甜瓜种皮颜色控制基因CmSC1的精细定位及候选基因分析

doi:10.3864/j.issn.0578-1752.2021.10.012

Abstract

Abstract:

【Objective】In this study, the genetic analysis and fine mapping of melon seed coat color gene were performed, and the candidate gene was isolated and the molecular marker developed, so as to provide a foundation for gene function and rational utilization. 【Method】The inbred line HP22 with white seed coat was crossed with the inbred line B8 and B150 with yellow seed coat, and the offspring population was obtained, respectively. The seed coat colors of offspring plants were investigated by visual inspection. The locus of gene was fine mapped through map-based cloning strategy, and the candidate target gene in the mapping region was determined by gene sequencing and function analysis of gene coding region. 【Result】The white seed coat was dominant to yellow, which was controlled by a single dominant gene locus, named CmSC1, showed delayed genetic characteristic. Using 368 yellow seed coat individuals from F₂ population, the target gene CmSC1 was delimited to a 95 kb interval flanked by markers S27 and S28 on chromosome 5 that contained twelve annotated open reading frames (ORFs). Among of them, the MELO3C014406 gene encoded a bHLH transcription factor protein which was homologous to the Arabidopsis AtTT8 gene. Sequence analysis of MELO3C014406 gene in the yellow seed coat inbred lines indicated that two types of sequence variation were identified. The first type was an A insertion at the 47th base pairs position downstream of ATG, and the other was a 14 bp deletion at the 260th base pair position downstream of ATG, which caused premature termination and produced truncated proteins, respectively. Furthermore, the sequencing analysis of 65 yellow seed coat accessions were detected by specific molecular marker YS, and the results showed that they all contained one mutation type of two variation types. These results suggested that the MELO3C014406 gene was an ideal candidate gene for CmSC1 controlling seed coat color.【Conclusion】In this study, the CmSC1 gene regulating seed coat color was finally mapped to a 95 kb interval, and the MELO3C014406 gene was presumed to be the candidate gene. In addition, a specific marker YS was developed based on the 14 bp deletion of MELO3C014406 gene.

Key words: melon (Cucumis melo L.), seed coat color, gene mapping, proanthocyanidins, basic helix-loop-helix transcription factor

MA Jian, LI CongCong, HUANG YaTing, XIE YuLi, CHENG LingLing, WANG JianShe. Fine Mapping and Candidate Gene Analysis of Seed Coat Color Gene CmSC1 in Melon[J].Scientia Agricultura Sinica, 2021, 54(10): 2167-2178.

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Figures/Tables 9

Table 1

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Table 2

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Table 5

Genotyping of the CmSC1 gene in 104 melon accessions"

