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作物学报 ›› 2020, Vol. 46 ›› Issue (11): 1667-1677.doi: 10.3724/SP.J.1006.2020.04043

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆分枝数相关分子标记开发及qBN-18位点精细定位

吴海涛1,3(), 张勇2, 苏伯鸿1,3, Lamlom F Sobhi3,4, 邱丽娟3,*()   

  1. 1 东北农业大学农学院, 黑龙江哈尔滨 150030
    2 黑龙江省农业科学院克山分院, 黑龙江齐齐哈尔 161606
    3 农作物基因资源与遗传改良国家重大科学工程 / 农业部种质资源利用重点实验室 / 中国农业科学院作物科学研究所, 北京 100081
    4 埃及亚历山大大学农业学院Saba Basha植物生产部, 埃及亚历山大市
  • 收稿日期:2020-02-25 接受日期:2020-06-02 出版日期:2020-11-12 网络出版日期:2020-06-22
  • 通讯作者: 邱丽娟
  • 作者简介:吴海涛, E-mail:1960478192@qq.com
  • 基金资助:
    本研究由“十三五”国家重点研发计划项目(2016YFD0100201);中国农业科学院科技创新工程和大豆种质资源保护与利用项目(2019NWB036-05)

Development of molecular markers and fine mapping of qBN-18 locus related to branch number in soybean (Glycine max L.)

WU Hai-Tao1,3(), ZHANG Yong2, SU Bo-Hong1,3, Lamlom F Sobhi3,4, QIU Li-Juan3,*()   

  1. 1 College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang, China
    2 Keshan Branch of Heilongjiang Academy of Agricultural Sciences, Qiqihar 161606, Heilongjiang, China
    3 National Key Facility for Gene Resources and Genetic Improvement / Key Laboratory of Crop Germplasm Utilization, Ministry of Agriculture / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    4 Plant Production Department, Faculty of Agriculture Saba Basha, Alexandria University, Alexandria, Egypt
  • Received:2020-02-25 Accepted:2020-06-02 Published:2020-11-12 Published online:2020-06-22
  • Contact: Li-Juan QIU
  • Supported by:
    The study was supported by the National Key Research and Development Program of China(2016YFD0100201);the Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences, and the Protection and Utilization of Soybean Germplasm Resources(2019NWB036-05)

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摘要：

分枝数是影响大豆产量的重要因素之一, 与植株结荚率直接相关; 同时也是决定大豆株型的重要组成因子, 并通过调节群体结构、种植密度等进一步影响产量。目前关于大豆分枝数QTL (quantitative trait loci)精细定位与图位克隆的报道极少。因此, 发掘参与调控大豆分枝的基因/QTL对于株型建成的基础研究和高产品种培育的应用研究都具有重要意义。本研究在少分枝品种垦丰19 (KF19)与多分枝品种垦农24 (KN24)组合F2的基础上, 培育出由606个株系组成的F7:8重组自交系(recombinant inbred lines, RIL)群体, 以及由1486个单株KF19-BC3F2和1150个单株KN24-BC2F2组成的2个回交群体。在18号染色体分枝数QTL新位点(qBN-18)的定位区间内筛选出多态性SSR标记11个, 利用RIL群体将qBN-18的定位区间由1.6 Mb缩小到113 kb。在定位区间内开发了2个InDel标记BR69与BR77。进一步利用回交群体筛选交换单株, 将qBN-18定位区间缩小到63.7 kb, 包括9个基因。本研究结果为大豆分枝数的基因图位克隆及分子标记辅助育种创造了条件。

关键词: 大豆, 分枝数, QTL定位, 候选基因

Abstract:

The branch number is one of the important factors influencing soybean yield, which is directly related to pod setting rate. At the same time, it is also an important component of soybean plant type, and further affects the yield by adjusting the population structure and planting density. At present, there is few report related to map-based cloning of genes related to branch number. Therefore, the discovery of genes/QTL involved in the regulation of soybean branching is of great significance for the basic research on the establishment of plant type and the applied research on the development of high-yielding varieties. In this study, based on the F2 of crossing low-branched variety Kenfeng 19 (KF19) and high-branched variety Kennong 24 (KN24), we developed the F7:8 recombinant inbred line (RIL) population, consisting of 606 lines, and two backcrossing populations consisting of 1486 individuals for KF19-BC3F2 and 1150 individuals for KN24-BC2F2. Within the localization interval of the new QTL of the branch number of chromosome 18 (qBN-18), 11 polymorphism SSR markers were screened out to identify the RIL population, and region of qBN-18 was reduced from 1.6 Mb to 113 kb. After developing two InDel markers BR69 and BR77 in the mapping region, the backcross population was used to screen the exchange individuals, the interval of qBN-18 was further reduced to 63.7 kb, including 9 genes. Those results provide the information for gene map-based cloning and molecular marker assisted breeding of branch number in soybean.

