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Quantitative Trait Loci Identification Uncovers Zinc Finger Protein CONSTANS-LIKE 4 as the Key Candidate Gene of Stigma Color in Watermelon (Citrullus lanatus)
Shuang Pei1, 2, Zexu Wu1, 2, Ziqiao Ji1, 2, Zheng Liu3, Zicheng Zhu1,  2#, Feishi Luan1,  2#, Shi Liu1, 2# 

1 Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Northeast Region), Ministry of Agriculture and Rural Affairs, Northeast Agricultural University, Harbin 150030, China

2 College of Horticulture and Landscape Architecture, Northeast Agricultural University, Harbin 150030, China

3Institute of Rural Revitalization Science and Technology, Heilongjiang Academy of Agricultural Sciences, Harbin 150028, China

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摘要  柱头颜色是西瓜重要的农艺性状,在传粉过程中起着重要作用。然而,迄今为止有关这方面的研究报道较少。本研究通过对ZXG1553P1,橙色柱头)和W1-17P2,黄色柱头)F2群体遗传分析表明,柱头颜色是一个数量性状,黄色对橙色表现为显性。BSA-seq (Bulk seq analysis sequencing) 测序分析发现在6号染色体3.75 Mb的区域存在与柱头颜色相关的染色体片段,并在CAPS标记Chr06_8338913Chr06_9344593之间检测到一个主效QTL位点Clqsc6.1,候选区间约1.01 Mb,包含51个注释基因。通过RNA-seq、实时荧光定量PCR和基因结构比对等分析,推测Cla97C06G117020 (Zinc finger protein CONSTANS-LIKE 4)为西瓜柱头颜色性状的候选基因,Cla97C06G117020在橙色柱头中表达量低于黄色柱头。利用9份西瓜材料重测序数据分析后发现,Cla97C06G117020编码区存在1个非同义SNP突变。柱头颜色的形成往往与类胡萝卜素有关,我们发现类胡萝卜素代谢途径中ClCHYB (Beta-carotene hydroxylase)的表达趋势与Cla97C06G117020一致,在橙柱头中表达量较少,推测Cla97C06G117020ClCHYB可能存在相互作用影响柱头颜色形成,该研究为西瓜柱头颜色基因精细定位和调控机制提供了理论依据。

Abstract  Stigma color is a critical agronomic trait in watermelon and plays an important role in pollination. However, there are few reports the regulation of this process to date. In this study, a genetic analysis of the F2 population derived from ZXG1553 (P1, with orange stigma) and W1-17 (P2, with yellow stigma) indicated that stigma color is a quantitative trait and orange stigma is recessive compared with yellow stigma. BSA-seq (Bulk segregant analysis sequencing) determined a chromosome segment related to stigma color in a 3.75 Mb on chromosome 6, and a major stable effective quantitative trait locus (QTL) Clqsc6.1 (QTL stigma color) was detected in two years between CAPS markers Chr06_8338913 and Chr06_9344593 spanning ~1.01 Mb interval harboring 51 annotated genes. Cla97C06G117020 (annotated as Zinc finger protein CONSTANS-LIKE 4) was identified as the best candidate gene for stigma color trait through RNA-seq, quantitative real-time (qRT)–polymerase chain reaction (PCR), and gene structure alignment analysis among the nature watermelon panel. The expression level of Cla97C06G117020 in orange stigma accession was lower than yellow stigma accessions with a significant difference. A nonsynonymous SNP site of Cla97C06G117020 coding region causing amino acid variation was related to stigma color variation among nine watermelon accessions according to their re-sequencing data. Stigma color formation is often related to carotenoid, and we also found that the expression trend of ClCHYB (annotated as Beta-carotene hydroxylase) in the carotenoid metabolic pathway was consistent with Cla97C06G117020, which was expressed in low amounts in the orange stigma accession. These data indicated that Cla97C06G117020 and ClCHYB may have interacted to form stigma color. This study provided a theoretical basis for gene fine mapping and mechanisms for the regulation of stigma color in watermelon. 
Keywords:  Watermelon       Stigma color        Gene mapping        Zinc finger protein CONSTANS-LIKE 4  
Fund: This work was supported by funding from the Natural Science Funds for Outstanding Youth of Heilongjiang Province [grant number YQ2022C011], National Natural Science Foundation of China [grant numbers 32172577], the China Agriculture Research System of MOF and MARA [grant number CARS-25], and the Taishan Industrial Leading Talents Project [grant number LJNY202112].
About author:  # Zicheng Zhu, Tel: +86-451-55190593, E-mail: zzc1983sc@163.com; Feishi Luan, Tel: + 86-451-55190836, E-mail: luanfeishi@neau.edu.cn; Shi Liu, Tel: +86-451-55190204, E-mail: shiliu@neau.edu.cn

Cite this article: 

Shuang Pei, Zexu Wu, Ziqiao Ji, Zheng Liu, Zicheng Zhu, Feishi Luan, Shi Liu. . Quantitative Trait Loci Identification Uncovers Zinc Finger Protein CONSTANS-LIKE 4 as the Key Candidate Gene of Stigma Color in Watermelon (Citrullus lanatus). Journal of Integrative Agriculture, Doi:10.1016/j.jia.2024.03.070

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