编号No.	材料名称 Accession	种皮颜色（F₂） Seed coat color (F₂)	基因型 Genotype		编号 No.	材料名称 Accession	种皮颜色（F₂） Seed coat color (F₂)	基因型 Genotype
编号No.	材料名称 Accession	种皮颜色（F₂） Seed coat color (F₂)	ATG47	ATG260	编号 No.	材料名称 Accession	种皮颜色（F₂） Seed coat color (F₂)	ATG47	ATG260
1	HP22	白色 White	― ―	― ―	53	H30	黄色 Yellow	― ―	-14 bp
2	B8	黄色 Yellow	+A	― ―	54	H32	黄色 Yellow	― ―	-14 bp
3	B150	黄色 Yellow	― ―	-14 bp	55	H33	黄色 Yellow	― ―	-14 bp
4	B1	白色 White	― ―	― ―	56	H34	黄色 Yellow	― ―	-14 bp
5	B2	黄色 Yellow	+A	― ―	57	H35	黄色 Yellow	― ―	-14 bp
6	B3	白色 White	― ―	― ―	58	H36	黄色 Yellow	― ―	-14 bp
7	B4	白色 White	― ―	― ―	59	H37	黄色 Yellow	― ―	-14 bp
8	B5	白色 White	― ―	― ―	60	H38	黄色 Yellow	― ―	-14 bp
9	B6	黄色 Yellow	+A	― ―	61	H41	黄色 Yellow	― ―	-14 bp
10	B7	白色 White	― ―	― ―	62	H50	黄色 Yellow	― ―	-14 bp
11	B9	白色 White	― ―	― ―	63	H53	黄色 Yellow	― ―	-14 bp
12	B10	白色 White	― ―	― ―	64	H56	黄色 Yellow	― ―	-14 bp
13	B11	黄色 Yellow	+A	― ―	65	H58	黄色 Yellow	― ―	-14 bp
14	B12	白色 White	― ―	― ―	66	H61	黄色 Yellow	― ―	-14 bp
15	B13	黄色 Yellow	+A	― ―	67	H68	黄色 Yellow	― ―	-14 bp
16	B14	白色 White	― ―	― ―	68	H74	黄色 Yellow	― ―	-14 bp
17	B15	白色 White	― ―	― ―	69	H76	黄色 Yellow	― ―	-14 bp
18	B16	黄色 Yellow	+A	― ―	70	H81	黄色 Yellow	― ―	-14 bp
19	B17	白色 White	― ―	― ―	71	H82	黄色 Yellow	― ―	-14 bp
20	B18	白色 White	― ―	― ―	72	H84	黄色 Yellow	― ―	-14 bp
21	B19	白色 White	― ―	― ―	73	H93	黄色 Yellow	― ―	-14 bp
22	B20	白色 White	― ―	― ―	74	H98	黄色 Yellow	― ―	-14 bp
23	B21	白色 White	― ―	― ―	75	H99	黄色 Yellow	― ―	-14 bp
24	B22	黄色 Yellow	+A	― ―	76	H101	黄色 Yellow	― ―	-14 bp
25	B23	黄色 Yellow	+A	― ―	77	H105	黄色 Yellow	― ―	-14 bp
26	B24	黄色 Yellow	― ―	-14 bp	78	H107	黄色 Yellow	― ―	-14 bp
27	B27	白色 White	― ―	― ―	79	H109	黄色 Yellow	― ―	-14 bp
28	B29	黄色 Yellow	― ―	-14 bp	80	H118	黄色 Yellow	― ―	-14 bp
29	B31	白色 White	― ―	― ―	81	H161	黄色 Yellow	― ―	-14 bp
30	B32	白色 White	― ―	― ―	82	HP1	白色 White	― ―	― ―
31	B33	白色 White	― ―	― ―	83	HP2	白色 White	― ―	― ―
32	B35	黄色 Yellow	+A	― ―	84	HP3	白色 White	― ―	― ―
33	B36	白色 White	― ―	― ―	85	HP4	白色 White	― ―	― ―
34	B37	白色 White	― ―	― ―	86	HP5	黄色 Yellow	― ―	-14 bp
35	B38	白色 White	― ―	― ―	87	HP6	黄色 Yellow	― ―	-14 bp
36	B39	白色 White	― ―	― ―	88	HP7	黄色 Yellow	― ―	-14 bp
37	B53	黄色 Yellow	+A	― ―	89	HP8	黄色 Yellow	― ―	-14 bp
38	B224	黄色 Yellow	― ―	-14 bp	90	HP9	黄色 Yellow	― ―	-14 bp
39	B98	黄色 Yellow	+A	― ―	91	HP10	黄色 Yellow	― ―	-14 bp
40	B133	黄色 Yellow	+A	― ―	92	HP11	黄色 Yellow	― ―	-14 bp
41	B134	黄色 Yellow	+A	― ―	93	HP12	黄色 Yellow	― ―	-14 bp
42	B196	黄色 Yellow	+A	― ―	94	H71	黄色 Yellow	― ―	-14 bp
43	B198	黄色 Yellow	+A	― ―	95	H72	黄色 Yellow	― ―	-14 bp
44	B220	黄色 Yellow	+A	― ―	96	1520A	白色 White	― ―	― ―
45	B241	黄色 Yellow	+A	― ―	97	Z8	白色 White	― ―	― ―
46	H7	黄色 Yellow	+A	― ―	98	BZ1119	褐色 Brown	+A	― ―
47	H16	黄色 Yellow	+A	― ―	99	BZ2659	褐色 Brown	+A	― ―
48	H17	黄色 Yellow	― ―	-14 bp	100	3A832	白色 White	― ―	― ―
49	H19	黄色 Yellow	― ―	-14 bp	101	雪丽脆 Xuelicui	白色 White	― ―	杂合 Hybrid
50	H23	黄色 Yellow	― ―	-14 bp	102	瑞金 Ruijin	白色 White	― ―	杂合 Hybrid
51	H24	黄色 Yellow	― ―	-14 bp	103	冀州鲜 Jizhouxian	白色 White	― ―	杂合 Hybrid
52	H29	黄色 Yellow	― ―	-14 bp	104	火焰二号F₁F₁of Huoyan 2	白色 White	― ―	杂合 Hybrid

