柚果肉颜色遗传变异分析及候选基因挖掘

doi:10.3864/j.issn.0578-1752.2023.17.015

摘要/Abstract

摘要： 【目的】 果肉颜色是柚类品种重要的外观性状以及品质性状，挖掘与柚果肉颜色显著相关的变异位点及相关基因，为进一步理解柚类品种果肉呈色机理以及分子标记辅助育种奠定基础。【方法】 通过表型调查及色差仪测量，对100份柚类种质的果肉颜色进行评价和分类，利用GBS（genotyping-by-sequencing）测序技术对所有材料进行简化基因组测序。将测序得到的基因型数据通过GCTA软件计算特征值及特征向量分析群体结构，利用plink 2.0软件计算不同果肉颜色群体的遗传分化系数（Fst），选用GEMMA软件中GLM模型进行全基因组关联分析，选取与颜色表型显著关联的变异位点进行等位变异分析，根据柑橘LD大小对变异位点邻近位置进行基因注释，筛选出与果肉颜色形成相关的候选基因，随机选择4份白肉柚和4份红肉柚材料在不同发育时期对候选基因表达进行初步验证。【结果】 根据果肉颜色将100份柚类种质分为白肉柚和红肉柚两大类，其中包含58份白肉柚和42份红肉柚。通过Fst遗传分化指数分析和GWAS全基因组关联分析共筛选到Fst系数大于0.4且-log₁₀（P）>9的位点6个，对6个变异位点进行基因分型，根据基因型能够预测柚品种的果肉颜色。对变异位点侧翼25 kb序列进行基因注释共筛选出14个基因，其中包含类异戊二烯合成、叶绿体氧化还原反应、质体发育、脱落酸信号调节、乙烯响应等相关基因和转录因子。基因表达分析显示，Cg7g013760（促分裂原活化蛋白激酶激酶激酶MAPKKK17）、Cg7g013840（叶绿体卟啉原脱氨酶）、Cg7g014020（转录因子TCP7）、Cg7g014120（叶绿体超氧化物歧化酶SOD3）、Cg7g014190（FAD合成酶2）与果肉颜色形成存在一定关联。【结论】 经过GWAS和Fst两种分析方式共鉴定到6个与果肉颜色显著关联的SNP位点，对该位点上下游25 kb的距离进行基因注释共筛选到了14个基因，通过基因表达模式分析初步判定其中5个基因参与了柚果肉颜色的形成。

关键词: 柚, 果肉颜色, Fst, 关联分析, 候选基因

Abstract:

【Objective】 The flesh color is an important appearance and quality trait of pomelo varieties. The aim of this study was to excavate variation loci and genes significantly related to the flesh color of pomelo varieties, so as to lay a foundation for further understanding the flesh color development mechanism and marker-assisted breeding of pomelo varieties. 【Method】 100 pomelo accessions were used to identify their flesh color with a color difference meter, and classified based on their phenotype results. The simplified genome sequencing of 100 pomelo accessions was carried out using GBS (genotyping-by-sequencing) technology. The eigenvalues and eigenvectors of the genotype data obtained from sequencing were calculated by GCTA software to analyze the population structure, and the genetic differentiation index (Fst) of two different flesh color populations were calculated using Plink 2.0 software. The GLM model in GEMMA software was adopted to perform genome-wide association analysis, and the variation loci significantly associated with color phenotype were identified for further allelic variation analysis. According to the citrus linkage disequilibrium (LD) distance, the candidate genes located in the 25 kb adjacent region around the variation loci that might be related to the forming of flesh color were screened out, four white and four red pomelo accessions were randomly selected to perform qPCR to obtain the candidate genes expression at different fruit development stages. 【Result】 According to the phenotype of flesh color, 100 accessions were divided into two categories corresponding to white and red pomelo accessions, including 58 white flesh pomelos and 42 red flesh pomelos. Six SNP sites with Fst index greater than 0.4 and -log₁₀(P)>9 were screened out after Fst genetic differentiation analysis and GWAS genome-wide association analysis. The genotypes of six variation SNPs sites were extracted from 100 pomelos accessions and the genotypes of these SNPs could be used to predict the flesh color of pomelos. A total of 14 genes that function annotated as isoprene synthesis, plastid development, abscisic acid signal regulation, ethylene response related genes and transcription factors were screened and selected. Genes expression analysis showed that Cg7g013760 (mitogen-activated protein kinase kinase MAPKKK17), Cg7g013840 (chloroplast porphyrinogen deaminase), Cg7g014020 (transcription factor TCP7), Cg7g014120 (chloroplast superoxide dismutase SOD3), Cg7g014190 (FAD synthetase 2) were related to the forming of flesh color in pomelos. 【Conclusion】 A total of 6 SNP sites significantly associated with flesh color in pomelos accessions were identified by GWAS and Fst analysis. A total of 14 genes within the 25 kb regions around the mutation SNPs site were screened and annotated. The gene expression analysis showed that five genes might be involved in the forming of flesh color in pomelo accessions.

