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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (4): 808-819.doi: 10.3724/SP.J.1006.2024.34106

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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 Online:2024-04-12 Published: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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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

Table S1

Primers used in this study"

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

Fig. 1

Morphological characteristics of mutant NT301 A: wild-type plants at seedling stage; B: mutant NT301 at seedling stage; C: wild-type plants at flowering stage; D: mutant NT301 at flowering stage; E: the transverse section of wild-type leaves; F: the transverse section of NT301 leaves. Bar: 1 cm (A, B); 5 cm (C, D); 200 μm (E, F). p: palisade parenchyma; s: spongy parenchyma; vb: vascular bundle."

Table 1

Chi-square tests of wild type and mutant plants of F2 and F2:3 generation in crosses"

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

Fig. 2

Mapping of two pairs of candidate genes of mutant NT301 A: mapping of rl1 on chromosome 18; B: fine mapping of rl2 on chromosome 8."

Table 2

Quality control of chip experiment"

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

Fig. 3

Cluster figure of differentially expressed genes in mutant NT301 and wild type The horizontal axis represents the sample names and clustering results of the samples, while the vertical axis represents the differentially expressed genes and clustering results of the genes. S1 represents the mutant, and S2 represents the wild type, three times repeat. Different rows represent different genes. Color represents the relative expression level of genes in the samples."

Fig. 4

KEGG enrichment map of differentially expressed genes A: the enrichment of upregulated genes in the KEGG; B: the enrichment of downregulated genes in the KEGG. The figure shows that the -log10 P is greater, the pathway is more significant. The horizontal axis represents the enrichment value, and the vertical axis represents the pathway of enrichment. The size of the circle indicates the number of genes."

Table 3

Annotation of rl2 candidate genes"

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

Fig. 5

Relative expression level of genes in the mapping interval of chromosome 8 A: the semi quantitative PCR analysis of genes in the mapping interval of chromosome 8; B: the quantitative RT-PCR analysis of genes in the mapping interval of chromosome 8. The red box indicates the most differentially expressed gene between wild type and mutant. The significant test was carried out by the student’s t-test (**: P ≤ 0.01). The error bars indicate the SDs (n = 3)."

Fig. S1

Clustering analysis of amino acid sequences of candidate genes within the mapping intervals on chromosomes 18 and 8 in the mutant NT301 The red box indicates the most differentially expressed gene Glyma.08G332500 on chromosome 8 and its homologous gene Glyma.18G085800 on chromosome 18."

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