中国农业科学 ›› 2019, Vol. 52 ›› Issue (16): 2743-2757.doi: 10.3864/j.issn.0578-1752.2019.16.001
收稿日期:
2019-03-25
接受日期:
2019-05-07
出版日期:
2019-08-16
发布日期:
2019-08-21
通讯作者:
张明荣
作者简介:
王嘉,E-mail:基金资助:
WANG Jia,ZENG ZhaoQiong,LIANG JianQiu,YU XiaoBo,WU HaiYing,ZHANG MingRong()
Received:
2019-03-25
Accepted:
2019-05-07
Online:
2019-08-16
Published:
2019-08-21
Contact:
MingRong ZHANG
摘要:
【目的】基于全基因组重测序结果,开发与高蛋白、耐荫、抗倒伏等性状紧密相关的分子标记,同时利用开发的分子标记构建遗传连锁图谱,并对籽粒蛋白质含量进行QTL定位,为后续高蛋白、耐荫、抗倒育种研究提供参考和分子标记资源。【方法】以大面积栽培品种南豆12和地方品种十月黄为亲本,构建F2分离群体。对亲本材料进行覆盖度约为40×的全基因组重测序,用BWA、GATK及Breakdancer等软件比对,检测亲本材料在全基因组范围内的突变类型,挖掘相关变异基因。结合种子不同发育时期和荫蔽处理获得的转录组数据,结合qRT-PCR对发生突变的储藏蛋白、环境适应相关基因进行表达规律分析。同时,基于重测序数据,挖掘亲本间存在于基因编码区的SNP位点,对其进行酶切位点分析,将SNP标记转化为CAPS或dCAPS标记。此外,搜索亲本间存在的插入/缺失变异位点,在插入/缺失位点两侧高度保守的区域设计引物开发InDel标记。对开发的CAPS标记和InDel标记进行多态性筛选,选取具有多态性的CAPS分子标记和InDel标记,对F2材料进行基因分型。根据分型结果,利用JoinMap 4.0软件进行遗传连锁图谱的构建。依据构建的遗传图谱,结合近红外分析获得F2材料的籽粒蛋白质含量数据,使用Windows QTL Cartographer V2.5软件对大豆籽粒蛋白质含量进行QTL分析。【结果】测序结果显示,南豆12大量储藏蛋白、环境适应相关的重要基因或同源基因发生突变。转录组数据分析结果显示部分变异基因呈现不同的表达模式且差异显著,qRT-PCR分析进一步验证了该结果。此外,经检测开发的540个CAPS分子标记中有332个具有酶切多态性,300对InDel引物中有201对引物能扩增出多态性。基于533个多态性分子标记构建了一张包含20个连锁群的遗传连锁图谱,覆盖长度2 973.87 cM,标记间平均遗传距离5.58 cM。利用此图谱对大豆籽粒蛋白质含量进行QTL定位,共检测到QTL位点6个,可解释4.68%—18.25%的表型变异。【结论】基于亲本间的变异位点,共开发了533个多态性分子标记(包含8个基因特异性分子标记),检测到6个大豆籽粒蛋白质含量QTL位点,其中,主效QTL位点1个(qSPC-6)。
王嘉, 曾召琼, 梁建秋, 于晓波, 吴海英, 张明荣. 基于全基因组重测序的大豆分子标记开发及籽粒蛋白质含量QTL定位[J]. 中国农业科学, 2019, 52(16): 2743-2757.
WANG Jia, ZENG ZhaoQiong, LIANG JianQiu, YU XiaoBo, WU HaiYing, ZHANG MingRong. Development New Molecular Markers for Quantitative Trait Locus (QTL) Analysis of the Seed Protein Content Based on Whole Genome Re-Sequencing in Soybean[J]. Scientia Agricultura Sinica, 2019, 52(16): 2743-2757.