Key words: pomelo, flesh color, Fst, GWAS, candidate genes

程莉, 杨胜男, 朱延松, 王旭, 赵婉彤, 李仁静, 李沛, 苑忠杰, 江东. 柚果肉颜色遗传变异分析及候选基因挖掘[J]. 中国农业科学, 2023, 56(17): 3420-3434.

CHENG Li, YANG ShengNan, ZHU YanSong, WANG Xu, ZHAO WanTong, LI RenJing, LI Pei, YUAN ZhongJie, JIANG Dong. Genetic Variation Analysis and Candidate Genes Mining of Regulating Flesh Color in Pomelo[J]. Scientia Agricultura Sinica, 2023, 56(17): 3420-3434.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2023.17.015

https://www.chinaagrisci.com/CN/Y2023/V56/I17/3420

图/表 14

表1

100份柚果肉颜色统计"

编号 Code	材料名称 Accession name	果肉颜色 Flesh color	编号 Code	材料名称 Accession name	果肉颜色 Flesh color
1	暹罗低酸柚 Siamese Acidless Pomelo	白 White	36	垫江黄沙白心柚 Dian Jiang Huang Sha Bai Pomelo	白 White
2	卫寺蜜柚 Wei Si Honey Pomelo	白 White	37	暹罗蜜柚 Xian Luo Mi Pomelo	白 White
3	白玉霜 Bai Yu Shuang Pomelo	白 White	38	晚白柚 Wan Bai Pomelo	白 White
4	晚白柚 Wan Bai Pomelo	白 White	39	安江香柚 An Jiang Xiang Pomelo	白 White
5	杭晚蜜柚实生2 Hang Wan Mi You Seedling No.2	白 White	40	水晶文旦 Suisyo Buntan	白 White
6	高浦柚 Kao Phuang Pomelo	白 White	41	垫江沙田柚 Dian Jiang Sha Tian You	白 White
7	琯溪蜜柚 Guan Xi Honey Pomelo	白 White	42	宜安倭柚 Yi An Wo You	白 White
8	福建文旦 Wen Dan Pomelo	白 White	43	脆柚 Cui You	白 White
9	沙田柚 Sha Tian You	白 White	44	垫江曾家白心柚 Dian Jiang Zeng Jia Bai You	白 White
10	东风早柚 Dong Feng Zao Pomelo	白 White	45	段氏柚 Duan Shi You	白 White
11	刘氏柚 Liu Shi Pomelo	白 White	46	岭南沙田柚 Ling Nan Sha Tian Pomelo	白 White
12	梁平柚1号 Liang Ping Pomelo No.1	白 White	47	14-4柚 Pomelo 14-4	白 White
13	岭南沙田柚 Ling Nan Sha Tian Pomelo	白 White	48	长寿沙田柚 Chang Shou Sha Tian You	白 White
14	通贤柚 Tong Xian Pomelo	白 White	49	古老钱沙田柚 Gu Lao Qian Sha Tian Pomelo	白 White
15	垫江白柚杂种-3 Hybrid of Dian Jiang Pomelo No.3	白 White	50	盘谷文旦 Pan Gu Wen Dan Pomelo	白 White
16	左氏柚 Zuo Shi Pomelo	白 White	51	太极图柚 Tai Ji Tu You	白 White
17	垫江白柚 Dian Jiang White Flesh Pomelo	白 White	52	古巴柚 Guba Pomelo	白 White
18	30-16柚 Pomelo 30-16	白 White	53	白玉霜 Bai Yu Shuang Pomelo	白 White
19	28-11柚 Pomelo 28-11	白 White	54	金沙柚 Jing Sha Pomelo	白 White
20	陈氏柚 Chen Shi You	白 White	55	无核沙田柚 Seedless Sha Tian You	白 White
21	华蓥山香柚 Hua Ying Shan Fragrant Pomelo	白 White	56	琯溪蜜柚 Guan Xi Honey Pomelo	白 White
22	段氏柚 Duan Shi You	白 White	57	玉环文旦柚 Yu Huan Wen Dan	白 White
23	泰国柚（暹罗低酸柚）Tailand Pomelo	白 White	58	安江无核蜜柚 An Jiang Seedless Honey Pomelo	白 White
24	玉环柚 Yu Huan Pomelo	白 White	59	垫江红心杂种3号 Dian Jiang Red Flesh pomelo Hybrid 3	红 Red
25	蒲莲柚(子代) Pu Lian Pomelo (Offspring tree)	白 White	60	垫江红心杂种2号 Dian Jiang Red Flesh pomelo Hybrid 2	红 Red
26	蒲莲柚（母树） Pu Lian Pomelo (Mother Tree)	白 White	61	红肉琯溪蜜柚 Red Flesh Mi You	红 Red
