基于二代测序技术的集团分离分析方法鉴定大豆类胡萝卜素基因关联区间和候选基因

doi:10.1016/j.jia.2024.02.005

Journal of Integrative Agriculture ›› 2025, Vol. 24 ›› Issue (6): 2063-2079.DOI: 10.1016/j.jia.2024.02.005

基于二代测序技术的集团分离分析方法鉴定大豆类胡萝卜素基因关联区间和候选基因

收稿日期:2023-08-18 修回日期:2024-02-03 接受日期:2023-12-12 出版日期:2025-06-20 发布日期:2025-05-12

Identification of genomic regions and candidate genes underlying carotenoid accumulation in soybean using next-generation sequen-cing based bulk segregant analysis

Berhane S. Gebregziabher^1,^2*, Shengrui Zhang^1*, Jing Li^1*, Bin Li^1#, Junming Sun¹^#

¹State 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
²Crop Sciences Research Department, Ethiopian Institute of Agricultural Research, Addis Ababa 1000, Ethiopia

Received:2023-08-18 Revised:2024-02-03 Accepted:2023-12-12 Online:2025-06-20 Published:2025-05-12
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.
Supported by:
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.

摘要/Abstract

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.

Key words: soybean (Glycine max L. Merrill) , carotenoid , bulk segregant analysis , next-generation sequencing , candidate genes

Berhane S. Gebregziabher, Shengrui Zhang, Jing Li, Bin Li, Junming Sun. 基于二代测序技术的集团分离分析方法鉴定大豆类胡萝卜素基因关联区间和候选基因[J]. Journal of Integrative Agriculture, 2025, 24(6): 2063-2079.

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

参考文献

Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H, Matsumura H, Yoshida K, Mitsuoka C, Tamiru M, Innan H, Cano L, Kamoun S, Terauchi R. 2012. Genome sequencing reveals agronomically important loci in rice using MutMap. Nature Biotechnology, 30, 174–178.

Altschul S F, Madden T L, Schäffer A A, Zhang J, Zhang Z, Miller W, Lipman D J. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Research, 25, 3389–3402.

Ampomah-Dwamena C, Thrimawithana A H, Dejnoprat S, Lewis D, Espley R V, Allan A C. 2019. A kiwifruit (Actinidia deliciosa) R2R3-MYB transcription factor modulates chlorophyll and carotenoid accumulation. New Phytologist, 221, 309–325.

Ashburner M, Ball C A, Blake J A, Botstein D, Butler H, Cherry J M, Davis A P, Dolinski K, Dwight S S, Eppig J T, Harris M A, Hill D P, Issel-Tarver L, Kasarskis A, Suzanna L, Matese J C, Richardson J E, Ringwald M, Rubin G M, Sherlock G. 2000. Gene Ontology: Tool for the unification of biology. Nature Genetics, 25, 25–29.

Azam M, Zhang S, Huai Y, Abdelghany A M, Shaibu A S, Qi J, Feng Y, Liu Y, Li J, Qiu L, Li B, Sun J. 2023. Identification of genes for seed isoflavones based on bulk segregant analysis sequencing in soybean natural population. Theoretical and Applied Genetics, 136, 13.

Das S, Upadhyaya H D, Bajaj D, Kujur A, Badoni S, Kumar V, Tripathi S, Gowda C L L, Sharma S, Singh S, Tyagi A K, Parida S K. 2015. Deploying QTL-seq for rapid delineation of a potential candidate gene underlying major trait-associated QTL in chickpea. DNA Research, 22, 193–203.

Dhanapal A P, Ray J D, Singh S K, Hoyos-Villegas V, Smith J R, Purcell L C, King C A, Fritschi F B. 2015. Association mapping of total carotenoids in diverse soybean genotypes based on leaf extracts and high-throughput canopy spectral reflectance measurements. PLoS ONE, 10, e0137213.

Gao J, Yang S, Tang K, Li G, Gao X, Liu B, Wang S, Feng X. 2021. GmCCD4 controls carotenoid content in soybeans. Plant Biotechnology Journal, 19, 801–813.

Gebregziabher B S, Zhang S, Azam M, Qi J I, Agyenim-boateng K G, Feng Y, Liu Y, Li J, Li B, Sun J M. 2023. Natural variations and geographical distributions of seed carotenoids and chlorophylls in 1 167 Chinese soybean accessions. Journal of Integrative Agriculture, 22, 2632–2647.

