番茄抗坏血酸含量相关QTL定位及候选基因鉴定

doi:10.16420/j.issn.0513-353x.2023-0659

园艺学报 ›› 2024, Vol. 51 ›› Issue (2): 219-228.doi: 10.16420/j.issn.0513-353x.2023-0659

• 遗传育种·种质资源·分子生物学 • 下一篇

番茄抗坏血酸含量相关QTL定位及候选基因鉴定

刘根忠¹^,², 李方曼², 葛平飞², 陶金宝², 张星雨², 叶志彪²^,³, 张余洋²^,³^,⁴^,⁵^,^*()

¹ 山东农业大学园艺科学与工程学院，山东泰安 271018
² 华中农业大学，果蔬园艺作物种质创新与利用全国重点实验室，武汉 430070
³ 湖北洪山实验室，武汉 430070
⁴ 华中农业大学深圳营养与健康研究院，武汉 430070
⁵ 中国农业科学院深圳农业基因组研究所，岭南现代农业科学与技术广东省实验室深圳分中心，广东深圳 518000

收稿日期:2023-09-03 修回日期:2024-01-15 出版日期:2024-02-25 发布日期:2024-02-26
通讯作者:
*E-mail：yyzhang@mail.hzau.edu.cn
基金资助:
国家重点研发计划项目(2022YFD1200502); 国家重点研发计划项目(2021YFD1200201); 国家自然科学基金项目(32372696); 国家自然科学基金项目(31991182); 湖北洪山实验室项目(2021hszd007); 武汉市生物育种重大专项(2022021302024852); 湖北省重点研发计划项目(2022BBA0062); 湖北省重点研发计划项目(2022BBA0066); 湖北省种业高质量发展资金项目(HBZY2023B004); 湖北省现代农业产业技术体系建设专项(2023HBSTX4-06); 华中农业大学与中国农业科学院深圳农业基因组所合作资金研发项目(SZYJY2023022)

QTL Mapping and Candidate Gene Identification Related to Ascorbic Acid Content in Tomato

LIU Genzhong¹^,², LI Fangman², GE Pingfei², TAO Jinbao², ZHANG Xingyu², YE Zhibiao²^,³, ZHANG Yuyang²^,³^,⁴^,⁵^,^*()

¹ College of Horticultural Science and Engineering，Shandong Agricultural University，Tai’an，Shandong 271018，China
² National Key Laboratory of Germplasm Innovation and Utilization of Fruit and Vegetable Horticultural Crops，Huazhong Agricultural University，Wuhan 430070，China
³ Hubei Hongshan Laboratory，Wuhan 430070，China
⁴ Shenzhen Institute of Nutrition and Health，Huazhong Agricultural University，Wuhan 430070，China
⁵ Shenzhen Branch of Guangdong Provincial Laboratory of Lingnan Modern Agricultural Science and Technology，Shenzhen Institute of Agricultural Genomics，Chinese Academy of Agricultural Sciences，Shenzhen，Guangdong 518000，China

Received:2023-09-03 Revised:2024-01-15 Published:2024-02-25 Online:2024-02-26

摘要/Abstract

摘要：

以番茄果实高抗坏血酸含量材料TS-226和低抗坏血酸含量材料TS-228为试验亲本杂交和自交创制F₂遗传分离群体，分别构建高抗坏血酸混池和低抗坏血酸混池，集团分离群体测序（bulked segregant analysis sequencing，BSA-seq）分析鉴定到与番茄果实抗坏血酸相关的主效QTL，进一步将与抗坏血酸相关的候选基因定位于番茄基因组8号染色体58.00 ~ 60.15 Mb区域内。结合∆SNP-index数据，推断SlPPO（Polyphenol oxidase）是参与调控番茄抗坏血酸含量的主效基因。以TS-228为背景材料，通过农杆菌介导方法进行遗传转化，获得SlPPO过表达和沉默株系，相比野生型，过表达株系OE-3和OE-18的红熟果实总抗坏血酸含量分别减少了18.89%和26.56%；沉默株系Ri-9和Ri-16红熟果实总抗坏血酸含量分别增加了37.53%和63.93%。综上所述，SlPPO为参与控制番茄红熟果实抗坏血酸含量的关键基因，对果实抗坏血酸含量起负向调控作用。