27	彭氏柚 Peng Shi pomelo	白 White	62	红琯溪蜜柚 Red Guan Xi Honey Pomelo	红 Red
28	越南小柚 Vietnam Small Pomelo	白 White	63	贡水红柚 Gong Shui Hong You	红 Red
29	合江柚 He Jiang You	白 White	64	平阳柚 Duong Da Lang Pomelo	红 Red
30	安江橙（安江香柚） An Jiang Cheng	白 White	65	菊花芯柚 Ju Hua Xin Pomelo	红 Red
31	正形沙田柚 Normal Shape Sha Tian You	白 White	66	光皮柚 Smooth Peel Pomelo	红 Red
32	梁砂柚 Liang Sha You	白 White	67	延城柚 Yan Cheng You	红 Red
33	北碚柚 Bei Bei Pomelo	白 White	68	23-13柚 Pomelo 23-13	红 Red
34	垫江周家白心柚 Dian Jiang Zhou Jia Bai You	白 White	69	22-13柚 Pomelo 22-13	红 Red
35	梅塆柚 Mei Wan Pomelo	白 White	70	强德勒柚-1 Chandler Pomelo No.1	红 Red
71	勐仑早柚 Meng Lun Early Pomelo	红 Red	86	彭县暹罗柚 Peng Xian Xian Luo You	红 Red
72	龙安柚 Long An Pomelo	红 Red	87	强德勒柚-2 Chandler Pomelo No.2	红 Red
73	红绵蜜柚 Hong Mian Honey Pomelo	红 Red	88	锅魁柚 Guo Kui Pomelo	红 Red
74	红绵蜜柚 Hong Mian Honey Pomelo	红 Red	89	江津红心柚 Jiang Jin Red Pomelo	红 Red
75	安农一号香柚 An Nong Xiang Pomelo No.1	红 Red	90	坪山柚 Ping Shan Pomelo	红 Red
76	垫江红心柚 Dian Jiang Hong Pomelo	红 Red	91	39-6柚 Pomelo 39-6	红 Red
77	29-12柚 Pomelo 29-12	红 Red	92	五布红心柚 Wu Bu Pomelo	红 Red
78	14-1柚 Pomelo 14-1	红 Red	93	毛化红柚 Mao Hua Hong You	红 Red
79	三红蜜柚 San Hong Honey Pomelo	红 Red	94	勐仑早柚 Meng Lun Early Pomelo	红 Red
80	红肉蜜柚 Red Flesh Mi You	红 Red	95	大庸菊花芯柚-1 Ju Hua Xin Pomelo No.1	红 Red
81	8088柚 8088 Pomelo	红 Red	96	金兰柚 Jin Lan Pomelo	红 Red
82	四季抛柚 Si Ji Pao You	红 Red	97	纳溪樱桃柚 Na Xi Ying Tao Pomelo	红 Red
83	14-7柚 Pomelo 14-7	红 Red	98	龙安柚 Long An Pomelo	红 Red
84	安农1号香柚 An Nong No.1 Pomelo	红 Red	99	大庸菊花芯柚-2 Ju Hua Xin Pomelo No.2	红 Red
85	缅甸柚 Burma Pomelo	红 Red	100	红肉柚 Red flesh Pomelo	红 Red

表1

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编号 Code	基因 Gene	正向引物序列 Forward primer sequences	反向引物序列 Reverse primer sequences
1	Cg7g013650	GCGGCAACAACAAACCATCT	CGTCCCGATCGGCTTATCAA
2	Cg7g013760	GTTGCCCTTTTGTGCTGGAG	GAACAACAGTTTCGGGTGCC
3	Cg7g013780	TTGGGTCAAGCTGCATCCAT	GCTTGCCCACTATACCCTCC
4	Cg7g013840	ATTGGTGGGAAGGGCTTGTT	CTTTCATCGAGTGCACAGCG
5	Cg7g013890	CATGCTCTTCCGGATACTACCC	ATCAATGCCAAAGAGCCACCTA
6	Cg7g013910	GGCGTCATCAAACCTGGAGA	TTGCCCTAGACCAACTGCAG
7	Cg7g014000	GCCCAGGAATACGGAGACAG	TCCTGCTTTCCGGAACTTCC
8	Cg7g014020	CCTCCTGGTAATGGCGTGTT	AGGGCTTATGTTCCAACGCA
9	Cg7g014120	CCACCTAGTACTCGTCTTCTGC	CGATGAAGCTCTCCCCAATGTA
10	Cg7g014130	GCTGGTTGGGCTTTCACGTA	AAACTCCTGGCCACCGAATT
11	Cg7g014190	AATGCCTACTCTCCCCAGCT	ACTATCCCTCCTGCCACTGA
12	Cg7g014220	AGCTCCAGTCATTTGCCGAA	ACTCGGCCGATGACTGATTC
13	Cg7g014430	GTCGTTTCGTCCGTGAGATT	AGGGGTTTTGGCTTTTGACT
14	Cg7g014530	ACTCCCCTTTCAAACACCCC	CCGGACGGTGAGAATGAAGT

