西瓜果肉柠檬黄色的遗传分析和基因定位

doi:10.3864/j.issn.0578-1752.2021.18.013

Abstract

Abstract:

【Objective】In order to find out the formation mechanism of watermelon canary yellow flesh color, the pigment composition, pigment contents, and the inheritance of white and canary yellow flesh watermelon color were studied in this study, and the candidate genes related to canary yellow flesh were predicted by BSA-seq. 【Method】Two six generations populations were constructed by crossing four materials, including Bingtangcui (white flesh) and Xihua (canary yellow flesh), as well as Sashengnaiyougua (white flesh) and Xinjinlanxuan (canary yellow flesh). High performance liquid chromatography (HPLC) was used to determine the carotenoid composition and content in flesh of four parents at four different development stages. Two BSA-seq populations (BSA-seq I and BSA-seq II) were initially located by using the bulked sergeant analysis (BSA), and then the candidate genes were screened according to the annotation information of watermelon reference genome ‘97103’ V2, and verified by real-time quantitative polymerase chain reaction (qRT-PCR).【Result】During the development of watermelon fruit, violaxanthin and lutein accumulated differently in both parents, and the content of violaxanthin was higher in canary yellow flesh than in white flesh. The content of violaxanthin in white and canary yellow flesh watermelon fruits were (10.96±4) μg·g ^-1DW and (22.84±2) μg·g^-1DW, respectively. The content of lutein in white and canary yellow flesh watermelon fruits were (2.23±1) μg·g^-1DW and (3.97±1) μg·g^-1DW, respectively. The content of violaxanthin was about 7 times of lutein. Two six generations segregation populations were analyzed, and the results showed that the flesh color of F₁ and Ⅰ BC₁P₁and Ⅱ BC₁P₁ populations were non-canary yellow, the segregation ratio of non-canary yellow and canary yellow in F₂ population were consistent with the Mendelian segregation ratio of 3:1, and the segregation ratio of non-canary yellow and canary yellow inⅠ F₁, Ⅱ F₁, Ⅰ BC₁P₂and Ⅱ BC₁P₂backcross populations were 1:1, so it could be concluded canary yellow was recessive to white in watermelon flesh. Through SNP and InDel association analysis of BSA-seq Ⅰ and BSA-seq Ⅱ data, the major locus which regulated canary yellow flesh color in watermelon fruit was mapped on chromosome 6 within a physical distance between 24.00 to 24.61 Mb, and there were 70 genes in this region. Combined with reference genomic annotation and qRT-PCR experiment, five genes that maybe related to canary yellow flesh color trait in watermelon fruit were obtained. Among them, Cla97C06G121680, Cla97C06G121700 and Cla97C06G121890 were all related to chloroplast biogenesis and chloroplast structure size. These three genes might affect fruit flesh color by affecting the formation of chromoplast; Cla97C06G121910 was an AP2 transcription factor in response to ethylene synthesis which related to watermelon fruit ripening, which might affect the accumulation of carotenoids in flesh by affecting fruit ripening and Cla97C06G122090 was described as a transmembrane transport effect, which played a role in the transmembrane transport of carotenoids.【Conclusion】The main pigments of white and canary yellow flesh watermelon were violaxanthin and lutein, and the pigment accumulation in canary yellow flesh was significantly higher than white flesh watermelon. Genetic analysis showed that canary yellow was recessive to white in watermelon flesh color. Based on BSA-seq analysis, a major locus was located in the region of 24.00 Mb to 24.61 Mb on chromosome 6, while Cla97C06G121680, Cla97C06G121700, Cla97C06G121890, Cla97C06G122090 and Cla97C06G121910 were predicted as candidate genes which related to canary yellow formation in watermelon flesh.

Key words: watermelon, flesh, canary yellow, carotenoids, gene mapping

DIAO WeiNan,YUAN PingLi,GONG ChengSheng,ZHAO ShengJie,ZHU HongJu,LU XuQiang,HE Nan,YANG DongDong,LIU WenGe. Genetic Analysis and Gene Mapping of Canary Yellow in Watermelon Flesh[J].Scientia Agricultura Sinica, 2021, 54(18): 3945-3958.

