番茄不规则裂果性状的QTL定位及候选基因分析

doi:10.16420/j.issn.0513-353x.2020-1032

摘要/Abstract

摘要：

为挖掘调控番茄不规则裂果性状的关键基因,利用番茄易发生不规则裂果的种质‘S189’和耐裂果的种质‘R91’杂交获得F₁代,之后F₁自交构建F₂群体。以F₂群体中挑选出的20株易裂果单株和20株耐裂果单株的DNA分别等量混合,构成易裂池和耐裂池,进行QTL-seq分析。共检测到2个调控番茄不规则裂果性状的QTL,分别位于2号染色体的38.75 ~ 42.14 Mb区域和5号染色体的49.07 ~ 49.48 Mb区域,暂命名为qCR2和qCR5。在qCR5的区域内仅检测到2个基因,利用2个亲本的红熟期果皮进行基因表达量分析,发现其差异均不显著。qCR2的区域较大,为快速有效地挖掘候选基因,利用43个多态性标记构建2号染色体标记连锁图,并结合F₂群体裂果率进行遗传连锁分析,在多态性标记sli2734和Bin3371之间检测到1个主效QTL,其LOD值为3.05,贡献率为7.05%,且其对应的物理位置与qCR2的区域相重合。联合QTL-seq分析和遗传连锁分析将候选区域缩小至39.55 ~ 39.94 Mb,记为qCR2.1。在qCR2.1的区域内共有53个基因,利用亲本红熟期果皮进行基因表达量分析,获得3个表达量差异显著的基因Solyc02g072400、Solyc02g072470和Solyc02g076780。其中Solyc02g076780表达量差异极显著,其与乙烯调控相关,推测其为控制番茄不规则裂果性状的关键候选基因。

关键词: 番茄, 不规则裂果, QTL定位, 候选基因

Abstract:

In order to excavate key regulatory genes for irregular fruit cracking in tomato,the F₁ and F₂ offspring were obtained by crossing the cracking-susceptible（CS）germplasm accession‘S189’and the cracking-resistant（CR）germplasm accession‘R91’and subsequent self-crossing. QTL-seq approach was applied to identify candidate QTLs. Genomic DNA of 20 CS and 20 CR F₂ individuals were evenly mixed as CS-pool and CR-pool,respectively. A total of two QTLs that regulated irregular fruit cracking in tomato were detected,which were physically located in the region of 38.75-42.14 Mb on chromosome 2 and the region of 49.07-49.48 Mb on chromosome 5. The two QTLs were temporarily designated as qCR2 and qCR5. In view of the fact that there were only two genes in the region of qCR5,the differences at gene expression levels were analyzed using the mature fruit pericarps from the parents. However,both the results showed no significant differences between the parents. Considering that the region of qCR2 was relatively large,in order to quickly and effectively mine candidate genes,the linkage map of chromosome 2 was constructed using 43 polymorphic markers. Afterwards,traditional QTL analysis was carried out based on the fruit cracking rate of F₂ population. A major QTL was detected between the polymorphic markers sli2734 and Bin3371,whose LOD score was 3.05 and contribution rate was 7.05%. The corresponding physical location of the major QTL was coincided with the qCR2’s region. According to the results from both QTL-seq and traditional QTL analysis,the candidate region was delimited between 39.55-39.94 Mb,eventually designated as qCR2.1. There were a total of 53 genes in qCR2.1’s region. Accordingly,the mature fruit pericarps from the parents were used to analyze the differences at gene expression levels and thus three genes with significantly different expression levels were obtained,namely Solyc02g072400,Solyc02g072470 and Solyc02g076780. Among them,the expression levels of Solyc02g076780 between the parents were extremely significantly different. Solyc02g076780 played a role in ethylene regulation,which could be a key candidate gene for irregular fruit cracking in tomato.

Key words: tomato, irregular fruit cracking, QTL mapping, candidate gene

中图分类号:

S641.2

陈斌, 吴震, 文军琴, 林昊维, 于璐, 薛灵姿, 周蓉, 蒋芳玲. 番茄不规则裂果性状的QTL定位及候选基因分析[J]. 园艺学报, 2021, 48(7): 1329-1339.

