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作物学报 ›› 2024, Vol. 50 ›› Issue (4): 808-819.doi: 10.3724/SP.J.1006.2024.34106

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆类病变皱叶突变体NT301遗传分析和2对基因定位

王亚琪1(), 徐海风1, 李曙光1, 傅蒙蒙1, 余希文1, 赵志鑫1, 杨加银1,*(), 赵团结2,*()   

  1. 1江苏徐淮地区淮阴农业科学研究所 / 淮安市农业生物技术重点实验室 / 农业农村部淮河下游种质创制重点实验室, 江苏淮安 223001
    2南京农业大学大豆研究所 / 国家大豆改良中心(南京) / 农业农村部大豆生物学与遗传育种重点实验室(综合) / 作物遗传与种质创新国家重点实验室, 江苏南京 210095
  • 收稿日期:2023-06-27 接受日期:2023-10-23 出版日期:2024-04-12 网络出版日期:2023-11-14
  • 通讯作者: * 赵团结, E-mail: tjzhao@njau.edu.cn;杨加银, E-mail: hynksyjy@163.com
  • 作者简介:E-mail: yqwang_01@126.com
  • 基金资助:
    国家自然科学基金项目(32201729);淮安市自然科学研究计划项目(联合专项, HABL202120)(HABL202120);淮安市农业科学研究院高层次引进人才科研启动发展基金(0112023014B);淮安市农业科学研究院科研发展基金(HNY202221);江苏省种业振兴揭榜挂帅项目(JBGS[2021]057);江苏省现代作物生产协同创新中心项目(JCIC-MCP)

Genetic analysis and two pairs of genes mapping in soybean mutant NT301 with disease-like rugose leaf

WANG Ya-Qi1(), XU Hai-Feng1, LI Shu-Guang1, FU Meng-Meng1, YU Xi-Wen1, ZHAO Zhi-Xin1, YANG Jia-Yin1,*(), ZHAO Tuan-Jie2,*()   

  1. 1Huaiyin Institute of Agricultural Sciences of Xuhuai Region in Jiangsu / Huai’an Key Laboratory for Agricultural Biotechnology / Key Laboratory of Germplasm Innovation in Lower Reaches of the Huaihe River, Ministry of Agriculture and Rural Affairs, Huai’an 223001, Jiangsu, China
    2Soybean Research Institute, Nanjing Agricultural University / National Center for Soybean Improvement (Nanjing) / Key Laboratory for Biology and Genetic Improvement of Soybean (General), Ministry of Agriculture and Rural Affairs / National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing 210095, Jiangsu, China
  • Received:2023-06-27 Accepted:2023-10-23 Published:2024-04-12 Published online:2023-11-14
  • Contact: * E-mail: tjzhao@njau.edu.cn; E-mail: hynksyjy@163.com
  • Supported by:
    National Natural Science Foundation of China(32201729);Natural Science Research Program of Huai’an (Joint Special Project, HABL202120)(HABL202120);Scientific Research Fund of Startup and Development for Introduced High-level Talents, Huai’an Academy of Agricultural Sciences(0112023014B);Research and Development Fund Project of Huai’an Academy of Agricultural Sciences(HNY202221);Core Technology Development for Breeding Program of Jiangsu Province(JBGS[2021]057);Jiangsu Collaborative Innovation Center for Modern Crop Production(JCIC-MCP)

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摘要：

通过研究类病变突变体, 挖掘抗病基因, 利用分子设计育种的方法快速培育优良抗病大豆新品种, 可减轻化学农药对环境的污染, 降低病害的抗药性。本研究以60Coγ诱变获得的类病变皱叶突变体NT301为父本, 分别与W82、KF1和KF35进行杂交, 构建了F2和F2:3分离群体, 通过SSR标记和SNP分析, 将目标基因1 (rl1)缩小到18号染色体937 kb区间内, 包含66个候选基因, 将目标基因2 (rl2)缩小到8号染色体130 kb区间内, 包含15个候选基因。接着, 本研究利用基因芯片技术对近等基因系进行了基因表达谱研究, 得到了差异表达基因参与的KEGG调控通路。另外对8号染色体的15个候选基因进行半定量与荧光定量RT-PCR表达分析发现, 只有基因Glyma. 08G332500在突变体NT301与野生型中的差异达到了4倍, 推测Glyma.08G332500基因是突变体NT301的候选基因。

关键词: 大豆, 类病变突变体, 皱叶, 基因表达谱

Abstract:

Research on lesion mimic mutant, mining resistance genes, and developing superior disease-resistant new soybean varieties by molecular design breeding methods can contribute to the alleviating the environmental pollution caused by chemical pesticides and drug resistance to disease. In this study, the disease-like rugose leaf mutant NT301 obtained by 60Coγ mutagenesis as the male parent was crossed with W82, KF1, and KF35, respectively, to construct F2 and F2:3 segregating populations. Using SSR and SNP markers, target gene 1 (rl1) was narrowed to 937 kb on chromosome 18 with 66 genes and target gene 2 (rl2) was narrowed to 130 kb on chromosome 8 with 15 genes. The gene expression patterns of the wild type and NT301 were compared using gene chip technology, and the KEGG pathways of the differentially expressed genes were assessed. Moreover, semi quantitative and quantitative RT-PCR methods were used to analyze the relative expression levels of candidate genes on chromosome 8. The results showed that the relative expression level of Glyma.08G332500 in NT301 was four times higher than the wild type. In contrast, the expression levels of other genes showed no more than double difference. Therefore, we suggest that Glyma.08G332500 may be a candidate gene for NT301.

