大豆红色种皮的色素鉴定和基因定位

doi:10.3864/j.issn.0578-1752.2023.14.002

Abstract

Abstract:

【Objective】To identify the key genes controlling anthocyanin synthesis and accumulation, to uncover changes in anthocyanin content of the seed coat during seed development, and the primary anthocyanin components responsible for the red seed coat of Taixingaijiaohong (TXAJH); and to lay the groundwork for a thorough understanding of the regulatory mechanism of red seed coat formation.【Method】Using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-ESI-MS/MS), the anthocyanin composition and concentration of the yellow seed coat of soybean Suinong 14 (SN14) and the red seed coat of soybean TXAJH at various developmental stages were identified. The potential areas of red testa-related genes were first identified using bulked segregant analysis (BSA) on the recombinant inbred lines (RILs) made by crossing SN14 and TXAJH. Based on this discovery, we performed marker linkage analysis to restrict the candidate intervals and predict the candidate genes, and qRT-PCR to confirm the expression of the anticipated candidate genes.【Result】When seed coats from the four developmental phases of SN14 and TXAJH were analyzed, a total of 12 anthocyanins were discovered. Cluster analysis of total anthocyanins revealed substantial changes in the seed coat's anthocyanin composition between TXAJH and SN14 as well as between TXAJH before and after color development. The anthocyanin content of the SN14 seed coat gradually decreased as the seed developed, whereas the TXAJH seed coat's content increased quickly and remained stable. After the development of the seed coat's color, the anthocyanin contents of SN14 and TXAJH showed highly significant differences, and at the mature stage, the TXAJH seed coat's anthocyanin content was more than 200 times that of SN14. The crimson coloring of the TXAJH seed coat was largely due to cyanidin-3-O-glucoside (Cy-3-glu), peonidin-3-O-glucoside (Pn-3-glu), and petunidin-3-O-glucoside (Pt-3-glu). The candidate interval for the red seed coat gene on chromosome 8 was discovered at 8.66 Mb by BSA-seq association analysis. 27 polymorphic markers were used in the marker linkage analysis, which produced 10 haplotypes and reduced the candidate interval to 702 kb. Nonsynonymous variations in 37 genes between the parents were found during this interval, these include the genes for encode the anthocyanin reductase 1 (Glyma.08g062000), the bHLH transcription factor (Glyma.08g061300 and Glyma.08g063900), and the MYB transcript factor (Glyma.08g059900). These genes may be involved in regulating the biosynthesis of anthocyanins, and anthocyanin reductase 1 can convert anthocyanins to proanthocyanidins (PA). The results of gene expression analysis revealed that candidate genes and genes related to the anthocyanin biosynthesis pathway had comparable expression patterns in SN14 and TXAJH, and both were expressed at lower levels in SN14 and at higher levels in TXAJH. It was discovered that there was a significant link between the principal constituents of seed coat anthocyanins and the level of candidate gene expression.【Conclusion】The anthocyanin makeup of SN14 and TXAJH's seed coats differed, and Cy-3-glu, Pn-3-glu, and Pt-3-glu may be to blame for the TXAJH's seed coat's red hue. According to predictions, Glyma.08g059900, Glyma.08g061300, Glyma.08g062000, and Glyma.08g063900 will likely be a candidate gene for the red seed coat, in which Glyma.08g059900, Glyma.08g061300, and Glyma.08g063900 may control a number of anthocyanin biosynthesis pathway genes.

Key words: soybean, seed coat color, anthocyanin, BSA-seq, gene mapping

CAO Jie, GU YongZhe, HONG HuiLong, WU HaiTao, ZHANG Xia, SUN JianQiang, BAO LiGao, QIU LiJuan. Pigment Identification and Gene Mapping in Red Seed Coat of Soybean[J].Scientia Agricultura Sinica, 2023, 56(14): 2643-2659.

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Figures/Tables 10

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Table 1

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Table 4

Candidate interval genes and functional notes"