关键词: 番茄, 集团分离群体测序, 抗坏血酸, QTL, SlPPO

Abstract:

The F₂ genetic segregating population was constructed via using high ascorbic acid（AsA）tomato accession TS-226 and low ascorbic acid tomato accession TS-228 as parents. The high-ascorbic acid and low-ascorbic acid pools were constructed respectively. Through bulked segregant analysis sequencing analysis（BSA-seq），the main QTL related to ascorbic acid was found，and the candidate gene related to ascorbic acid was located in the 58.00-60.15 Mb region of tomato chromosome 8. Furthermore，the ∆SNP-index data were analyzed to determine the main candidate gene SlPPO（Polyphenol oxidase）that controls the ascorbic acid content of tomato fruit. Using TS-228 as the background plant，transgenic lines with SlPPO overexpression and silencing were obtained by Agrobacterium-mediated transformation. Compared with the wild type，the total ascorbic acid content in the red ripe fruits of the overexpression lines OE-3 and OE-18 decreased by 18.89% and 26.56%，respectively. The total ascorbic acid content in the red ripe fruits of the silenced lines Ri-9 and Ri-16 increased by 37.53% and 63.93% relative to wild type，respectively. Taken together，SlPPO is the key gene to control the ascorbic acid content of tomato red ripe fruit，and plays a negative role in regulating fruit ascorbic acid content.

Key words: tomato, BSA-seq, ascorbic acid, QTL, SlPPO

刘根忠, 李方曼, 葛平飞, 陶金宝, 张星雨, 叶志彪, 张余洋. 番茄抗坏血酸含量相关QTL定位及候选基因鉴定[J]. 园艺学报, 2024, 51(2): 219-228.

LIU Genzhong, LI Fangman, GE Pingfei, TAO Jinbao, ZHANG Xingyu, YE Zhibiao, ZHANG Yuyang. QTL Mapping and Candidate Gene Identification Related to Ascorbic Acid Content in Tomato[J]. Acta Horticulturae Sinica, 2024, 51(2): 219-228.

图/表 9

表1 番茄抗坏血酸相关基因引物

Table 1 Primers for the genes related to the tomato ascorbic acid

目的 Purpose	正向引物（5′-3′） Forward primer	反向的引物（5′-3′） Reverse primer
SlPPO for qRT-PCR	TGCCAACCAAAACAATGGTGA	CAGCATAGGCTAAGGGTGCT
SlPPO for Ri	GGGGACAAGTTTGTACAAAAAAGCA GGCTGAGTCGGAGACTGCTTGGATACA	GGGGACCACTTTGTACAAGAAAGCT GGGTGCATTCACATTGTCATCCTCGTT
SlPPO for OE	AAAAAGCAGGCTATGTCTTCTTCTT TTTGGAGTAATTC	AGAAAGCTGGGTTCAACAGTCCTCC AAAGATTCTGTA

图1 番茄亲本、F1和F2代群体红熟果实总抗坏血酸含量的分布不同小写字母表示差异显著（P < 0.05）。

Fig. 1 Total ascorbic acid content of red ripe tomato fruit in parents, F1 and F2 population Different lowercase letters indicate significant differences（P < 0.05）.