CHEN Bin, WU Zhen, WEN Junqin, LIN Haowei, YU Lu, XUE Lingzi, ZHOU Rong, JIANG Fangling. QTL Mapping and Candidate Genes Analysis of Irregular Fruit Cracking in Tomato[J]. Acta Horticulturae Sinica, 2021, 48(7): 1329-1339.

图/表 7

图1 亲本果实性状

Fig.1 Fruit traits of parents

表1 部分标记的引物序列

Table 1 Primer sequences of partial markers

标记类型	标记	正向引物（5′-3′）	反向引物（5′-3′）
Protocol	Marker	Forward primer	Reverse primer
SSR	SSR50	CCGTGACCCTCTTTACAAGC	TTGCTTTCTTCTTCGCCATT
	SSR287	GCATCCCAAACAATCCAATC	TCCACTTTCAAGATCAGAGCAA
	TES373	GCCAATGGCTTTGTTGAAGGT	ATGGTGACTTTGCCCTTGTC
	TES992	GATGGACTGGCTCTGGCTCTA	TCACCATCACTGCTTCCATC
InDel	C02M36734	GACAAATATCATTCGTTTTAATG	CCAAAACACCAAATAATTACTAA
	C02M52141	TTCTTATGTCCTCCGAACAAC	GGACTACACCCTACTTAGGCTA
	Bin3361	CAGGGGAACTCCAAATTTCA	CACATGGTATCAAAGCACGG
	Bin3371	GAAGTTGACACAATCCCGCT	TCGAAATCAGATCCAACTGC
	sli2734	GGTAGGGATCTTGAGCATGG	GGAGCCAGAGGGATCAAAAG
	sli2763	AAGGTCTCTCCCTTTGAGCTG	TTGATCATGAGGGTAGCCTTG

图2 番茄F2群体裂果率频率分布直方图

Fig.2 Frequency distribution histogram of fruit cracking rate in F2 population of tomato

图3 Δ（SNP-index）（A）、易裂池SNP-index（B）和耐裂池SNP-index（C）在全基因组上的分布图中散点表示计算得到ΔSNP-index和SNP-index值;黑色曲线表示相应的拟合值;红色虚线表示LOESS设置的99%阈值线。

Fig. 3 Distribution of Δ（SNP-index）（A）,WB-SNP-index（B）and MB-SNP-index（C）in the whole genome The scattered points in the figure represent the calculated values of ΔSNP-index and SNP-index;The black curve represents the corresponding fitting value;The red dotted line represents 99% threshold line set by LOESS.

图4 多态性标记（sli2747）条带图

Fig.4 Polymorphic marker（sli2747）strip chart

图5 番茄2号染色体QTL定位

Fig.5 QTL mapping on tomato chromosome 2

图6 显著差异表达基因

Fig. 6 Significantly differentially expressed genes * P < 0.05;** P < 0.01;*** P < 0.001.