Key words: soybean, disease-like mutant, rugose leaf, gene expression profiles

附表1

本研究所用到的引物"

引物名称
Primer name		 正向引物
Forward primer (5°-3°)		 反向引物
Reverse primer (5°-3°)
BARCSOYSSR_18_0415 GCTTGGCGAATTCCATCTAA ATTCATTTCACATCCCAGGC
BARCSOYSSR_18_0419 TTTATTATGGGGATCAAATTAAAACT TCGGAGTTAGCCAAATCAAAT
BARCSOYSSR_18_0485 AAGGATTGGCAAAGCGATTA AAAAACAGGGTTTGGTGGGT
BARCSOYSSR_18_0444 TTGACGGCCTTATTTTGGAC GGTGGCTGAATCCAAGACAT
BARCSOYSSR_18_0481 ATCTCAAGGATCTGGCAAGC AATATTCTTTGGCGGTGGTG
BARCSOYSSR_08_1700 CCTTTAATCAACTCTGTGAGATCG GCATAATGCTATTCTCCGCA
BARCSOYSSR_08_1800 ACAAGGAGATTTGGCTTTGC CCGGAGATCGATAAGTTGCT
BARCSOYSSR_08_1724 CATTGGAGCTTGGTAAGGGA GGGAGCAGAGAACTTGAGCA
BARCSOYSSR_08_1736 TCCTTTTTGAAAACGAAATTCA GAGTACATTGGAATAACTGTGCAA
BARCSOYSSR_08_1724 CATTGGAGCTTGGTAAGGGA GGGAGCAGAGAACTTGAGCA
BARCSOYSSR_08_1738 TGTTAGGGACACACTCAACCC CCACGAGATAAGACGAGCAA
BARCSOYSSR_08_1753 CCAGCCACTTCACAGACTGA TTGGGTTATCTGTTTGCTTTGTT
SSR18 GGCTTGACTCTCTGAATCTGTT TAAACAACTTTGAGCCAACGGC
SSR40 ACAAACCTGGTTCGGCTATGT TTACCAAAGCAGTGGGAGCC
Satt409 CCTTAGACCATGAATGTCTCGAAGATA CTTAAGGACACGTGGAAGATGACTAC
SNP1 TTCGAAGGAACTTACTCATTA CCAACATTCTCAAGCCCAAGG
SNP2 AGCACGATTCTACCTCCGAAT GGTAGGCAATCTGAACTCATCA
SNP3 AGGGATATGGTTCATCTTTCATCT ATGATGTGTTATGTGCATAT
Glyma.08g332000QRT GGCGTCCAGGATTTACAAAGT TGATATTGCACCAGGCTTTGA
Glyma.08g332100QRT CATGGCTGGCTTTAAGAGGA AGTATCAAAGAAGGAGCCGTT
Glyma.08g332200QRT ACTACGGTGCATCAGAGATTC TCAGTGCAATCATAACTGTGGTA
Glyma.08g332300QRT GGCCGCTACCATCAACTTCA AGCTCTAGTGAACTACGGCATT
Glyma.08g332400QRT TGCCTTCACACATTATAAACAGG TATGGTTTCCACTTCCGACCC
Glyma.08g332500QRT AATGAGAGAACAAAGGGAAGAGG GAGTCACAAAGCAACCCACAG
Glyma.08g332600QRT GATGTGGTGCAGAGCAAACC CACCATCGTCAAGTTGTGGC
Glyma.08g332700QRT TGGTGATGAAGAACAACAGC ACAAGAAGTCCTGGCCTAGC
Glyma.08g332800QRT TGCTGAATCTGGCATGAACC GACGGATGGCGCAAAACAAG
Glyma.08g332900QRT TTGTTGACAGTGGTATTGGCA AACCCCACACCAAACTGTCC
Glyma.08g333000QRT CGGTTTGTCTATATTGTTGAAGG TTTCTCCACTGAACTGGTCCAC
Glyma.08g333100QRT CCGCCCAAACCTCTGAAGAT CAGACTGGAAGTTGCCAGGTA
Glyma.08g333200QRT CAAGAACTGCAACTGAAAATGGTTG CATGCCAAAACTGTACACATCAC
Glyma.08g333300QRT CGCCAGAACTTTGCAAGGAA ACCCGAACCATTCACGACTT
Glyma.08g333400QRT TGAAGCATGGCGAAGCAGTA CGAACACGTGGGTCATGGAA
GmActin GGTGGTTCTATCTTGGCATC CTTCGCTTCAATAACCCTA

