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Acta Agronomica Sinica ›› 2024, Vol. 50 ›› Issue (5): 1091-1103.doi: 10.3724/SP.J.1006.2024.34142

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

Identification and gene mapping of soybean mutant Mrstz in root-stem transition zone

MIAO Long1**(), SHU Kuo1**(), LI Juan1, HUANG Ru1, WANG Ye-Xing1, Soltani Muhammad YOUSOF1, XU Jing-Hao1, WU Chuan-Lei1, LI Jia-Jia1, WANG Xiao-Bo1,*(), QIU Li-Juan2,*()   

  1. 1College of Agriculture, Anhui Agricultural University, Hefei 230036, Anhui, China
    2Institute of Crop Sciences, Chinese Academy of Agricultural Sciences / the National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI), the Ministry of Agriculture and Rural Affairs / Key Laboratory of Crop Gene Resource and Germplasm Enhancement, the Ministry of Agriculture and Rural Affairs, Beijing 100081, China
  • Received:2023-07-20 Accepted:2024-01-12 Online:2024-05-12 Published:2024-02-08
  • Contact: E-mail: wxbphd@163.com; E-mail: qiulijuan@caas.cn E-mail:lmiao5@163.com;1125974604@qq.com;wxbphd@163.com;qiulijuan@caas.cn
  • About author:First author contact:

    **Contributed equally to this work

  • Supported by:
    National Key Research and Development Program of China(2021YFD1201605);Natural Science Foundation of Anhui Province(2308085MC88);Natural Science Foundation of Anhui Province(2108085QC114);Natural Science Foundation Project of Anhui Agricultural University(k2031005)

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Abstract:

Root-stem transition zone (RSTZ) connects roots and stems, and its morphology modifies the structure of aerial part and lodging resistance potential in soybean. In this study, a soybean mutant Mrstz, appearing with curved or rotated RSTZ, was obtained by EMS mutagenesis. Its morphological characteristics were stably inherited and could provide specific sources for exploring the development patterns of soybean stems. Therefore, this mutant was crossed with Zhonghuang 13 to construct a recombinant inbred line population. By comparing the anatomical structure of vertical RSTZ and curved RSTZ, the wider vascular cambium, more secondary xylem cell layers and irregular cell shape were identified in curved lines, indicating that vascular cambium differentiation may be one of the important factors leading to the difference of RSTZ morphology. Subsequently, the chemical compositions of lines with vertical RSTZ or curved RSTZ were determined, respectively. It was found that the higher lignin and crude fiber content in the RSTZ, the more difficult to bend. The RSTZ with curved or vertical lines were further selected for BSA-seq. SNP-index and InDel-index methods were employed to mine a significant association region, Chr. 19: 43,030,943-45,849,854 containing 319 genes, which may regulate RSTZ morphology. Combined bioinformatics analysis, gene annotation information and expression abundance analysis, seven candidate genes (Glyma.19G170200, Glyma.19G201500, Glyma.19G187800, Glyma.19G178200, Glyma.19G197000, Glyma.19G179100, Glyma.19G196900) were screened. Among them, Glyma.19G187800, Glyma.19G178200, and Glyma.19G196900 potentially affected RSTZ morphogenesis in soybean domestication. This study not only provides germplasm resources for the understanding of soybean RSTZ tissue formation and its genetic basis, but also provides new insights for further exploration of genes regulating soybean stalk development.

Key words: soybean, root and stem transition zone, mutant, BSA-seq, candidate genes

Fig. 1

Comparison of plants with vertical or curved in the root-stem transition zone and its anatomical structures A: soybean plants with vertical or curved RSTZ at the VC, V6, R5, and R8 stages; B: the anatomical structure of hypocotyl for vertical and curved lines at R5 stage; Vc: vascular cambium; Sx: secondary xylem; Sp: secondary phloem. Bar: 2 cm (A) and 50 μm (B). VC: at cotyledon stage; V6: at six leaves stage; R5: at beginning seed stage; R8: at maturity stage."

Fig. 2

Distribution and comparison of chemical components in the vertical and curved root-stem transition zone A, B, and C are the distribution maps of chemical components of crude fiber, cellulose, and lignin in the vertical and curved root-stem transition zone, respectively; D, E, F: histogram represents mean ± SD (*: P < 0.05, **: P < 0.01, t-test)."