参考文献 37

[1]	Abbott J D, Peet M M, Willits D H, Sanders D C, Gough R E. 1986. Effects of irrigation frequency and scheduling on fruit production and radial fruit cracking in greenhouse tomatoes in soil beds and in a soil-less medium in bags. Scientia Horticulturae, 28(3):209-217. doi: 10.1016/0304-4238(86)90002-6 URL
[2]	Capel C, Yuste-Lisbona F J, Lopez-Casado G, Angosto T, Cuartero J, Lozano R, Capel J. 2017. Multi-environment QTL mapping reveals genetic architecture of fruit cracking in a tomato RIL Solanum lycopersicum × S-pimpinellifolium population. Theoretical and Applied Genetics, 130(1):213-222. doi: 10.1007/s00122-016-2809-9 URL
[3]	Cuartero J, Palomares G, Balasch S, Nuez, F. 1981. Tomato fruit cracking under plastic-house and in the open air. ii. general and specific combining abilities. BMC Infectious Diseases, 15(1):1-13. doi: 10.1186/s12879-014-0722-x URL
[4]	Gine-Bordonaba J, Echeverria G, Ubach D, Aguilo-Aguayo I, Lopez M L, Larrigaudiere C. 2017. Biochemical and physiological changes during fruit development and ripening of two sweet cherry varieties with different levels of cracking tolerance. Plant Physiology and Biochemistry, 111:216-225. doi: 10.1016/j.plaphy.2016.12.002 URL
[5]	Hovav R, Chehanovsky N, Moy M, Jetter R, Schaffer A A. 2007. The identification of a gene(Cwp1),silenced during Solanum evolution,which causes cuticle microfissuring and dehydration when expressed in tomato fruit. Plant Journal, 52(4):627-639. pmid: 17877702
[6]	Hudson I W. 1956. The inheritance of resistance to fruit cracking in the tomato(Lycopersicon esculentum L.)[M. D. Dissertation]. Corvallis,US:Oregon State University.
[7]	Jiang F L, Lopez A, Jeon S, de Freitas S T, Yu Q H, Wu Z, Labavitch J M, Tian S K, Powell A L T, Mitcham E. 2019. Disassembly of the fruit cell wall by the ripening-associated polygalacturonase and expansin influences tomato cracking. Horticulture Research, 6:17. doi: 10.1038/s41438-018-0105-3 URL
[8]	Kadambari G, Vemireddy L R, Srividhya A, Nagireddy R, Jena S S, Gandikota M, Patil S, Veeraghattapu R, Deborah D A K, Reddy G E, Shake M, Dasari A, Ramanarao P V, Durgarani C V, Neeraja C N, Siddiq E A, Sheshumadhav M. 2018. QTL-Seq-based genetic analysis identifies a major genomic region governing dwarfness in rice(Oryza sativa L.). Plant Cell Reports, 37(4):677-687. doi: 10.1007/s00299-018-2260-2 URL
[9]	Khadivi-Khub A. 2015. Physiological and genetic factors influencing fruit cracking. Acta Physiologiae Plantarum, 37(1):1718. doi: 10.1007/s11738-014-1718-2 URL
[10]	Kunwar S, Iriarte F, Fan Q R, da Silva E E, Ritchie L, Nguyen N S, Freeman J H, Stall R E, Jones J B, Minsavage G V, Colee J, Scott J W, Vallad G E, Zipfel C, Horvath D, Westwood J, Hutton S F, Paret M L. 2018. Transgenic expression of EFR and Bs2 genes for field management of bacterial wilt and bacterial spot of tomato. Phytopathology, 108(12):1402-1411. doi: 10.1094/PHYTO-12-17-0424-R pmid: 29923802
[11]	Leclercq J, Adams-Phillips L C, Zegzouti H, Jones B, Bouzayen M. 2002. Lectr1,a tomato ctr1-like gene,demonstrates ethylene signaling ability in Arabidopsis and novel expression patterns in tomato. Plant Physiology, 130(3):1132-1142. pmid: 12427980
[12]	Li Hui-jia. 2016. Genetic analysis and QTL mapping in tomato cracking[M. D. Dissertation]. Harbin:Northeast Agricultural University. (in Chinese)