图1

突变体NT301的形态特征 A: 苗期野生型植株; B: 苗期NT301植株; C: 开花期野生型植株; D: 开花期NT301植株; E: 野生型叶片横切面; F: NT301叶片横切面。标尺: 1 cm (A, B); 5 cm (C, D); 200 μm (E, F)。p: 栅栏组织; s: 海绵组织; vb: 维管束。"

表1

杂交组合F2和 F2:3群体野生型和突变体植株分离率适合性检验"

杂交组合
Cross		 世代Generation 单株或株系数目No. of plants or lines 卡方测验Chi-square tests
总计
Total		 野生型
Wild type		 分离
Segregation		 突变体
Mutant		 期望比
Expected ratio		 卡方值
χ2		 P值
P-value
KF1×NT301 F1 2 2 0 0 — — —
F2 451 430 0 21 15:1 0.50 0.48
F2:3 lines 182 88 94 — 7:8 0.15 0.70
KF35×NT301
 F1 2 2 0 0 — — —
F2 362 342 0 20 15:1 0.06 0.80
F2:3 lines 176 87 89 — 7:8 0.44 0.51

图2

突变体NT301 2对候选基因定位 A: 18号染色体rl1定位; B: 8号染色体rl2精细定位。"

表2

芯片试验质控情况"

样品名称
Sample name		 背景值
Background value		 BioB检出情况
BioB detection status		 Beta-actin
3°/5°*		 GAPDH
3°/5°*		 检出率
Detection rate (%)*
1-1 32.67 + — — 53.18
1-2 32.57 + — — 53.01
1-3 35.58 + — — 51.37
2-1 32.49 + — — 49.75
2-2 33.40 + — — 50.43
2-3 31.90 + — — 50.23

图3

突变体NT301与野生型差异表达基因的聚类图 横坐标代表样品名称及样品的聚类结果, 纵坐标代表差异基因及基因的聚类结果。S1为突变体, S2为野生型, 各3个重复, 不同的行代表不同的基因。颜色代表了基因在样品中的表达量水平。"

图4

差异表达基因KEGG富集图 A: 上调基因富集的KEGG通路; B: 下调基因富集的KEGG通路。图中-log10 P越大, 表示该通路越显著。横坐标表示富集值, 纵坐标表示富集的通路。圆圈大小表示基因数量。"

表3

rl2位点候选基因功能注释"

序号
No.		 基因名称
Gene name		 起始-终止位置Start-Stop (bp) 注释信息
Annotation
1 Glyma.08G332000 44937110-44943950 S-腺苷-L-蛋氨酸依赖性甲基转移酶超家族蛋白S-adenosyl-L-methionine-dependent
2 Glyma.08G332100 44947629-44951967 ABC-2 型转运蛋白家族ABC-2 type transporter family protein
3 Glyma.08G332200 44955377-44958957 —
4 Glyma.08G332300 44964341-44968328 RING/U-box超家族蛋白RING/U-box superfamily protein
5 Glyma.08G332400 44969692-44971581 丝氨酸蛋白相关Serine-rich protein-related
6 Glyma.08G332500 44972649-44974052 UDP 糖基转移酶超家族蛋白UDP-glycosyltransferase superfamily protein
7 Glyma.08G332600 44975232-44979003 胚胎缺陷1303 Embryo defective 1303
8 Glyma.08G332700 44980398-44987729 WW 结构域蛋白WW domain-containing protein
9 Glyma.08G332800 44992809-44997458 钙调神经磷酸酶B Calcineurin B-like 3
10 Glyma.08G332900 44997823-45000952 热激蛋白81.4 Heat Shock Protein 81.4
11 Glyma.08G333000 45003016-45005379 类FKBP肽基脯氨酰顺反异构酶FKBP-like peptidyl-prolyl cis-trans isomerase
12 Glyma.08G333100 45006829-45012886 类Got1/Sft2维管转运蛋白家族Got1/Sft2-like vescicle transport protein family
13 Glyma.08G333200 45024206-45028194 富亮氨酸类受体蛋白激酶家族Leucine-rich receptor-like protein kinase family
14 Glyma.08G333300 45033130-45043479 类驱动蛋白1 Kinesin-like protein 1
15 Glyma.08G333400 45064017-45071298 类尿苷激酶4 Uridine kinase-like 4

图5

8号染色体定位区间内基因的表达分析 A: 8号染色体定位区间内基因半定量PCR分析; B: 8号染色体定位区间内基因荧光定量PCR分析。红色方框表示野生型和突变体表达量差异最大的基因, 采用学生t测验进行显著性检验(**: P ≤ 0.01), 误差线表示标准差(n = 3)。"

附图1

突变体NT301 18号和8号染色体定位区间内候选基因氨基酸序列聚类分析 红色方框表示8号染色体表达量差异最大的基因Glyma.08G332500和18号染色体的同源基因Glyma.18G085800。"

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