Table 5

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引物名称 Primer	正向引物 Forward primer (5'-3')	反向引物 Reverse primer (5'-3')
ID1716	TTCCACGAACTCAGGAGCTG	AGTAGCATGAGGCTAGACTTGA
S24	TGAACTCGTGTCTAACGTACCA	ACTCCACTCTCGTATCCAGT
S26	GCAATGGAGGTGAGTGCCAA	GCATGTCTTTGCCATGTTGTGT
S27	ATGACCAAACAATGGTGCTGTG	GCTTGTGTGAGAGTAAATCAAGGT
S2	AGAGGGAAGCCATCAAGCAA	ATTGTACATACTGTCTAGGGTTTCT
S28	AAACCATTGACACAAGCTCCA	ACATCACATGTATCAAGTGCCT
S29	ATTCCATTTTCGTCAAACAACTTTC	GTCCATCATGATCTATCGCAAAA
S30	GGCTTTCCTTTGTCAGATTCCA	CCAAAGTGGGGTAGGAGCATT
S33	GGTTTATGATGAAAACGACCGA	GAGGTGACGAGGTCCAAATAA
S34	CACCCACTTAGGGTTGAAGAA	GTTTGGAATTGAAGTACACACCT
S35	GAGCATTAAGACCAAAGACACAA	TAAGTAACGAGTGAGGTTGGG
ID3716	GCTACAAGCCATGTTGAACTCT	TGAAGAAGCGAGGAAAGAATAGG
YS	CTTATCCGCCTCCGACACC	AGTTAACGCACCCGAACCC
CmSC1	AGACCCATTTGTTTCACTTTCACC	CAGAGAAGCTTCCACTCCCA

亲本或组合 Parents or generation	白色种皮株数 White seed color individuals	黄色种皮株数 Yellow seed color individuals	分离比 Expected ratio	χ²_0.05值 χ²_0.05
HP22 (P₁)	5	0
B8 (P₂)	0	5
B150 (P₃)	0	5
F₁(P₁^♂×P₂^♀)	0	10
F₁(P₁^♀×P₂^♂)	10	0
F₁(P₁^♂×P₃^♀)	0	10
F₁(P₁^♀×P₃^♂)	10	0
F₁(P₂^♂×P₃^♀)	0	10
F₁(P₂^♀×P₃^♂)	0	10
F₂(P₁×P₂)	10	0
F₂(P₂×P₃)	0	10
F_2:3(P₁×P₂)	1029	368	3:1	1.41
F_2:3(P₁×P₃)	358	112	3:1	0.28
F_2:3(P₂×P₃)	0	115

预测基因编号 Predicted ORF code	基因名称 Gene name	预测基因功能 Putative function
1	MELO3C014412	铝激活的类苹果酸转运蛋白12 Aluminum-activated malate transporter 12-like
2	MELO3C014411	富含半胱氨酸的类受体激酶蛋白 Cysteine-rich receptor-kinase-like protein
3	MELO3C014408	类bHLH69转录因子 Transcription factor bHLH69-like isoform X1
4	MELO3C014407	醛糖/酮还原酶家族蛋白 Aldo/keto reductase family protein
5	MELO3C031122	碱性螺旋-环-螺旋转录因子 Basic helix-loop-helix transcription factor
6	MELO3C031072	碱性螺旋-环-螺旋转录因子 Basic helix-loop-helix transcription factor
7	MELO3C014405	未知功能蛋白 Unknown protein
8	MELO3C014409	未知功能蛋白 Unknown protein
9	MELO3C014403	蛋白前转位酶亚基SCY2 Preprotein translocase subunit SCY2
10	MELO3C014402	FANTASTIC FOUR 2蛋白 Protein FANTASTIC FOUR 2
11	MELO3C014401	硫胺素磷酸合成酶 Thiamine phosphate synthase
12	MELO3C014400	硫胺素磷酸合成酶 Thiamine phosphate synthase

基因名称 Gene	变异位点 Variation site
基因名称 Gene	DHL92	HP22	B8
MELO3C014412	2750429A (I65)	2750429C (L65)	2750429A (I65)
MELO3C014408	2765792-, 2766079A (T104)	2765792A (移码突变 Code shift mutation), 2766079G (A104)	2765792-, 2766079A (T104)
MELO3C014409	2755903T	2755903A (终止密码 Termination coden)	2755903T
MELO3C031122	2781790-	2781790-	2781790A (终止密码 Termination coden)

Fine Mapping and Candidate Gene Analysis of Seed Coat Color Gene CmSC1 in Melon

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