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作物学报 ›› 2023, Vol. 49 ›› Issue (2): 310-320.doi: 10.3724/SP.J.1006.2023.24015

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆蛋白含量主效位点qPRO-20-1的精细定位

杨硕1,3(), 武阳春4, 刘鑫磊2, 唐晓飞2, 薛永国2, 曹旦2, 王婉3, 刘亭萱3, 祁航3, 栾晓燕2,*, 邱丽娟1,3,*()   

  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, 黑龙江哈尔滨 150030
    3农作物基因资源与遗传改良国家重大科学工程 / 农业农村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吉林农业大学生命科学学院, 吉林长春 130000
  • 收稿日期:2022-01-10 接受日期:2022-06-07 出版日期:2022-07-08 网络出版日期:2022-07-08
  • 通讯作者: 栾晓燕,邱丽娟
  • 作者简介:杨硕, E-mail: 857813782@qq.com第一联系人：

    **同等贡献

  • 基金资助:
    国家自然科学基金项目(31960408);中央级公益性科研院所基本科研业务费专项(S2022ZD02);中国农业科学院科技创新工程项目资助。

Fine mapping of qPRO-20-1 related to high protein content in soybean

YANG Shuo1,3(), WU Yang-Chun4, LIU Xin-Lei2, TANG Xiao-Fei2, XUE Yong-Guo2, CAO Dan2, WANG Wan3, LIU Ting-Xuan3, QI Hang3, LUAN Xiao-Yan2,*, QIU Li-Juan1,3,*()   

  1. 1College of Agriculture, Northeast Agricultural University, Harbin 150030, Heilongjiang, China
    2Heilongjiang Academy of Agricultural Sciences, Harbin 150030, Heilongjiang, China
    3National Key Facility for Gene Resources and Genetic Improvement / Key Laboratory of Crop Germplasm Utilization, Ministry of Agriculture and Rural Affairs / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    4College of Life Sciences, Jilin Agricultural University, Changchun 130000, Jinlin, China
  • Received:2022-01-10 Accepted:2022-06-07 Published:2022-07-08 Published online:2022-07-08
  • Contact: LUAN Xiao-Yan,QIU Li-Juan
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    National Natural Science Foundation of China(31960408);Central Public-interest Scientific Institution Basal Research Fund(S2022ZD02);Agricultural Science and Technology Innovation Program

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摘要：

蛋白质含量是大豆重要的品质性状, 受多基因控制, 定位大豆蛋白质含量相关位点并挖掘候选基因, 对定向培育高蛋白含量大豆品种具有重要意义。本研究以优良品种黑农88作为母本与高蛋白优异种质P73-6B作为父本杂交, 构建了一个由265个单株组成的F2群体, 利用中豆芯1号对F2群体进行基因型鉴定并构建图谱, 结合蛋白质含量表型数据, 采用IciMapping 4.2软件在20号染色体上定位了一个QTL, 物理距离为2.46 Mb, 在区间附近筛选出11个多态性SSR标记并分析群体, 将定位区间从2.46 Mb缩小至100.8 kb。增加Gm20_28349696、Gm20_30805913、Gm20_31341532和Gm20_31483719共4个SNP位点, 进一步将区间缩小到95.8 kb。对区间内包含的4个基因的9个不同组织在Phytozome v13.1和PPRD RNA-seq 2个数据库中的表达量分析得到了2个候选基因, 分别为Glyma.20g081800和Glyma.20g082000基因, 本试验结果为大豆蛋白质含量基因克隆及蛋白质调控机制研究提供了理论基础, 为大豆高蛋白分子标记育种提供材料和技术支撑。

关键词: 大豆, 蛋白质含量, QTL定位, 候选基因

Abstract:

Protein content is a crucial quality trait of soybean, which is controlled by multiple genes. It is of great significance to locate soybean protein content-related loci and mine candidate genes for directional breeding of soybean varieties with high protein content. In this study, an F2 population consisting of 265 individual plants was constructed by crossing the excellent variety Heinong 88 as the female parent with the high-protein germplasm P73-6B as the male parent. The genotypes of F2 population were identified by using high-density SNP chip of “ZDX1” and the physical map was constructed. Combined with the protein content phenotypic data, the initial mapping interval of a 2.46 Mb QTL was located on chromosome 20 using the IciMapping 4.2 software. Using 11 polymorphic SSR markers screened out, the mapping range was narrowed from 2.46 Mb to 100.8 kb. Adding four SNP markers (Gm20_28349696, Gm20_30805913, Gm20_31341532, and Gm20_31483719), the interval was further reduced to 95.8 kb. The relative expression levels of the four genes contained in the interval in nine different tissues in both databases Phytozome v13.1 and PPRD RNA-seq yielded two candidate genes (Glyma.20g081800 and Glyma.20g082000). These results provide a theoretical basis for soybean protein content gene cloning and protein regulation mechanism research, as well as elite material and molecular marker for breeding high protein soybean.