基因编号 Gene ID	位置 Location (bp)	功能注释 Function
Glyma.08g058100	Chr.08: 4460549-4465659	单链核糖核酸内切酶Single-stranded-nucleate endonuclease
Glyma.08g059300	Chr.08: 4530151-4536884	吡啶核苷酸-二硫化物氧化还原酶Pyridine nucleotide-disulphide oxidoreductase
Glyma.08g059800	Chr.08: 4601723-4608768	囊泡相关蛋白Vesicle-associated protein
Glyma.08g059900	Chr.08: 4616457-4618234	MYB转录因子MYB transcription factor
Glyma.08g060400	Chr.08: 4648226-4658991	PLAC8蛋白家族PLAC8 family
Glyma.08g060500	Chr.08: 4664282-4669458	酪氨酸蛋白激酶Tyrosine protein kinase
Glyma.08g060600	Chr.08: 4673233-4676958	含五肽重复的蛋白质Proteins containing pentapeptide repeats
Glyma.08g060800	Chr.08: 4681172-4690671	苏氨酸激酶Threonine kinase
Glyma.08g060900	Chr.08: 4691761-4694059	苏氨酸蛋白激酶Threonine-protein kinase
Glyma.08g061000	Chr.08: 4694260-4698526	酪氨酸蛋白激酶Tyrosine protein kinase
Glyma.08g061100	Chr.08: 4708531-4710950	含U-BOX结构域的蛋白质8 U-box domain-containing protein 8
Glyma.08g061200	Chr.08: 4718616-4721895	NAD(P)H脱氢酶NAD(P)H dehydrogenase
Glyma.08g061300	Chr.08: 4731510-4735504	bHLH转录因子bHLH transcription factor
Glyma.08g061500	Chr.08: 4745228-4747004	PPR重复系列PPR repeat
Glyma.08g061600	Chr.08: 4751073-4754299	橙花醇合酶Nerolidol synthase
Glyma.08g062000	Chr.08: 4783724-4786913	花青素还原酶1 Anthocyanidin reductase 1
Glyma.08g062400	Chr.08: 4805184-4806757	--
Glyma.08g062500	Chr.08: 4809724-4816932	驱动蛋白Kinesin
Glyma.08g062600	Chr.08: 4817430-4821371	安全家族蛋白Safe family protein
Glyma.08g062700	Chr.08: 4829862-4832511	酪氨酸蛋白激酶Tyrosine protein kinase
Glyma.08g062800	Chr.08: 4833755-4836533	苏氨酸蛋白激酶Threonine-protein kinase
Glyma.08g062900	Chr.08: 4837205-4840140	酪氨酸蛋白激酶Tyrosine protein kinase
Glyma.08g063200	Chr.08: 4848292-4850826	蛋氨酸转氨酶Methionine transaminase
Glyma.08g063300	Chr.08: 4851595-4856852	蛋氨酸转氨酶Methionine transaminase
Glyma.08g063600	Chr.08: 4883287-4887296	MuDR家族转座酶MuDR family transposase
Glyma.08g063700	Chr.08: 4888519-4892619	角鲨烯单加氧酶Squalene monooxygenase
Glyma.08g063800	Chr.08: 4897354-4900790	L-苹果酸脱氢酶L-Malate dehydrogenase
Glyma.08g063900	Chr.08: 4905921-4906926	bHLH转录因子bHLH transcription factor
Glyma.08g064000	Chr.08: 4910549-4911502	--
Glyma.08g064200	Chr.08: 4915590-4916430	--
Glyma.08g064300	Chr.08: 4917314-4927545	蛋白质S-酰基转移酶Protein S-acyltransferase
Glyma.08g064400	Chr.08: 4927906-4929447	--
Glyma.08g064500	Chr.08: 4932437-4940287	鞘脂转运蛋白同源物Sphingolipid transporter spinster homolog
Glyma.08g064600	Chr.08: 4943335-4953605	K⁺外向逆流转运蛋白K⁺ efflux antiporter
Glyma.08g064700	Chr.08: 4956340-4959142	鸟氨酸环脱氨酶相关Ornithine cyclodeaminase-related
Glyma.08g064800	Chr.08: 4963573-4966706	蛋白质毛状体双折射Protein trichome birefringence-like
Glyma.08g065000	Chr.08: 4977696-4979377	PB1域PB1 domain

Table 4

Fig. 6

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样品 Sample	清洁read对数 Clean reads	质量值>30碱基 Q30 (%)	可定位清洁read Mapped (%)	平均覆盖深度 Ave depth	至少覆盖1次的碱基 Cov≥1× (%)
SN	73658060	92.50	99.03	16	97.94
TX	74839493	93.45	99.19	19	97.75
Y	110060520	94.05	99.25	29	99.03
R	109932563	93.05	99.41	30	99.03

关联分析类型 Type of correlation analysis	候选区域位置 Location of candidate areas (bp)	区域长度 Size of area (Mb)	基因总数 Gene number
SNP-ED	Chr.08: 3500000-12530000	9.03	1174
SNP-index	Chr.06: 32790000-32800000	0.01	1
	Chr.06: 32940000-33020000	0.08	2
	Chr.08: 3530000-12280000	8.75	1138
SNP分析结果的交集 Intersection of the results of SNP analyses	Chr.08: 3530000-12280000	8.75	1138
InDel-ED	Chr.08: 3290000-12530000	9.24	1201
InDel-index	Chr.08: 3510000-12190000	8.68	1132
InDel分析结果的交集 Intersection of the results of InDel analyses	Chr.08: 3510000-12190000	8.68	1132
所有关联分析结果的交集 Intersection of all correlation analysis results	Chr.08: 3530000-12190000	8.66	1127

Pigment Identification and Gene Mapping in Red Seed Coat of Soybean

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