Gebregziabher B S, Zhang S, Ghosh S, Shaibu A S, Azam M, Abdelghany A M, Qi J, Agyenim-boateng K G, Htway H T P, Feng Y, Ma C, Li Y, Li J, Li B, Qiu L, Sun J. 2022. Origin, maturity group and seed coat color influence carotenoid and chlorophyll concentrations in soybean seeds. Plants, 11, 848.

Gebregziabher B S, Zhang S, Qi J, Azam M, Ghosh S, Feng Y, Huai Y, Li J, Li B, Sun J. 2021. Simultaneous determination of carotenoids and chlorophylls by the HPLC-UV-VIS method in soybean seeds. Agronomy, 11, 758.

Ghosh S, Zhang S, Azam M, Gyapong K, Boateng A, Qi J, Feng Y, Li Y, Li J, Li B, Sun J. 2022. Identification of genomic loci and candidate genes related to seed tocopherol content in soybean. Plants, 11, 1703.

Giovannoni J J, Wing R A, Ganal M W, Tanksley S D. 1991. Isolation of molecular markers from specific chromosomal intervals using DNA pools from existing mapping populations. Nucleic Acids Research, 19, 6553–6568.

Gonzalez-Jorge S, Mehrshahi P, Magallanes-Lundback M, Lipka A E, Angelovici R, Gore M A, DellaPenna D. 2016. ZEAXANTHIN EPOXIDASE activity potentiates carotenoid degradation in maturing seed. Plant Physiology, 171, 1837–1851.

Jiang C, Zhang Y, Lin Y, Chen Y, Lu X. 2019. Illumina® sequencing reveals candidate genes of carotenoid metabolism in three pummelo cultivars (Citrus maxima) with different pulp color. International Journal of Molecular Sciences, 20, 2246.

Jin H, Martin C. 1999. Multifunctionality and diversity within the plant MYB-gene family. Plant Molecular Biology, 41, 577–585.

Kim J, DellaPenna D. 2006. Defining the primary route for lutein synthesis in plants: The role of Arabidopsis carotenoid β-ring hydroxylase CYP97A3. Proceedings of the National Academy of Sciences of the United States of America, 103, 3474–3479.

Kosugi S, Natsume S, Yoshida K, MacLean D, Cano L, Kamoun S, Terauchi R. 2013. Coval: Improving alignment quality and variant calling accuracy for next-generation sequencing data. PLoS ONE, 8, e75402.

Li H, Durbin R. 2009. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics, 25, 1754–1760.

Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R. 2009. The sequence alignment/map format and SAMtools. Bioinformatics, 25, 2078–2079.

Li P, Xia E, Fu J, Xu Y, Zhao X, Tong W, Tang Q, Tadege M, Fernie A R, Zhao J. 2022. Diverse roles of MYB transcription factors in regulating secondary metabolite biosynthesis, shoot development, and stress responses in tea plants (Camellia sinensis). The Plant Journal, 110, 1144–1165.

Li Z, Xu Y. 2022. Bulk segregation analysis in the NGS era: A review of its teenage years. Plant Journal, 109, 1355–1374.

Liang T, Chi W, Huang L, Qu M, Zhang S, Tang W, Chen S. 2020. Bulked segregant analysis coupled with whole-genome sequencing (BSA-Seq) mapping identifies a novel pi21 haplotype conferring basal resistance to rice blast disease. International Journal of Molecular Sciences, 21, 2162.

Liu S, Liu Z, Hou X, Li X. 2023. Genetic mapping and functional genomics of soybean seed protein. Molecular Breeding, 43, 29.

Liu Y, Jiang T, Chen Y, Gu Y, Song F, Sun J, Luo J. 2021. Identification of candidate genes associated with hypoxia tolerance in Trachinotus blochii using bulked segregant analysis and RNA-Seq. Frontiers in Genetics, 12, 811685.

Lu L, Wei W, Li Q T, Bian X H, Lu X, Hu Y, Cheng T, Wang Z Y, Jin M, Tao J J, Yin C C, He S J, Man W Q, Li W, Lai Y C, Zhang W K, Chen S Y, Zhang J S. 2021. A transcriptional regulatory module controls lipid accumulation in soybean. New Phytologist, 231, 661–678.

Magwene P M, Willis J H, Kelly J K. 2011. The statistics of bulk segregant analysis using next generation sequencing. PLoS Computational Biology, 7, e1002255.

Majeed A, Johar P, Raina A, Salgotra R K, Feng X, Bhat J A. 2022. Harnessing the potential of bulk segregant analysis sequencing and its related approaches in crop breeding. Frontiers in Genetics, 13, 944501.

Mansfeld B N, Grumet R. 2018. QTLseqr: An R package for bulk segregant analysis with next generation sequencing. The Plant Genome, 11, 180006.

McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K, Kernytsky A, Garimella K, Altshuler D, Gabriel S, Daly M, DePristo M A. 2010. The genome analysis toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data. Genome Research, 20, 1297–1303.

Michelmore R W, Paran I, Kesseli R V. 1991. Identification of markers linked to disease-resistance genes by bulked segregant analysis: A rapid method to detect markers in specific genomic regions by using segregating populations. Proceedings of the National Academy of Sciences of the United States of America, 88, 9828–9832.

Niu G, Niu G, Guo Q, Guo Q, Wang J, Wang J, Zhao S, He Y, Liu L. 2020. Structural basis for plant lutein biosynthesis from α-carotene. Proceedings of the National Academy of Sciences of the United States of America, 117, 14150–14157.

Ramakrishna G, Kaur P, Nigam D, Chaduvula P K, Yadav S, Talukdar A. 2018. Genome-wide identification and characterization of InDels and SNPs in Glycine max and Glycine soja for contrasting seed permeability traits. BMC Plant Biology, 18, 141.

Ramos A, Fu Y, Michael V, Meru G. 2020. QTL-seq for identification of loci associated with resistance to Phytophthora crown rot in squash. Scientific Reports, 10, 5326.

Rosello S, Adalid A, Cebolla-Cornejo J, Nuez F. 2011. Evaluation of the genotype, environment and their interaction on carotenoid and ascorbic acid accumulation in tomato germplasm. Journal of the Science of Food and Agriculture, 91, 1014–1021.

Sagawa J M, Stanley L E, Lafountain A M, Frank H A, Liu C, Yuan Y. 2016. An R2R3-MYB transcription factor regulates carotenoid pigmentation in Mimulus lewisii flowers. New Phytologist, 3, 1049–1057.

Saini R K, Nile S H, Park S W. 2015. Carotenoids from fruits and vegetables: Chemistry, analysis, occurrence, bioavailability and biological activities. Food Research International, 76, 735–750.

Seto Y, Yasui R, Kameoka H, Tamiru M, Cao M, Terauchi R, Sakurada A, Hirano R, Kisugi T, Hanada A, Umehara M, Seo E, Akiyama K, Burke J, Takeda-Kamiya N, Li W, Hirano Y, Hakoshima T, Mashiguchi K, Noel J P, et al. 2019. Strigolactone perception and deactivation by a hydrolase receptor DWARF14. Nature Communications, 10, 191.

Shen F, Huang Z, Zhang B, Wang Y, Zhang X, Wu T, Xu X, Zhang X, Han Z. 2019. Mapping gene markers for apple fruit ring rot disease resistance using a multi-omics approach. G3 (Bethesda), 9, 1663–1678.

da Silva M P, Zaccaron A Z, Bluhm B H, Rupe J C, Wood L, Mozzoni L A, Mason R E, Yingling S, Pereira A. 2020. Bulked segregant analysis using next-generation sequencing for identification of genetic loci for charcoal rot resistance in soybean. Physiological and Molecular Plant Pathology, 109, 101440.

Song J, Li Z, Liu Z, Guo Y, Qiu L. 2017. Next-generation sequencing from bulked-segregant analysis accelerates the simultaneous identification of two qualitative genes in soybean. Frontiers in Plant Science, 8, 919.

Stanley L, Yuan Y W. 2019. Transcriptional regulation of carotenoid biosynthesis in plants: So many regulators, so little consensus. Frontiers in Plant Science, 10, 1017.

Stanley L E, Ding B, Sun W, Mou F, Hill C, Chen S, Yuan Y W. 2020. A tetratricopeptide repeat protein regulates carotenoid biosynthesis and chromoplast development in monkeyflowers (Mimulus). The Plant Cell, 32, 1536–1555.

Stracke R, Werber M, Weisshaar B. 2001. The R2R3-MYB gene family in Arabidopsis thaliana. Current Opinion in Plant Biology, 4, 447–456.

Sun T, Rao S, Zhou X, Li L. 2022. Plant carotenoids: Recent advances and future perspectives. Molecular Horticulture, 2, 3.

Supek F, Bošnjak M, Kunca N S, Šmuc T. 2011. REVIGO summarizes and visualizes long lists of Gene Ontology terms. PLoS ONE, 6, e21800.

Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, Uemura A, Utsushi H, Tamiru M, Takuno S, Innan H, Cano L M, Kamoun S, Terauchi R. 2013. QTL-seq: Rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. The Plant Journal, 74, 174–183.