Key words: soybean, protein content, QTL mapping, candidate genes

表1

精细定位所用SSR标记"

引物名称
Primer name		 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (5°-3°)
BARCSOYSSR_20_0608 GTGTTCCACTCCACGTTTCC CATTTCCCCTTTCACAATCG
BARCSOYSSR_20_0613 AACCGAGTTTGGTTCGATTC TGCTGCTTGATGATGAGGAC
BARCSOYSSR_20_0616 CCATCTTATGGACTTGTTTGGA GCCAAGAATGACCATTATGC
BARCSOYSSR_20_0618 TCACTAATCACAACAACCCAAA CGACCGGTGTGTTTAAGGTC
BARCSOYSSR_20_0619 TCAGTCGCAGATTGATCAGG CCCAATTGTATCCATCAACG
BARCSOYSSR_20_0629 AACCTAGCATTGCAACCTGC TCATCACCCCTTATCCGTTC
BARCSOYSSR_20_0636 AAAACGAGGCCTTAATCGAAA AAACCAAAGAATACCGTGAAAAA
BARCSOYSSR_20_0638 CGAAATGCCACCTTTTCAAT AGCAAACTAAGGTCGTTTTCG
BARCSOYSSR_20_0644 GCAGTTGTGCGTGGGAGAGAG GCGACATAGCTAATTAAGTAAGTT
BARCSOYSSR_20_0647 GCGTGGTGCACGATCATATAGA GCGTCTCCTTCGCTATCTCAAAC
BARCSOYSSR_20_0649 CCAGGAATGCAGGTTTCTCT CGTGACTCTTCTTCCTTTCCA

图1

亲本及F2群体蛋白质含量频率分布直方图 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2

亲本及F2群体蛋白质含量统计分析"

群体
Population		 母本
Female parent
(mean±SD, %)		 父本
Male parent
(mean±SD, %)		 F2分离群体F2 segregation population
变异范围
Range		 平均数±标准差
(mean±SD, %)		 变异系数
CV (%)		 峰度
Kurtosis		 偏度
Skewness
黑农88×P73-6B
Heinong 88×P73-6B		 43.67±0.67 49.64±2.50 39.22-53.25 47.01±3.98 8.48 2.94 0.26

图2

用黑农88和P73-6B构成的F2群体在大豆20号染色体定位蛋白质QTL"

表3

在黑农88×P73-6B F2群体鉴定到的QTL"

群体
Population		 染色体
Chr.		 标记区间
Marker interval		 LOD得分
LOD score		 表型贡献率
R2		 加性效应
Additive
effect		 显性效应
Dominant
effect
F2 20 Gm20_28349696-Gm20_30805913 12.23 19.31 -1.79 0.19

图3

蛋白含量QTL qPRO-20-1的精细定位 A: 利用HN88×P73-6B F2 200K芯片测序数据遗传图谱构建定位的QTL位点qPRO-20-1在SNP2839696~SNP30805913之间。B: 利用BARCSOYSSR_20_0608、BARCSOYSSR_20_0613、BARCSOYSSR_20_0616、BARCSOYSSR_20_0618、BARCSOYSSR_20_0619、BARCSOYSSR_20_0629、BARCSOYSSR_20_0636、BARCSOYSSR_20_0638、BARCSOYSSR_20_0644、BARCSOYSSR_20_0647、BARCSOYSSR_20_0649共11个标记鉴定5个交换单株(297、272、68、72和209)的基因型, 并验证并初定位。C: 利用芯片数据SNP位点标记28349696、30805913、31343532、31483719和3个SSR标记BARCSOYSSR_20_0636、BARCSOYSSR_20_0647、BARCSOYSSR_20_0649将区间精细定位至标记30805913~BARCSOYSSR_20_0649之间。D: 获得4个候选基因Glyma.20g081800、Glyma.20g081900、Glyma.20g082000、Glyma.20g082100。"

图4

利用7个分子标记定位黑农88×P73-6B大豆F2群体20号染色体的蛋白质QTL"

表4

在黑农88×P73-6B F2群体验证得到的QTL"

群体
Population		 染色体
Chr.		 标记区间
Marker interval		 LOD得分
LOD score		 表型贡献率
R2		 加性效应
Additive effect		 显性效应
Dominant effect
F2 20 Gm20_30805913-BARCSOYSSR_20_0649 12.75 21.44 -2.03 0.56

图5

F2群体中qPRO-20-1位点两侧标记Gm20_30805913与BARCSOYSSR_20_0649的基因型与蛋白质表型相关性分析 A: Gm20_30805913的基因型与表型相关性分析; B: BARCSOYSSR_20_0649的基因型与表型相关性分析。图中横坐标为基因型, 低蛋白为a, 高蛋白为b, 纵坐标为蛋白质含量。***: P < 0.001。"

表5

定位区间内候选基因注释"

基因
Gene name		 基因注释
Gene annotation
Glyma.20g081800 具有RNA结合(RRM-RBD-RNP基序)域的核运输因子2 (NTF2)家族蛋白
Nuclear transport factor 2 (NTF2) family protein with RNA binding (RRM-RBD-RNP motifs) domain
Glyma.20g081900 PTHR32246:SF16与钙相关的脂质结合蛋白
PTHR32246:SF16-calcium-dependentlipid-binding domain-containing protein-related
Glyma.20g082000 未知
Unknown
Glyma.20g082100 PantherFam黄嘌呤-尿嘧啶/维生素C渗透酶家族成员
PantherFam Xanthine-uracil/Vitamin C permease family member

图6

用Phytozome v13.1获得4个基因的表达谱"

图7

用PPRD数据库获得4个基因在大豆9个不同组织的表达量"

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