Key words: soybean, branch number, QTL mapping, candidate genes

表1

精细定位所用分子标记"

引物名称
Primer name		 物理位置
Physical position (bp)		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')
BARCSOYSSR_18_1777 54,744,147-54,744,204 CGTTCTTGCATTAAAGGTGGA AACTTCATTGAATTGACGGTGA
BARCSOYSSR_18_1791 54,975,880-54,975,949 TGACCAGTCAATTGTTCATTCTTT TTTACTCAACCATCTCCGCA
BARCSOYSSR_18_1797 55,076,726-55,076,764 AAGCAAAGAGAACCAAAGCG AAAACACGAAAAGGAAGGCA
BARCSOYSSR_18_1803 55,244,106-55,244,173 TTTCGCACTCAATGTCCGTA GTTTCCAAACCACATGGACC
BARCSOYSSR_18_1825 55,701,309-55,701,352 GAATCCACCATCACCAAACC CAATGGCAACCCAGTAAGGT
BARCSOYSSR_18_1827 55,714,562-55,714,637 GCCCCACTCGATGAAATAAA GCTTTGGCAGAAATTCAAGG
BARCSOYSSR_18_1831 55,763,781-55,763,814 TGTTTTTGTTAAATCTTTTGTTTGG TGTGTATGTTTGTGTGTGCACTT
BARCSOYSSR_18_1832 55,775,899-55,775,922 AAAAGCTGAGAGCACAAGGC CGTGCTTTTTCAGTCCCATT
BARCSOYSSR_18_1834 55,877,756-55,877,781 TTGAAGAGGAGAGAAGAATGGTG GGATGTGATTGTTAGAAAAGAAGAA
BARCSOYSSR_18_1841 55,990,705-55,990,750 TGCACGAGGCCATTACATAG TGAAGCGCATATGACTCAACTT
BARCSOYSSR_18_1847 56,075,087-56,075,116 TGGTAGGAGTACTCTGAAGTCATTTTT AGCGCTCAAATGAGATTCCT
BARCSOYSSR_18_1852 56,172,124-56,172,157 TGTGTGCGTAAGGGAGATCA CTACCAACCTCCGCATGTCT
BARCSOYSSR_18_1856 56,223,172-56,223,217 TGGCCATATGCCTAGCTGAT ATGGTGAGCAAACGTCATTG
BARCSOYSSR_18_1875 56,797,313-56,797,366 TGAAAAAGAACGTGTTCAAAATG CGAGTTTCATTCTCGGAAGC

表2

两个InDel标记的位置、引物序列及预期扩增片段长度"

引物名称
Primer name		 物理位置
Physical
position (bp)		 正向引物
Forward primer (5'-3')		 反向引物
Reverse primer (5'-3')		 片段长度Product size (bp)
垦农24
Kennong 24		 垦丰19
Kenfeng 19
BR69 55,882,229-55,882,378 AGTTGACAGGAACTAAAGTC GATAATTCAAGTAAATAGCGA 156 15
BR77 55,946,063-55,946,342 TCTCTTTGTGTATGTCTTCTCC TTGTTGCATCCAAATGAGAG 268 280

图1

垦丰19、垦农24及群体分枝数鉴定 A: 亲本植株; B~C: 分别为2018年与2019年KN24与KF19分枝数表型; D~E: 分别为F7与F7:8分枝数频率分布直方图; F: 2018年回交群体F2分枝数频率分布直方图。垂直虚线表示两亲本表型的平均值和标准差。曲线代表密度图。***: P<0.001。"

表3

2018年、2019年KF19和KN24及其F7、F7:8群体的分枝数(BN)统计分析"

年份
Year		 分枝数的平均值±标准差
Mean±SD of BN		 群体的分枝数参数
Parameters of BN in populations
KN24 KF19 平均值±标准差
Mean±SD		 范围
Range		 偏度
Skewness		 峰度
Kurtosis
2018 5.5±1.0 0.5±0.5 4.5±1.9 0-10 0.2740 -0.3887
2019 6.8±1.2 0.7±0.6 3.4±1.5 0-8 0.3390 -0.5720

图2

用垦丰19和垦农24构成的RIL群体在大豆18号染色体定位分枝数QTL qBN-18"

表4

KF19×KN24的F7:8群体中鉴定到的分枝数QTL"

群体
Population		 染色体
Chr.		 标记区间
Marker interval		 区间物理位置
Physical position of
interval (bp, Wm82.a2.v1)		 LOD值
LOD value		 表型变异率
PVE (%)		 加性效应
Additive effect
F7:8 18 BARCSOYSSR_18_1834-BARCSOYSSR_18_1841 55,877,756-55,990,750 19.33 14.24 0.75

图3

RIL群体中标记 BR69与BR77的基因型与表型相关性分析 A: BR69的基因型与表型相关性分析; B: BR77的基因型与表型相关性分析。图中横坐标为基因型, 多分枝记为a, 少分枝记为b, 纵坐标为分枝数。***P < 0.001。"

图4

qBN-18位点的验证 A: 利用KF19-BC3F2的1486个单株和KN24-BC2F2的1150个单株鉴定了标记BARCSOYSSR_18_1777和BARCSOYSSR_18_1875之间的qBN-18位点; B: 利用BARCSOYSSR_18_1791、BARCSOYSSR_18_1803、BARCSOYSSR_18_1827与BARCSOYSSR_18_1856 4个标记鉴定BARCSOYSSR_18_1777和BARCSOYSSR_18_1875之间的568个重组单株; C: qBN-18位于标记BR69与标记BR77之间。H: 杂合基因型。"

表5

定位区间内基因的同源基因及基因注释"

基因
Gene		 同源基因
Homologous gene		 基因注释
Gene annotation
Glyma.18g276900 AT5G11390.1 WPP domain-interacting protein 1
Glyma.18g277000 AT3G02030.1 Pentatricopeptide repeat (PPR) superfamily protein
Glyma.18g277100 AT3G30530.1 Basic leucine-zipper 42
Glyma.18g277300 AT3G02060.1 6-phosphogluconate dehydrogenase family protein
Glyma.18g277600 AT4G25760.1 Glutamine dumper 2
Glyma.18g277700 AT4G15733.1 SCR-like 11

图5

用Phytozome v12.1获得9个基因的表达谱"

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