Teng W L, Feng W J, Zhang J Y, Xia N, Guo J, Li W, Wu D P, Zhao X, Han Y P. 2017. Identification of quantitative trait loci underlying lutein content in soybean seeds across multiple environments. Journal of Agricultural Science, 155, 1–9.

Wang J, Mao L, Zeng Z, Yu X, Lian J, Feng J, Yang W, An J, Wu H, Zhang M, Liu L. 2021. Genetic mapping high protein content QTL from soybean ‘Nanxiadou 25’ and candidate gene analysis. BMC Plant Biology, 21, 388.

Wang J, Niu G, Guo Q, Liu L. 2022. Production and structural characterization of the cytochrome P450 enzymes in carotene ring hydroxylation. Methods in Enzymology, 671, 223–241.

Wang K, Li M, Hakonarson H. 2010. ANNOVAR: Functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Research, 38, e164.

Wang R K, Wang C E, Fei Y Y, Gai J Y, Zhao T J. 2013. Genome-wide identification and transcription analysis of soybean carotenoid oxygenase genes during abiotic stress treatments. Molecular Biology Reports, 40, 4737–4745.

Wang Z, Yu A, Li F, Xu W, Han B, Cheng X, Liu A. 2021. Bulked segregant analysis reveals candidate genes responsible for dwarf formation in woody oilseed crop castor bean. Scientific Reports, 11, 6277.

Watanabe S, Tsukamoto C, Oshita T, Yamada T, Anai T, Kaga A. 2017. Identification of quantitative trait loci for flowering time by a combination of restriction site-associated DNA sequencing and bulked segregant analysis in soybean. Breeding Science, 67, 277–285.

Whent M, Ha J, Slavin M, Zhou M, Song J, Kenworthy W I, Yu L L. 2009. Effect of genotype, environment, and their interaction on chemical composition and antioxidant properties of low-linolenic soybeans grown in Maryland. Journal of Agricultural and Food Chemistry, 57, 10163–10174.

Win K T, Vegas J, Zhang C, Song K, Lee S. 2017. QTL mapping for downy mildew resistance in cucumber via bulked segregant analysis using next-generation sequencing and conventional methods. Theoretical and Applied Genetics, 130, 199–211.

Wu S, Qiu J, Gao Q. 2019. QTL-BSA: A bulked segregant analysis and visualization pipeline for QTL-seq. Interdisciplinary Sciences (Computational Life Sciences), 11, 730–737.

Xing S, Zhu H, Zhou Y, Xue L, Wei Z, Wang Y, He S, Zhang H, Gao S, Zhao N, Zhai H, Liu Q. 2022. A cytochrome P450 superfamily gene, IbCYP82D47, increases carotenoid contents in transgenic sweet potato. Plant Science, 318, 111233.

Xu J, Wang X Y, Guo W Z. 2015. The cytochrome P450 superfamily: Key players in plant development and defense. Journal of Integrative Agriculture, 14, 1673–1686.

Ye J, Hu T, Yang C, Li H, Yang M, Ijaz R, Ye Z, Zhang Y. 2015. Transcriptome profiling of tomato fruit development reveals transcription factors associated with ascorbic acid, carotenoid and flavonoid biosynthesis. PLoS ONE, 10, e0130885.

Yin Y, Guo C, Shi H, Zhao J, Ma F, An W, He X, Luo Q, Cao Y, Zhan X. 2022. Genome-wide comparative analysis of the R2R3-MYB gene family in five Solanaceae species and identification of members regulating carotenoid biosynthesis in wolfberry. International Journal of Molecular Sciences, 23, 2259.

Zhang J, Panthee D R. 2020. PyBSASeq: A simple and effective algorithm for bulked segregant analysis with whole-genome sequencing data. BMC Bioinformatics, 21, 99.

Zhang S, Abdelghany A M, Azam M, Qi J, Li J, Feng Y, Liu Y, Feng H, Ma C, Gebregziabher B S, Ghosh S, Agyenim-boateng K G, Shaibu A S, Thet H, Htway P, Wu T, Li B, Qiu L, Sun J. 2023. Mining candidate genes underlying seed oil content using BSA-seq in soybean. Industrial Crops & Products, 194, 116308.

Zou C, Wang P, Xu Y. 2016. Bulked sample analysis in genetics,genomics and crop improvement. Plant Biotechnology Journal, 14, 1941–1955.

基于二代测序技术的集团分离分析方法鉴定大豆类胡萝卜素基因关联区间和候选基因

Identification of genomic regions and candidate genes underlying carotenoid accumulation in soybean using next-generation sequen-cing based bulk segregant analysis

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