表1
qRT-PCR验证基因引物信息"
基因ID Gene ID | 基因名称 Gene name | 正向引物 Forward primer (5'-3') | 反向引物 Reverse primer (5'-3') |
---|---|---|---|
Glyma.18G176100 | ABI3 | CTCATCATGCAAATCCTACAGC | CTATCTGATGCAACTCGACTCT |
Glyma.13G123500 | Gy5 | CAGATATAGAGCACCCAGAGAC | GGTGTTGAAGGATTGTGCTAAG |
Glyma.18G175500 | MAG2 | CAAATTTGGGGCATGGCTAATA | CTTGAAAATTGTTTCGGTTGCG |
Glyma.18G226500 | CRA1 | CTGCAAGAAGTGGTCGTTTTTA | ATTTCAGTTGTTCCATGTTCCG |
Glyma.02g248300 | PAT2 | AAAGGCGTTGAAGCTGGATATA | AATCTCCTATCTGGATCTCCGA |
Glyma.11g112800 | BBX21 | CACAACAGGTTTCTTCTCACTG | TGTTCTTTCTTCAATGGCAGTG |
Glyma.14g062100 | COI1 | GTACATACACGACTCCAAGGAC | TTTCCCTTCAGGTTTAACGACT |
Glyma.05g062700 | SAV1 | GTGGGTCTCCAATGTTTTGTAC | GCAAATTGTTTCAATGGGTGTG |
Glyma.18g043000 | KAN | TTGGAGTTCACATTAGGGAGAC | AAGTTTGTCACATGTTACCGTG |
Glyma.03g227300 | PHYA | CAAAGTCTGATAGAGCAGCAAC | CTCACTGTAAGCGATGACCTTA |
ACT11 | CGGTGGTTCTATCTTGGCATC | GTCTTTCGCTTCAATAACCCTA |
表2
南豆12和十月黄之间出现非同义替换最多的前10个基因"
基因ID Gene ID | 基因名称 Gene name | 注释 Annotation | 基因区域内的SNP数目 Genic SNP | CDS中的SNP SNP in CDS | 非同义替换SNP Non-syn SNP |
---|---|---|---|---|---|
Glyma.18G176100 | ABI3 | ABI3-like转录因子家族成员,促进种子储藏蛋白的积累 A member of ABI3-like transcription factor family, promoting the accumulation of seed storage proteins | 72 | 31 | 20 |
Glyma.13G123500 | Gy5 | 大豆球蛋白基因家族成员之一,编码一个11S球蛋白亚基 Encodes an 11S globulin subunit, a member of the soybean globulin gene family | 46 | 33 | 18 |
Glyma.11G112800 | BBX21 | 编码B-box锌指转录因子bbx21,与cop1基因相互作用调节避荫 Encodes a B-box zinc finger transcription factor BBX21, genetically interacts with COP1 to regulate shade avoidance. | 30 | 29 | 16 |
Glyma.18G175500 | MAG2 | 参与种子贮藏蛋白从内质网到液泡的运输 Involved in transportation of seed storage proteins from the ER to the vacuole | 36 | 27 | 14 |
Glyma.13G335200 | OBP3 | 编码一个主要在根中表达的包含转录因子的核定位Dof域 Encodes a nuclear localized Dof domain containing transcription factor expressed primarily in roots. | 35 | 35 | 14 |
Glyma.02G057500 | SAV1 | 编码一个油菜素类固醇生物合成途径的限速酶22α羟化酶 Encodes a 22α hydroxylase whose reaction is a rate-limiting step in brassinosteroid biosynthetic pathway | 51 | 29 | 12 |
Glyma.08G171800 | GA2OX4 | 编码作用于赤霉素C19的赤霉素2-氧化酶 Encodes a gibberellin 2-oxidase that acts on C19 gibberellins | 88 | 26 | 11 |
Glyma.20G048500 | VPS26B | 细胞内蛋白转运 Intracellular protein transport | 32 | 25 | 11 |
Glyma.18G226500 | CRA1 | 编码一个12S种子储存蛋白 Encodes a 12S seed storage protein | 72 | 26 | 10 |
Glyma.14G062100 | COI1 | 避荫 Shade avoidance | 28 | 25 | 10 |
表4
利用复合区间作图法检测到的籽粒蛋白质含量QTL"
数量性状座位 QTL | 染色体 Chromosome | 标记区间 Position (cM) | 置信区间 Confidence interval | LOD | 加性效应 Additive | 贡献率 R2 (%) |
---|---|---|---|---|---|---|
qSPC-1 | Gm06(C2) | 34.01 | GmIn063—GmCA130 | 8.09 | -0.23 | 5.39 |
qSPC-2 | Gm07(M) | 46.71 | GmCA168—GmCA171 | 5.26 | 0.22 | 5.36 |
qSPC-3 | Gm15(E) | 26.51 | GmIn149—GmIn150 | 4.53 | 0.21 | 4.68 |
qSPC-4 | Gm15(E) | 72.21 | GmIn152—GmIn163 | 9.93 | -0.29 | 9.32 |
qSPC-5 | Gm20(I) | 35.61 | GmCA493—GmIn285 | 8.40 | -0.47 | 12.04 |
qSPC-6 | Gm20(I) | 103.41 | GmdCA522—GmCA537 | 15.06 | -0.68 | 18.86 |
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