Table 1

Quality evaluation of sample sequencing data"

样本类型
Sample type		 纯化后读数
Clean reads		 纯化后碱基数
Clean base		 GC
(%)		 Q30
(%)		 比对上的序列
Mapped reads (%)
中黄13 Zhonghuang 13 37,644,247 11,293,274,100 36.24 94.85 98.40
根茎过渡区弯曲突变体MRstz 43,314,597 12,994,379,100 35.16 94.54 98.32
RSTZ直立型株系Vertical RSTZ lines 126,937,624 38,081,287,200 35.10 94.94 97.99
RSTZ弯曲型株系Curved RSTZ lines 130,489,056 39,146,716,800 35.88 94.56 98.14

Fig. 3

Distribution of SNP index and InDel-index association values on chromosomes Delta-SNP-index (A) and delta-InDel-index (B) fitting plots for two types of RSTZ. The abscissa indicates the position of chromosomes and the black curve represents the associated values of each site, while the top red line indicates the threshold line with the confidence level of 99%. The red arrow points to the candidate region for positioning."

Table 2

Candidate genomic regions of soybean root and stem transition zone morphylogy by BSA-seq correlation technique"

关联分析方法
Association analysis methods		 染色体
Chr.		 起点
Start
(bp)		 终点
End
(bp)		 区间大小
Region size (Mb)		 基因数
Number of genes		 已报道倒伏性状相关候选位点
Candidate loci for inversion reported
Delta-SNP-index 19 43,030,943 46,037,125 3.01 346 Lodging 1-1, Lodging 4-2, Lodging 4-3, Lodging 8-4, Lodging 9-5[32]
Delta-InDel-index 19 42,975,089 45,849,854 2.87 323 Lodging 1-1, Lodging 4-2, Lodging 4-3, Lodging 8-4, Lodging 9-5[32]
Delta-SNP-index and delta-InDel-index 19 43,030,943 45,849,854 2.82 319 Lodging 1-1, Lodging 4-2, Lodging 4-3, Lodging 8-4, Lodging 9-5[32]

Fig. 4

GO annotation classification of genes in candidate regions The horizontal axis indicates the GO classification terms, while the left and right of the vertical axis represent the percentage of genes and the number of genes, respectively."

Fig. 5

Clustering heatmap of expressive abundance of all genes within the consistent association interval in different soybean tissues"

Fig. 6

Comparison of candidate gene expression levels in curved and vertical RSTZ of lines The relative expression levels of candidate genes in different lines were tested by t-test (*: P < 0.05, **: P < 0.01, and ***: P < 0.001) using GmCYP2 as the internal reference."

Table 3

Genetic diversity analysis of morphological candidate genes in roots and stem transition in soybean"

基因
Gene		 单倍型
Haplotype		 频率* Frequency* 核苷酸多态性 π 遗传
分化指数 FST
野生品种
Wild		 当地品种
Landrace		 育成品种
Cultivar		 野生大豆
Wild soybean		 栽培大豆
Cultivated soybean
Glyma.19G170200 Hap0-Hap6 0.095, 0.05, 0.139, 0.275, 0.160, 0.142, 0.139 0.616, 0.352, 0.032, 0, 0, 0, 0 0.719, 0.262, 0.020, 0, 0, 0, 0 0.00364 0.00198 0.22
Glyma.19G201500 Hap0-Hap6 0.305, 0.006, 0.013, 0.242, 0.082, 0.198, 0.154 0.524, 0.408, 0.052, 0, 0.013, 0, 0.002 0.612, 0.323, 0.055, 0, 0.001, 0, 0 0.00257 0.00171 0.16
Glyma.19G187800 Hap0-Hap3 0.212, 0.361, 0.206, 0.222 0.737, 0.126, 0.137, 0 0.862, 0.099, 0.039, 0 0.00373 0.00173 0.26
Glyma.19G178200 Hap0-Hap9 0.075, 0.024, 0.008, 0.012, 0.811, 0.004, 0.028, 0.004, 0.035, 0 0.258, 0.066, 0.171, 0.136, 0.002, 0.116, 0.116, 0.073, 0.021, 0.041 0.338, 0.316, 0.123, 0.082, 0.003, 0.075, 0.008, 0.026, 0.019, 0.012 0.00170 0.00107 0.32
Glyma.19G197000 Hap0-Hap2 0.695, 0.194, 0.111 0.83, 0.168, 0.002 0.727, 0.273, 0 0.00322 0.00258 0.16
Glyma.19G179100 Hap0-Hap2 0.46, 0.003, 0.537 0.965, 0.035, 0 0.937, 0.062, 0 0.00223 0.00069 0.17
Glyma.19G196900 Hap0-Hap4 0.05, 0.528, 0.142, 0.184, 0.096 0.437, 0.385, 0.174, 0.003, 0.002 0.512, 0.212, 0.275, 0, 0 0.00195 0.00123 0.28
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