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作物学报 ›› 2019, Vol. 45 ›› Issue (6): 807-817.doi: 10.3724/SP.J.1006.2019.81090

• 作物遗传育种·种质资源·分子遗传学 •    下一篇

小麦芒长抑制基因B2的精细定位与候选基因分析

金迪1,*,王冬至2,*,王焕雪3,李润枝3,陈树林1,阳文龙2,张爱民2,刘冬成2,4,*(),詹克慧1,*()   

  1. 1 河南农业大学农学院, 河南郑州 450002
    2 中国科学院遗传与发育生物学研究所 / 植物细胞与染色体工程国家重点实验室, 北京 100101
    3 北京农学院植物科学技术学院, 北京 102206
    4 北京科技大学生物与农业研究中心, 北京 100024
  • 收稿日期:2018-12-19 接受日期:2019-01-19 出版日期:2019-06-12 网络出版日期:2019-06-12
  • 通讯作者: 金迪,王冬至,刘冬成,詹克慧
  • 作者简介:金迪, E-mail: jindizheng@qq.com|王冬至, E-mail: wangdongzhi1990@163.com
  • 基金资助:
    本研究由国家重点研发计划项目(2016YFD0101800);国家自然科学基金项目(31571643)

Fine mapping and candidate gene analysis of awn inhibiting gene B2 in common wheat

Di JIN1,*,Dong-Zhi WANG2,*,Huan-Xue WANG3,Run-Zhi LI3,Shu-Lin CHEN1,Wen-Long YANG2,Ai-Min ZHANG2,Dong-Cheng LIU2,4,*(),Ke-Hui ZHAN1,*()   

  1. 1 Agronomy College of Henan Agricultural University, Zhengzhou 450002, Henan, China
    2 State Key Laboratory of Plant Cell and Chromosome Engineering / Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
    3 College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China
    4 Biology and Agriculture Research Center, University of Science and Technology Beijing, Beijing 100024, China
  • Received:2018-12-19 Accepted:2019-01-19 Published:2019-06-12 Published online:2019-06-12
  • Contact: Di JIN,Dong-Zhi WANG,Dong-Cheng LIU,Ke-Hui ZHAN
  • Supported by:
    This study was supported by the National Key Research and Development Program of China(2016YFD0101800);the National Natural Science Foundation of China(31571643)

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摘要：

芒是小麦重要的穗部器官和形态特征, 是小麦长期进化和适应环境的结果, 对产量和抗旱性等具有重要影响。目前, 对麦芒的遗传与发育还缺乏系统的研究, 相关基因克隆或精细定位的研究尚未见报道。本试验利用短芒材料‘六柱头’与长芒材料‘石矮1号’构建的F2群体(SL-F2)对芒的遗传与发育进行了研究。细胞学观察表明, 短芒主要是由细胞长度变短引起; 遗传分析表明, ‘六柱头’的短芒由显性单基因控制; 借助Wheat660K SNP芯片的BSA分析和SL-F2群体的精细定位, 确定‘六柱头’的芒长抑制基因是前人报道的B2位点, 并将其定位到6B染色体4.84 Mb的物理区间(471.28~476.12 Mb)内, 该区段在中国春与矮抗58间具有良好的共线性。在B2定位区间共有61个基因, 其中5个在中国春穗部特异表达, TraesCS6B02G264400在中国春和Azhurnaya幼穗表达差异显著。这些研究结果为B2基因的克隆、小麦芒形成机理的解析及育种中的应用奠定了基础。

关键词: 小麦, 芒, B2, 精细定位, BSA, Wheat660K

Abstract:

Awn is one of the important photosynthetic organs in common wheat and plays a vital role in yield potential and environmental adaptation. At present, the inheritance and development of wheat awn have been not systematically studied, and cloning or fine mapping of related genes are seldom reported. In this study, the genetics and candidate genes conferring the short awn of a Chinese wheat landrace ‘Liuzhutou’ were investigated. Longitudinal section of awn showed cell size of short awn was much shorter than that of long awn. Using Wheat660K SNP chip based bulked segregant analysis (BSA) and SL-F2 population derived from ‘Liuzhutou’ (short awn) and modern cultivar ‘Shiai 1’ (long awn), the awn inhibiting gene in ‘Liuzhutou’ was mapped in a 4.84 Mb interval on chromosome 6BL and predicted as previously characterized B2. A good micro-collinearity of B2 region was observed among chromosomes 6B, 6A, 6D of Chinese Spring and chromosome 6B of AK58, there were 61 genes annotated in the 4.84 Mb B2 region, five of which were specifically expressed in the developing spike of Chinese Spring, and TraesCS6B02G264400 was differentially expressed between Chinese Spring and Azhurnaya. These data provide important clues for cloning the B2 gene, dissecting the developing mechanism of wheat awn and its application in molecular breeding.