果肉性状 Pulp color trait	颜色参数 Color index	最小值 Min.	最大值 Max.	平均值 Mean	标准差 SD	变异系数 CV (%)
白肉 White pulp	L	46.00	77.00	59.68	7.92	13.26
	A	-2.00	10.00	3.29	2.58	78.37
	B	3.25	20.25	11.26	3.42	30.42
	C	6.14	45.97	18.20	9.40	51.60
	CI	-3.27	29.81	20.88	15.29	73.21
红肉 Red pulp	L	29.50	65.25	48.11	7.55	15.69
	A	8.75	39.50	22.19	8.82	39.73
	B	4.75	29.50	13.68	5.08	37.15
	C	13.10	45.97	26.59	8.74	32.86
	CI	18.37	65.00	35.42	9.70	27.38

编号 Code	染色体 Chromosome	物理位置 Physical position (bp)	遗传分化系数 Fst	基因型 Genotype
1	7	15825706	0.43	A/C
2	7	15987387	0.43	C/T
3	7	15795993	0.42	C/T
4	7	15755408	0.42	C/T
5	7	16111951	0.41	C/T
6	7	15744770	0.40	G/A

编号 Code	染色体 Chrmosome	位置 Physical position (bp)	-log₁₀(P)值 -log₁₀(P) value	基因型 Genotype
1	7	15825706	13.62	A/C
2	7	15932621	12.00	A/C
3	7	4757754	10.69	A/C
4	7	15987387	10.69	C/T
5	7	15795993	10.21	C/T
6	7	16560622	10.14	A/C
7	7	15755408	9.87	C/T
8	7	15816519	9.60	G/T
9	7	15744770	9.60	G/A
10	7	16111951	9.57	C/T
11	7	5240390	9.43	C/A
12	7	15286146	9.30	A/C

编号 Code	基因 Gene	基因位置 Gene position	基因注释 Gene annotation
1	Cg7g013650	15559599...15563683	4-羟基苯甲酸聚壬基转移酶4-hydroxybenzoate polyprenyltransferase
2	Cg7g013760	15674549…15675866	丝裂原活化蛋白激酶激酶激酶17 Mitogen-activated protein kinase kinase kinase 17 (MAPKKK17)
3	Cg7g013780	15693273…15695508	线粒体ATP依赖性蛋白CLPP2 ATP-dependent Clp protease proteolytic subunit 2
4	Cg7g013840	15729956…15733863	胆色素原脱氨酶PBGD Porphobilinogen deaminase
5	Cg7g013890	15751668…15754112	伴侣蛋白dnaJ C76 Chaperone protein dnaJ C76
6	Cg7g013910	15765878…15770273	琥珀酰辅酶A Succinate-CoA ligase
7	Cg7g014000	15830671…15833556	硫氧还蛋白X Thioredoxin X
8	Cg7g014020	15845810…15846907	转录因子TCP7 Transcription factor TCP7
9	Cg7g014120	15926265…15929264	超氧化物歧化酶SOD3 Superoxide dismutase [Fe] 3
10	Cg7g014130	15929785…15930656	蛋白质S-异丙基半胱氨酸O-甲基转移酶A Protein-S-isoprenylcysteine O-methyltransferase A
11	Cg7g014190	15964951...15968470	FAD合成酶2 FAD synthetase 2
12	Cg7g014220	15980384...15986623	叶绿体外膜蛋白80 Outer envelope protein 80
13	Cg7g014430	16176520…16177533	ERF119乙烯应答转录因子 Ethylene-responsive transcription factor
14	Cg7g014530	16270001…16274137	丙酮酸脱氢酶复合物的二氢脂赖氨酸残基乙酰转移酶组分4 Dihydrolipoyllysine-residue acetyltransferase component 4 of pyruvate dehydrogenase complex