表2 测序数据统计

Table 2 Summary of whole-genome re-sequencing data

样品名称 Sample	Q20比率/% Q20 rate	高质量读长数/条 Clean reads	高质量碱基数/bp Clean bases	读长数比对率/% Mapping rate of reads	基因组测序深度/× Sequencing depth
低AsA池 Low AsA pool	93.62	143 821 112	21 573 166 800	83.95	26.18
高AsA池 High AsA pool	93.67	144 629 966	21 694 494 900	82.94	26.33

图2 基于SNP-index对番茄果实抗坏血酸含量相关QTL定位

Fig. 2 QTL mapping of AsA content in tomato fruit based on SNP-index

表3 番茄8号染色体目标区间内候选基因变异分析

Table 3 Variation analysis of candidate genes in the target region of tomato chromosome 8

多酚氧化酶基因 Polyphenol oxidase gene	SNP位置 SNP location	参考碱基 Reference base	低AsA池Low AsA pool		高AsA池High AsA pool		△SNP-index
多酚氧化酶基因 Polyphenol oxidase gene	SNP位置 SNP location	参考碱基 Reference base	Base	SNP-index	Base	SNP-index	△SNP-index
Solyc08g074620	58745745	C	C	0	Y	0.792	0.792
	58746330	C	Y	0.208	Y	0.833	0.625
Solyc08g074630	58756071	G	S	0.312	S	0.778	0.466
	58757391	A	W	0.346	W	0.762	0.416
Solyc08g074640	58771375	G	G	0	R	0.750	0.750
	58772995	T	T	0	Y	0.769	0.769
Solyc08g074650	58785356	A	A	0	G	0.938	0.938
	58785423	C	C	0	T	0.905	0.905
Solyc08g074680	58830789	G	S	0.296	C	0.929	0.633
	58841121	T	Y	0.292	Y	0.857	0.565
Solyc08g074690	58851893	A	W	0.250	W	0.714	0.464
	58852031	G	G	0	R	0.696	0.696

图3 6个多酚氧化酶蛋白功能域预测

Fig. 3 Prediction of six polyphenol oxidase proteins functional domains

图4 番茄多酚氧化酶基因进化树（A）和组织表达（B）分析

Fig. 4 Phylogenetic tree（A）and expression analysis（B）of tomato polyphenol oxidase genes

图5 番茄抗坏血酸高含量TS-265和低含量TS-228的红熟果实中多酚氧化酶活性和SlPPO表达量

Fig. 5 Determination of polyphenol oxidase activity and SlPPO gene expression in red ripe fruit of TS-265 with high AsA and TS-228 with low AsA

图6 番茄TS-228的SlPPO过表达（OE）和沉默（Ri）植株红熟果实中SlPPO的表达量和抗坏血酸含量

Fig. 6 SlPPO expression and ascorbic acid content in red ripe fruits of SlPPO overexpression and silencing lines