Key words: common wheat, awn, B2, fine mapping, BSA, Wheat660K

表1

本研究所用到的引物"

引物名称
Primer ID		 正向引物序列
Forward sequence (5'-3')		 反向引物序列
Reverse sequence (5'-3')		 内切酶
Restriction enzyme		 片段大小
Size (bp)
B2精细定位 Markers for mapping B2
Xgwm88 * CACTACAACTATGCGCTCGC TCCATTGGCTTCTCTCTCAA — 120
SSR43 ACTATCACAAGCCGGAGTAA AGTGGTTAAATCGCCACTAA — 172
SSR122 CATGTTACCACCAAAGGATT CTATGTGGCTGACCGTTACT — 205
EST-499300LC TCCAAAGAATCCTGGAGGTG TGTTCTGACCATCCCACGTA Pvu II 393/270
dCAPS-18 GACCACCCGACACCGCCGCTGCA GCTCATCTCCAATAGCTCGC Pst I 204/184
dCAPS-19 CAAAACTTCTTGGGAGAATATAC CCCTGACATCGTCTCGAACT Mlu I 192/172
dCAPS-4 GTAAGCAGCGAGGTTAGCACTGC CTGGTGCTCATGAGTTGTGG Pst I 296/276
EST-263300 CGTGTGCTTAAATGCTCCATGG GCTGACTTATGCTGCTTTACTTTCTAA Taq I 521/472
CAPS-12 ATTGATGCAAAGGAACCAGG GAGTTCCATGGCCTCATTGT Eco RI 308/236
CAPS-14 GGAAGGTCCACTTTGCATGT CACCACAGGGACGAAATTCT Bam HI 281/265
dCAPS-39 GTTTTCTTTGTAGAAGTCCCATA AAACACCTGAAAATCGTGGC Nde I 185/165
EST-265400 CCATCAGTCCAACCATGACTTGT TAACATAAGCGTCGCTGTGGC Pst I 246/(111+135)
CAPS-19 TGTCCTTGAAGGGGGTAGTG TCCTGCATTATGCCACAAAA Nde I 483/(217+266)
WABM232658 * AAGTTCGCCTCTTCACCAGT TCTGCCCCTACATCTGTTGC — 130
SSR74 CATAATCACAATTCATCGGA TTCATACCTGACCCATCTTC — 214
SSR116 AGAATCAGTTTTCAGCCAGA AACTATCCCGTATACTTGCG — 168
WABM214868 * GGGTGCCTGAACATTGATGC CCCCAAGTGCTGTCGTGTAT — 126
候选基因克隆 Gene cloning primers
6B01G262600 AGCGAAGCAGTCAGTACTCAGT CTTGCATGGATAATCGAAACATAGGA — 1041
6B01G263300 CGTGTGCTTAAATGCTCCATGG GCTGACTTATGCTGCTTTACTTTCTAA — 1110
6B01G264600 GCCCATTTTTGCAGTCATGACATCA CGGGATGAAAACCTGCTCTTC — 1760
6B01G265400 CCATCAGTCCAACCATGACTTGT TAACATAAGCGTCGCTGTGGC — 1071
6B01G504500LC CACACACACACACACCAAAACTC GGCATACTGCGAACAACCCA — 985
6B01G505500LC AGCAGCTGGGTGAAGTTAACTA TTCCAGCCCCCTTTTACAAGAT — 1437

图1

长芒材料(A)和短芒材料(B)芒的石蜡切片"

图2

SL-F2群体的构建及Wheat660K芯片分型结果分析 (A) 群体构建和芒长表现; (B) SL-F2群体混池间差异SNP标记的染色体分布; (C) SL-F2群体混池6B染色体差异SNP标记的分布。"

表2

SL-F2群体的遗传分析及卡方检验结果"

单株数
No. of F2 plants		 观察值 Observed value 理论值 Expected value 理论比值
Expected ratio		 χ2值
χ2-value		 P值
P-value
长芒
Long awn		 短芒
Short awn		 长芒
Long awn		 短芒
Short awn
1413 350 1063 353 1060 3:1 0.04 0.84

图3

B2基因的精细定位 (A) SL-F2群体B2位点的遗传图谱; (B) SL-F2群体B2位点的物理图谱; (C) SL-F2群体关键重组体的标记基因型及子代验证表型。"

图4

B2区段的基因微共线性分析 自左向右依次为矮抗58的6B染色体及中国春6B、6A和6D染色体; 图中标为红色和青色的基因分别表示在中国春6B和矮抗58的6B染色体中没有同源基因。"

图5

中国春B2区间基因注释与表达谱分析 热图中蓝色越深代表其表达量(TPM)越低, 红色越深则表达量越高, 标红色的基因代表在中国春中Fold-Change (TPMSpike/TPMMean(Leaf, Root, Grain)) ≥ 2, 基因名称后面为其基因注释信息。"

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