temperature
(℃)		 产物大小
Product size (bp)		 用途
Purpose
GLK2_5UTR_F1 TGCCATCGTCAAGTCAGCTG 60.12 746 覆盖exon1的片段扩增
Amplification of fragments covering exon1
GLK2_IN1_R1 GTGAGAGAATGCAGGGCAGTAC 58.95
GLK2_IN1_F1 CAGTGAGGCGCTCGATTTAG 58.62 521 覆盖exon2的片段扩增
Amplification of fragments covering exon2
GLK2_IN2_R1 GCGTACGTCCGTGTGAATTT 58.45
GLK2_IN2_F1 TATATGAGATTGCATTTGTGGTCG 59.82 1456 覆盖exon3-6的片段扩增
Amplification of fragments covering exon3-6
GLK2_3UTR_R1 TGCACGTAGGTACAGACGGAC 58.43
3H135348068-F CTCCATCTACCCATCGATCAAGA 60.75 178 检测基因型与表型关联性的KASP标记
KASP markers were used to detect genotype-phenotype associations
3H135348068-RA GAAGGTCGGAGTCAACGGATTGGCCGGACTAGGGCTTTA 61.11
3H135348068-RG GAAGGTGACCAAGTTCATGCTGGCCGGACTAGGGCTTTG 60.33

图4

alh.a突变体中功能缺失型变异位点发展的KASP标记与稃壳白化表型共分离检测"

图5

HvGLK2在大麦全生育期的时空表达模式 FPKM: 每千个碱基的转录每百万映射读取的片段。ROO1: 10 cm高幼苗的根; LEA: 10 cm高幼苗的芽; INF: 发育中的5 mm幼穗; INF2: 发育中的1.0~1.5 cm穗; NOD: 发育中主分蘖的第3节间; CAR5: 授粉5 d发育中的籽粒; CAR15: 授粉15 d发育中的籽粒; ETI: 播种后黑暗条件下10 d的白化幼苗; LEM: 播种后42 d的外稃; LOD: 播种后42 d的浆片; EPI: 播种后28 d的叶脉; RAC: 播种后35 d的花序轴; ROO2: 播种后28 d的根; SEN: 播种后56 d的衰老叶。"

图6

不同物种的GLK基因系统进化树 对大麦(Hv)、水稻(Os)、玉米(Zm)、六倍体小麦(Ta)、高粱(Sb)、谷子(Si)、拟南芥(At)、棉花(Gr)、大豆(Gm)和番茄(Sl)的GLK2和GLK1基因氨基酸序列进行多重比对, 并构建系统进化树。"

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