	李会佳. 2016. 番茄裂果性状的遗传分析及QTL定位[硕士论文]. 哈尔滨:东北农业大学.
[13]	Li R, Sun S, Wang H J, Wang K T, Yu H, Zhou Z, Xin P Y, Chu J F, Zhao T M, Wang H Z, Li J Y, Cui X. 2020. FIS1 encodes a GA2-oxidase that regulates fruit firmness in tomato. Nature Communications, 11(1):5844. doi: 10.1038/s41467-020-19705-w URL
[14]	Liao N Q, Hu Z Y, Li Y Y, Hao J F, Chen S N, Xue Q, Ma Y Y, Zhang K J, Mahmoud A, Ali A, Malangisha G K, Lyu X L, Yang J H, Zhang M F. 2020. Ethylene-responsive factor 4 is associated with the desirable rind hardness trait conferring cracking resistance in fresh fruits of watermelon. Plant Biotechnology Journal, 18(4):1066-1077. doi: 10.1111/pbi.v18.4 URL
[15]	Liu Qiang, Zhang Gui-you, Kazuo S. 2000. The plant mitogen-activated protein(MAP)kinase. Acta Botanica Sinica, 42(7):661-667. (in Chinese)
	刘强, 张贵友, Kazuo S. 2000. 植物促分裂原活化蛋白(MAP)激酶. 植物学报, 42(7):661-667.
[16]	Liu Zhong-qi, Xue Jun, Jin Feng-mei, Bai Yan-ling. 2007. Relationoship between fruit crack and fruit skin architecture and their heterosis in tomato(Lycopersicon esculentum Mill.). Acta Agriculturae Boreai-Sinica, 22(3):141-147. (in Chinese)
	刘仲齐, 薛俊, 金凤媚, 白艳玲. 2007. 番茄裂果与果皮结构的关系及其杂种优势表现. 华北农学报, 22(3):141-147.
[17]	Livak K J, Schmittgen T D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method. Methods, 25(4):402-408. pmid: 11846609
[18]	Lopez-Casado G, Matas A J, Dominguez E, Cuartero J, Heredia A. 2007. Biomechanics of isolated tomato(Solanum lycopersicum L.)fruit cuticles:the role of the cutin matrix and polysaccharides. Journal of Experimental Botany, 58(14):3875-3883. doi: 10.1093/jxb/erm233 URL
[19]	Lu H, Lin T, Klein J, Wang S, Qi J, Zhou Q, Sun J, Zhang Z, Weng Y, Huang S. 2014. QTL-seq identifies an early flowering QTL located near Flowering Locus T in cucumber. Theoretical and Applied Genetics, 127(7):1491-1499. doi: 10.1007/s00122-014-2313-z URL
[20]	Lu K, Li T, He J, Chang W, Zhang R, Liu M, Yu M N, Fan Y H, Ma J Q, Sun W, Qu C M, Liu L Z, Li N N, Liang Y, Wang R, Qian W, Tang Z L, Xu X F, Lei B, Zhang K, Li J A. 2018. qPrimerDB:a thermodynamics-based gene-specific qPCR primer database for 147 organisms. Nucleic Acids Research, 46(D1):D1229-D1236. doi: 10.1093/nar/gkx725 URL
[21]	Moctezuma E, Smith D L, Gross K C. 2003. Antisense suppression of a beta-galactosidase gene(TBG6)in tomato increases fruit cracking. Journal of Experimental Botany, 54(390):2025-2033. pmid: 12867545
[22]	Murray M, Thompson W F. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8(19):4321-4326. pmid: 7433111
[23]	Pandey M K, Khan A W, Singh V K, Vishwakarma M K, Shasidhar Y, Kumar V, Garg V, Bhat R S, Chitikineni A, Janila P, Guo B, Varshney R K. 2017. QTL-seq approach identified genomic regions and diagnostic markers for rust and late leaf spot resistance in groundnut(Arachis hypogaea L.). Plant Biotechnology Journal, 15(8):927-941. doi: 10.1111/pbi.2017.15.issue-8 URL
[24]	Peet M M. 1992. Fruit cracking in tomato. Horttechnology, 2(2):216-223. doi: 10.21273/HORTTECH.2.2.216 URL
[25]	Singh V K, Khan A W, Jaganathan D, Thudi M, Roorkiwal M, Takagi H, Garg V, Kumar V, Chitikineni A, Gaur P M, Sutton T, Terauchi R, Varshney R K. 2016. QTL-seq for rapid identification of candidate genes for 100-seed weight and root/total plant dry weight ratio under rainfed conditions in chickpea. Plant Biotechnology Journal, 14(11):2110-2119. doi: 10.1111/pbi.2016.14.issue-11 URL
