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Journal of Integrative Agriculture  2025, Vol. 24 Issue (6): 2063-2079    DOI: 10.1016/j.jia.2024.02.005
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Identification of genomic regions and candidate genes underlying carotenoid accumulation in soybean using next-generation sequen-cing based bulk segregant analysis
Berhane S. Gebregziabher1, 2*, Shengrui Zhang1*, Jing Li1*, Bin Li1#, Junming Sun1# 

1 State Key Laboratory of Crop Gene Resources and Breeding/National Engineering Center for Crop Molecular Breeding, Ministry of Agriculture and Rural Affairs/Key Laboratory of Soybean Biology (Beijing)/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

2 Crop Sciences Research Department, Ethiopian Institute of Agricultural Research, Addis Ababa 1000, Ethiopia

 Highlights 
Sixteen candidate genomic loci underlying carotenoids were determined by BSA-seq analysis.
Five candidate genes regulating carotenoids were identified by ANNOVAR and haplotype analysis.
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摘要  
由于类胡萝卜素对人类健康和营养平衡具有重要的价值,因此改良大豆籽粒中类胡萝卜素含量具有重要研究意义。目前大豆类胡萝卜素生物合成的遗传基础尚不完全清楚。本研究利用基于二代测序技术的集团分离分析方法,从1551份大豆种质中鉴定出调控类胡萝卜素含量的基因关联区间。通过对高/低类胡萝卜素含量的大豆种质DNA极端分组混池进行二代测序,获得了125.09 Gb的clean碱基。采用G'值方法对测序数据进行分析,发现16个基因位点与类胡萝卜素关联,总计物理距离为20.41 Mb。特别是6号染色体18.53-22.67 Mb区段,以及19号染色体的8.36-10.94、12.06-13.79和18.45-20.26 Mb区段为热点候选基因区间。针对这些区间的基因进行分析,发现包含250个预测基因,显著富集在90个基因功能注释上。根据方差分析,筛选出50个潜在的候选基因。进一步通过基因注释信息和单倍型分析,最终筛选出5个关键候选基因。综上,通过基于二代测序技术的集团分离分析方法解析了大豆类胡萝卜素积累的遗传基础,同时所鉴定出的关键基因也为高类胡萝卜素分子育种提供了新的视角。


Abstract  

The improvement of soybean seed carotenoid contents is very important due to the beneficial role of carotenoids in human health and nutrition.  However, the genetic architecture underlying soybean carotenoid biosynthesis remains largely unknown.  In the present study, we employed next generation sequencing-based bulked-segregant analysis to identify new genomic regions governing seed carotenoids in 1,551 natural soybean accessions.  The genomic DNA samples of individual plants with extreme phenotypes were pooled to form two bulks with high (50 accessions) and low (50 accessions) carotenoid contents for Illumina sequencing.  A total of 125.09 Gb of clean bases and 89.82% of Q30 were obtained, and the average alignment efficiency was 99.45% with an average coverage depth of 62.20× and 99.75% genome coverage.  Based on the G prime statistic algorithm (G´) method analysis, 16 candidate genomic loci with a total length 20.41 Mb were found to be related to the trait.  Of these loci, the most significant regions displaying the highest elevated G´ values were found in chromosome 06 at a position of 18.53–22.67 Mb, and chromosome 19 at genomic region intervals of 8.36–10.94, 12.06–13.79 and 18.45–20.26 Mb.  These regions were then used to identify the key candidate genes.  In these regions, 250 predicted genes were found and analyzed to obtain 90 significantly enriched (P<0.05) Gene Ontology (GO) terms.  Based on ANNOVAR analysis, 50 genes with non-synonymous and stopgained mutations were preferentially selected as potential candidate genes.  Of those 50 genes, following their gene annotation functions and high significant haplotype variations in various environments, five genes were identified as the most promising candidate genes regulating soybean seed carotenoid accumulation, and they should be investigated in further functional validation studies.   Collectively, understanding the genetic basis of carotenoid pigments and identifying genes underpinning carotenoid accumulation via a bulked-segregant analysis-based sequencing (BSA-seq) approach provide new insights for exploring future molecular breeding efforts to produce soybean cultivars with high carotenoid content.


Keywords:  soybean (Glycine max L. Merrill)       carotenoid       bulk segregant analysis       next-generation sequencing       candidate genes  
Received: 18 August 2023   Online: 03 February 2024   Accepted: 12 December 2023
Fund: 
This work was financially supported by the National Natural Science Foundation of China (32161143033, 32272178, and 32001574), National Key Research and Development Program of China (2021YFD1201605) and the Agricultural Science and Technology Innovation Project of CAAS.
About author:  Berhane S. Gebregziabher, E-mail: berhane76@gmail.com; Shengrui Zhang, E-mail: zhangshengrui@caas.cn; Jing Li, E-mail: lijing02@caas.cn; #Correspondence Junming Sun, Tel/Fax: +86-10-82105805, E-mail: sunjunming@caas.cn; Bin Li, Tel/Fax: +86-10-82105805, E-mail: libin02@caas.cn * These authors contributed equally to this study.

Cite this article: 

Berhane S. Gebregziabher, Shengrui Zhang, Jing Li, Bin Li, Junming Sun. 2025. Identification of genomic regions and candidate genes underlying carotenoid accumulation in soybean using next-generation sequen-cing based bulk segregant analysis. Journal of Integrative Agriculture, 24(6): 2063-2079.

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