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References 44

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引物名称 Primer name	引物序列 Sequence of primer	葫芦科基因组数据库登录号 Cucurbits genomics database accession No.
G1	F: TTGCTGGATTCTTTGCAGCAT R: GCTGCTCGTCTCAATTCCAC	Cla97C06G121680
G2	F: CAGGGCAGAGTTTGATGGCT R: CTTCTGATGTGCGTCCTGGT	Cla97C06G121700
G3	F: GGCAATGGAAATATGACAGTGCT R: GTGCTTGTACACAGGGACCA	Cla97C06G121890
G4	F: TACATCGGGTCGTTGAGAAGAA R: GCGTCGCATATGTTCCAAGA	Cla97C06G121910
G5	F: TCAATTCAGTCTGGGTGCTG R: TATCCAACACTCCACCCTTGC	Cla97C06G122090
G6	F: ACTCTGCAAGGAGAAATGGCA R: AATTAACGATGGCACTGGCG	Cla97C06G122110
CIACT	F: CCTACAACTCAATTATGAAGTGTG R: GAAATCCACATCTGCTGGAAGGTG	Cla97C02G026960

世代 Generation	植株总数 Number of total plants	非柠檬黄色果肉 Non-canary yellow flesh	柠檬黄色果肉 Canary yellow flesh	非柠檬黄色果肉﹕柠檬黄色果肉 Non-canary yellow flesh﹕Canary yellow flesh	卡方值 χ² value
Ⅰ P₁冰糖脆×Ⅰ P₂ 喜华 Ⅰ P₁ Bingtangcui×Ⅰ P₂Xihua
Ⅰ P₁	8	8	-
Ⅰ P₂	8	-	8
Ⅰ F₁	10	10	-
Ⅰ F₂	156	115	41	2.80:1	0.13
Ⅰ BC₁P₁	51	51	-
Ⅰ BC₁P₂	104	56	48	1.16:1	0.62
Ⅱ P₁ 萨省奶油瓜×Ⅱ P₂ 新金兰选 Ⅱ P₁Sashengnaiyougua×Ⅱ P₂Xinjinlanxuan
Ⅱ P₁	10	10	-
Ⅱ P₂	8	-	8
Ⅱ F₁	10	10	-
Ⅱ F₂	343	256	87	2.94:1	0.02
Ⅱ BC₁P₁	81	81	-
Ⅱ BC₁P₂	77	39	38	1.02:1	0.03

BSA测序 BSA-seq	染色体 Chromosome_ID	开始 Start	结束 End	大小 Size (Mb)	基因数目 Gene numbers
BSA-seq Ⅰ	Cla97Chr06	24000000	24610000	0.610	70
BSA-seq Ⅱ	Cla97Chr06	23800000	26330000	2.530	264

基因Gene ID	基因注释 Gene annotation
Cla97C06G121680	APO蛋白2 APO protein 2
Cla97C06G121700	核酸相关蛋白,叶绿体 Nucleoid-associated protein, chloroplastic
Cla97C06G121890	TPR类超家族蛋白亚型1 Tetratricopeptide repeat (TPR) like superfamily protein isoform 1
Cla97C06G121910	AP2类乙烯响应转录因子家族 AP2 like ethylene-responsive transcription factor family
Cla97C06G122090	TET 8 Tetraspanin-8-like
Cla97C06G122110	WAT 1相关蛋白 Wall are thin 1-WAT 1 related protein

Genetic Analysis and Gene Mapping of Canary Yellow in Watermelon Flesh

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功能注释数据库 Annotated database	注释基因数目 Annotated gene numbers	存在非同义突变的基因数 Number of genes with nonsynonymous mutations	存在移码突变的基因数 Number of genes with frameshift mutations
NT	69	8	1
NR	54	6	1
trEMBL	54	6	1
SwissProt	35	5	1
GO	30	2	1
KEGG	15	2	1
COG	20	1	0
合计 Total	70	8	1

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