[26]	Sonderby I E, Geu-Flores F, Halkier B A. 2010. Biosynthesis of glucosinolates-gene discovery and beyond. Trends in Plant Science, 15(5):283-290. doi: 10.1016/j.tplants.2010.02.005 URL
[27]	Song Dong-hui, Song Feng-ming, Zheng Zhong. 2004. Function of MAP kinases in plant signal transduction pathway networks. Journal of Zhejiang University(Agriculture and Life Sciences), 30(2):119-126. (in Chinese)
	宋东辉, 宋凤鸣, 郑重. 2004. MAP激酶在植物信号传递网络中的功能. 浙江大学学报(农业与生命科学版), 30(2):119-126.
[28]	Tong V G. 2016. Development of InDel markers and fine-mapping of locule number 2.2 in tomato[Ph. D. Dissertation]. Beijing:Chinese Academy of Agricultural Sciences. (in Chinese)
	Tong Van Giang. 2016. 番茄InDel分子标记的开发与果实心室数量主效QTL lcn2.2的精细定位[博士论文]. 北京:中国农业科学院.
[29]	Walter J M. 1967. Hereditary resistance to disease in tomato. Annual Review of Phytopathology, 5(1):131-160. doi: 10.1146/annurev.py.05.090167.001023 URL
[30]	Wang J G, Gao X M, Ma Z L, Chen J, Liu Y N, Shi W Q. 2019. Metabolomic and transcriptomic profiling of three types of litchi pericarps reveals that changes in the hormone balance constitute the molecular basis of the fruit cracking susceptibility of Litchi chinensis cv. Baitangying. Molecular Biology Reports, 46(5):5295-5308. doi: 10.1007/s11033-019-04986-2 URL
[31]	Wei Kai, Liu Xiaoyan, Cao Xue, Liu Xiaolin, Wang Xiaotian, Yang Mengxia, Wang Jing, Wang Xiaoxuan, Guo Yanmei, Du Yongchen, Li Junming, Liu Lei, Shu Jinshuai, Qin Yong, Huang Zejun. 2020. Identification of quantitative trait loci controlling tomato fruit weight by QTL-seq. Acta Horticulturae Sinica, 47(3):571-580. (in Chinese)
	魏凯, 刘晓燕, 曹雪, 刘晓林, 王晓甜, 杨孟霞, 王净, 王孝宣, 国艳梅, 杜永臣, 李君明, 刘磊, 舒金帅, 秦勇, 黄泽军. 2020. 利用QTL-seq定位番茄果实质量QTL. 园艺学报, 47(3):571-580.
[32]	Wen J Q, Jiang F L, Weng Y Q, Sun M T, Shi X P, Zhou Y Z, Yu L, Wu Z. 2019. Identification of heat-tolerance QTLs and high-temperature stress-responsive genes through conventional QTL mapping,QTL-seq and RNA-seq in tomato. BMC Plant Biology, 19(1):398. doi: 10.1186/s12870-019-2008-3 URL
[33]	Xue L Z, Sun M T, Wu Z, Yu L, Yu Q H, Tang Y P, Jiang F L. 2020. LncRNA regulates tomato fruit cracking by coordinating gene expression via a hormone-redox-cell wall network. BMC Plant Biology, 20(1):162. doi: 10.1186/s12870-020-02373-9 URL
[34]	Yamaguchi-Shinozaki K, Shinozaki K. 2006. Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annual Review of Plant Biology, 57:781-803. pmid: 16669782
[35]	Yang J J, Wang Y Y, Shen H L, Yang W C. 2014. In silico identification and experimental validation of insertion-deletion polymorphisms in tomato genome. DNA Research, 21(4):429-438. doi: 10.1093/dnares/dsu008 URL
[36]	Yang Ze-en. 2015. Screening of agronomic characters related to tomato fruit cracking and the study on mechanism of tomato fruit cracking based on the pericarp[M. D. Dissertation]. Nanjing:Nanjing Agricultural University. (in Chinese)
	杨泽恩. 2015. 番茄裂果相关指标筛选及基于果皮特征的裂果机理研究[硕士论文]. 南京:南京农业大学.
[37]	Zhu Yu, Fan Lina, Huang Zejun, Du Yongchen, Guo Yanmei, Li Junming, Liu Lei, Shu Jinshuai, Wang Xiaoxuan. 2020. Fine mapping of fruit cracking-resistant gene Cr3a in tomato. Acta Horticulturae Sinica, 47(2):275-286. (in Chinese)
	朱玉, 范丽娜, 黄泽军, 杜永臣, 国艳梅, 李君明, 刘磊, 舒金帅, 王孝宣. 2020. 番茄耐裂果基因Cr3a的精细定位. 园艺学报, 47(2):275-286.