参考文献 26

[1]	Akram N A, Shafiq F, Ashraf M. 2017. Ascorbic acid-a potential oxidant scavenger and its role in plant development and abiotic stress tolerance. Frontiers in Plant Science, 8:613. doi: 10.3389/fpls.2017.00613 pmid: 28491070
[2]	Bao Tian-li, Liang Yin-li, Li Wen-ping, Mu Lan, Gao De-kai. 2015. Effect of soil type on vitamin C content and activities of related enzymes in cherry tomato. Food Science, 36 (5):24-28. (in Chinese) doi: 10.7506/spkx1002-6630-201505005
	包天莉, 梁银丽, 李文平, 穆兰, 高德凯. 2015. 土壤类型对樱桃番茄VC含量及其相关酶活性的影响. 食品科学, 36 (5):24-28. doi: 10.7506/spkx1002-6630-201505005
[3]	Chen C, Zhang M, Zhang M Y, Yang M M, Dai S S, Meng Q W, Lv W, Zhuang K Y. 2023. ETHYLENE-INSENSITIVE 3-LIKE 2 regulates β-carotene and ascorbic acid accumulation in tomatoes during ripening. Plant Physiology, 192:2067-2080. doi: 10.1093/plphys/kiad151 pmid: 36891812
[4]	Cheng L N, Zhu Z W, Sun D W. 2021. Impacts of high pressure assisted freezing on the denaturation of polyphenol oxidase. Food Chemistry, 335:127485.
[5]	Gregory V, Kyle E L, Michael M, Michael A G, Christine D S. 2021. A combined BSA-Seq and linkage mapping approach identifies genomic regions associated with Phytophthora root and crown rot resistance in squash. Theoretical and Applied Genetics, 134:1015-1031. doi: 10.1007/s00122-020-03747-1 pmid: 33388885
[6]	Hou X Q, Feng L W, Liu G Z, Saeed D A, Li H X, Zhang Y Y, Ye Z B. 2015. The influence of growth media pH on ascorbic acid accumulation and biosynthetic gene expression in tomato. Scientia Horticulturae, 197:637-643.
[7]	Landi M, Degl'Innocenti E, Guglielminetti L, Guidi L. 2013. Role of ascorbic acid in the inhibition of polyphenol oxidase and the prevention of browning in different browning-sensitive Lactuca sativa var. capitata(L.)and Eruca sativa(Mill.) stored as fresh-cut produce. Journal of the Science of Food and Agriculture, 93:1814-1819.
[8]	Li M X, Wang H Y, Tian S L, Zhu Y, Zhang Y J. 2023. Triticeae-BGC:a web-based platform for detecting,annotating and evolutionary analysis of biosynthetic gene clusters in Triticeae. Journal of Genetics and Genomics, 50:921-923.
[9]	Li Y, Chu Z N, Luo J Y, Zhou Y H, Cai Y J, Lu Y G, Xia J H, Kuang H H, Ye Z B, Ouyang B. 2018. The C2H2 zinc-finger protein SlZF3 regulates AsA synthesis and salt tolerance by interacting with CSN5B. Plant Biotechnology Journal, 16:1201-1213.
[10]	Liu G Z, Yu H Y, Yuan L, Li C X, Ye J, Chen W F, Wang Y, Ge P F, Zhang J H, Ye Z B, Zhang Y Y. 2021. SlRCM1,which encodes tomato Lutescent1,is required for chlorophyll synthesis and chloroplast development in fruits. Horticulture Research, 8:128.
[11]	Ouyang Bo. 2003. Studies on Agrobacterium-mediated transformation of tomato with several pathogenesis-related genes[Ph. D. Dissertation]. Wuhan: Huazhong Agricultural University. (in Chinese)
	欧阳波. 2003. 几种病程相关蛋白基因转化番茄的研究[博士论文]. 武汉: 华中农业大学.
[12]	Page M, Sultana N, Paszkiewicz K, Florance H, Smirnoff N. 2012. The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana:further evidence for redox control of anthocyanin synthesis. Plant Cell and Environment, 35:388-404.
[13]	Saleem M S, Anjum M A, Naz S, Ali S, Hussain S, Azam M, Sardar H, Khaliq G, Canan I, Ejaz S. 2021. Incorporation of ascorbic acid in chitosan-based edible coating improves postharvest quality and storability of strawberry fruits. International Journal of Biological Macromolecules, 189:160-169. doi: 10.1016/j.ijbiomac.2021.08.051 pmid: 34411616
[14]	Stevens R, Buret M, Garchery C, Carretero Y, Causse M. 2006. Technique for rapid,small-scale analysis of vitamin C levels in fruit and application to a tomato mutant collection. Journal of Agricultural and Food Chemistry, 54:6159-6165.
[15]	Wang J, Yu Y W, Zhang Z J, Quan R D, Zhang H W, Ma L G, Deng X W, Huang R F. 2013. Arabidopsis CSN5B interacts with VTC1 and modulates ascorbic acid synthesis. Plant Cell, 25:625-636.
[16]	Wang X, Gao L, Jiao C, Stravoravdis S, Hosmani P S, Saha S, Zhang J, Mainiero S, Strickler S R, Catala C, Martin G B, Mueller L A, Vrebalov J, Giovannoni J J, Wu S, Fei Z J. 2020. Genome of Solanum pimpinellifolium provides insights into structural variants during tomato breeding. Nature Communications, 11 (1):5817. doi: 10.1038/s41467-020-19682-0 pmid: 33199703
[17]	Winzer T, Gazda V, He Z, Kaminski F, Kern M, Larson T R, Li Y, Meade F, Teodor R, Vaistij F E, Walker C, Bowser T A, Graham I A. 2012. A papaver somniferum 10-gene cluster for synthesis of the anticancer alkaloid noscapine. Science, 336:1704-1708. doi: 10.1126/science.1220757 pmid: 22653730
[18]	Xiong C, Luo D, Lin A H, Zhang C L, Shan L B, He P, Li B, Zhang Q M, Hua B, Yuan Z L, Li H X, Zhang J H, Yang C X, Lu Y E, Ye Z B, Wang T T. 2019. A tomato B-box protein BBX20 modulates carotenoid biosynthesis by directly activating PHYTOENE SYNTHASE1,and is targeted for 26S proteasome-mediated degradation. New Phytologist, 221:279-294.
[19]	Ye J, Wang X, Wang W Q, Yu H Y, Ai G, Li C X, Sun P Y, Wang X Y, Li H X, Ouyang B, Zhang J H, Zhang Y Y, Han H Y, Giovannoni J J, Fei Z J, Ye Z B. 2021. Genome-wide association study reveals the genetic architecture of 27 agronomic traits in tomato. Plant Physiology, 186:2078-2092. doi: 10.1093/plphys/kiab230 pmid: 34618111
[20]	Yu K B, Zhou L, Sun Y F, Zeng Z C, Chen H W, Liu J P, Zou L Q, Liu W. 2021. Anti-browning effect of on apple juice and identification of polyphenol oxidase inhibitors. Food Chemistry, 359:15.
[21]	Zhan C S, Shen S Q, Yang C K, Liu Z H, Fernie A R, Graham I A, Luo J. 2022. Plant metabolic gene clusters in the multi-omics era. Trends in Plant Science, 27:981-1001. doi: 10.1016/j.tplants.2022.03.002 pmid: 35365433
[22]	Zhang Feng-xia, Tao Pei-wen, Li Han-xia, Ye Zhi-biao, Zhang Yu-yang. 2016. Research progress in tomato ascorbic acid biosynthesis and metabolism. China Vegetables,(1):18-23. (in Chinese)
	张凤霞, 陶佩文, 李汉霞, 叶志彪, 张余洋. 2016. 番茄抗坏血酸合成代谢研究进展. 中国蔬菜,(1):18-23.
[23]	Zhang Y T, Ntagkas N, Fanourakis D, Tsaniklidis G, Zhao J T, Cheng R F, Yang Q C, Li T. 2021. The role of light intensity in mediating ascorbic acid content during postharvest tomato ripening:a transcriptomic analysis. Postharvest Biology and Technology, 180:111622.
[24]	Zhang Z J, Wang J, Zhang R X, Huang R F. 2012. The ethylene response factor AtERF 98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis. Plant Journal, 71:273-287.
[25]	Zheng X Z, Gong M, Zhang Q D, Tan H Q, Li L P, Tang Y W, Li Z G, Peng M C, Deng W. 2022. Metabolism and regulation of ascorbic acid in fruits. Plants-Basel, 11 (12):1602.
[26]	Zhou D, Li L, Wu Y W, Fan J F, Ouyang J. 2015. Salicylic acid inhibits enzymatic browning of fresh-cut Chinese chestnut(Castanea mollissima)by competitively inhibiting polyphenol oxidase. Food Chemistry, 171:19-25. doi: 10.1016/j.foodchem.2014.08.115 pmid: 25308637

番茄抗坏血酸含量相关QTL定位及候选基因鉴定

QTL Mapping and Candidate Gene Identification Related to Ascorbic Acid Content in Tomato

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 26

相关文章 15

编辑推荐

Metrics

本文评价

访问总数:　当日访问总数:　当前在线人数:

[1]	韩荧, 段颖, 牛一杰, 李衍素, 贺超兴, 孙敏涛, 王君, 李强, 陈双臣, 闫妍. 腐殖酸生物降解地膜提高番茄品质的转录代谢机制研究[J]. 园艺学报, 2024, 51(8): 1758-1772.
[2]	龚小雅, 李贤, 周新刚, 吴凤芝. 分蘖洋葱伴生番茄诱导的根际微生物对根结线虫病的影响[J]. 园艺学报, 2024, 51(8): 1913-1926.
[3]	孟思达, 韩磊磊, 相恒佐, 朱美玉, 冯珍, 叶云珠, 孙美华, 李艳冰, 赵利萍, 谭昌华, 齐明芳, 李天来. 番茄心室数的调控机制研究进展[J]. 园艺学报, 2024, 51(7): 1649-1664.
[4]	赵崇斌, 张苇胤, 李舒庆, 郭乙含, 徐红霞, 陈俊伟, 杨向晖, 彭泽. 枇杷果、叶、花相关性状QTL定位分析[J]. 园艺学报, 2024, 51(6): 1189-1200.
[5]	马星云, 范冰丽, 唐光彩, 贾芝琪, 李营, 薛东齐, 张世文. DXR调控番茄叶绿体发育、花色与果实着色机制初探[J]. 园艺学报, 2024, 51(6): 1241-1255.
[6]	张文静, 徐大勇, 吴倩琳, 杨佛, 信丙越, 曾昕, 李峰. 拮抗番茄灰霉病的贝莱斯芽孢杆菌XDY66基因组分析[J]. 园艺学报, 2024, 51(6): 1413-1425.
[7]	王永珍, 张剑国, 刘彩虹, 李思蓓, 吕甜甜. 番茄新品种‘圆红212’[J]. 园艺学报, 2024, 51(6): 1435-1436.
[8]	刘泽营, 孙帅, 刘志强, 崔霞, 李仁. 番茄尖果脐突变体的生理特性及其候选基因分析[J]. 园艺学报, 2024, 51(5): 982-992.
[9]	李品, 甘宁, 陈家伟, 项思翔, 沈静漪, 欧阳波, 卢永恩. 番茄自然群体磷利用效率分析及耐低磷种质筛选[J]. 园艺学报, 2024, 51(5): 993-1004.
[10]	杨婷, 席德慧, 夏明, 李佳楠. α-苦瓜素基因提高番茄对烟草花叶病毒抗性的机理研究[J]. 园艺学报, 2024, 51(5): 1126-1136.
[11]	胡志峰, 邵景成, 张莉. 番茄新品种‘陇番15号’[J]. 园艺学报, 2024, 51(4): 917-918.
[12]	董舒超, 洪骏, 凌嘉怡, 谢紫欣, 张胜军, 赵丽萍, 宋刘霞, 王银磊, 赵统敏. 番茄抗旱性的全基因组关联分析[J]. 园艺学报, 2024, 51(2): 229-238.
[13]	徐琴, 王嘉颖, 张曼楠, 萧志浩, 郑涵楷, 卢永恩, 王涛涛, 张余洋, 张俊红, 叶志彪, 叶杰. 番茄苗期耐盐相关遗传位点鉴定及分子标记开发[J]. 园艺学报, 2024, 51(2): 239-252.
[14]	杨亮, 刘欢, 马燕勤, 李菊, 王海娥, 周玉洁, 龙海成, 苗明军, 李志, 常伟. 利用CRISPR/Cas9技术创制高番茄红素番茄新材料[J]. 园艺学报, 2024, 51(2): 253-265.
[15]	闫超凡, 孙雪梅, 钟启文, 邵登魁, 邓昌蓉, 文军琴. 番茄20S蛋白酶体基因家族鉴定及生物信息学分析[J]. 园艺学报, 2024, 51(2): 266-280.

番茄抗坏血酸含量相关QTL定位及候选基因鉴定

QTL Mapping and Candidate Gene Identification Related to Ascorbic Acid Content in Tomato

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 9

参考文献 26

相关文章 15

编辑推荐

Metrics

本文评价

访问总数: 当日访问总数: 当前在线人数:

访问总数:　当日访问总数:　当前在线人数: