宽皮柑橘褐斑病抗性的全基因组关联分析

doi:10.3864/j.issn.0578-1752.2023.18.012

摘要/Abstract

摘要：

【目的】挖掘宽皮柑橘褐斑病抗性基因及抗病资源中的相关基因型，为柑橘品种抗褐斑病改良提供依据。【方法】在夏秋两季对136份宽皮柑橘离体叶片进行褐斑病病菌接种试验，将两次试验结果取交集，得到121份宽皮柑橘的褐斑病感抗结果。用121份宽皮柑橘表型对前人开发的CAPS进行验证，再对这121份宽皮柑橘的表型结果与利用简化基因组获得的SNP基因型结果进行主成分分析、GWAS分析和F_st分析，获得宽皮柑橘褐斑病抗性相关SNP位点。对通过GWAS获得的SNP进行基因型分析，在所有候选SNP的上下游25 kb范围内选取候选基因，根据phytozome注释对候选基因进行筛选。在感病资源‘秤砣红橘’和抗病资源‘新克里曼丁’接种褐斑病病菌24、48和72 h后，利用实时荧光定量PCR对筛选出的基因进行表达量分析。【结果】发现在121份宽皮柑橘中，温州蜜柑和克里曼丁类等67份资源表现抗褐斑病，大部分红橘和椪柑等54份资源感褐斑病。本研究发现前人开发的CAPS准确率为76.86%，其相关性为中等程度相关。以-log₁₀(P)>4.5为标准，GWAS筛选出6个强关联SNP；以F_st>0.38为标准，F_st筛选出8个SNP。GWAS筛选出的6个SNP的基因型与宽皮柑橘抗褐斑病表现为强相关，其中位于3号染色体上24 838 146 bp位置处Ciclev10021676的SNP1基因型对宽皮柑橘抗褐斑病区分能力最强。从14个SNP中筛选出Ciclev10018604、Ciclev10023485、Ciclev10023486、Ciclev10024586和Ciclev10019874等5个基因。这5个基因在‘秤砣红橘’接种病原菌后表达量上调，且都在48 h后表达量达到最高，上调倍数高达90倍。【结论】通过GWAS挖掘出3号染色体上24 838 146 bp位置的SNP与宽皮柑橘褐斑病抗病性相关性最显著，相关系数为0.641。并且该位置的基因型能比较有效地将抗性资源和感病资源进行区分。挖掘到Ciclev10019874、Ciclev10018604、Ciclev10023485、Ciclev10024586、Ciclev10023486等5个调控宽皮柑橘抗褐斑病的候选基因。

关键词: 宽皮柑橘, 褐斑病, 分子标记, 全基因组关联分析, 群体遗传分化指数分析

Abstract:

【Objective】The study objective was to explore candidate genes associated with resistance to Alternaria brown spot disease in citrus mandarin, so as to provide the basis for developing suitable molecular markers for further citrus resistance breeding work. 【Method】In summer and autumn of 2022, the young leaves of 136 citrus mandarin accessions were picked and inoculated with the fungus mycelium of Alternaria alternata in the laboratory. The combined results of two experiments were used to obtain the reliable results of 121 citrus mandarins responding to Alternaria brown spot. The phenotypes of 121 citrus mandarins were selected to verify the effectiveness of the CAPS marker developed in a previous study. Then, in order to obtain the candidate SNPs related to Alternaria brown spot resistance, the phenotypes of 121 citrus mandarins and corresponding SNP genotype data from GBS sequencing were analyzed with PCA, GWAS, and F_st methods, respectively. Candidate genes had been selected from the flanking region of 25 kb sequences surrounding the candidate SNPs site, and then they were screened out according to phytozome annotations. The expressions of candidate genes were analyzed after inoculating with the pathogen fungus on leave of Chengtuo hongju and Clementina (Algeria) for 24, 48, and 72 h. 【Result】Among 121 citrus mandarins, 67 varieties such as Clementine and Satsuma were resistant to Alternaria brown spot disease, whereas 54 varieties such as Hong Ju and Ponkan were susceptible. Some varieties resistance to Alternaria brown spot disease could discriminated by CAPS, but its accuracy only accounted for 76.86% in the study. GWAS analysis identified six significant SNPs highly associated with disease resistance, among which SNP1 located in Ciclev10021676 at 24 838 146 bp of chromosome 3 could be used to predicate the resistance of varieties, and their genotype showed a strong correlation with phenotype. Eight significant SNPs highly associated with disease resistance selected by F_st analysis. Finally, five genes of Ciclev10018604, Ciclev10023485, Ciclev10023486, Ciclev10024586 and Ciclev10019874 were screened out. The expression levels of these five genes in Chengtuo Hongju were up-regulated extensively after inoculating leaves with the pathogen fungus, and their expression levels reached the highest 48 hours later. 【Conclusion】Through GWAS, the SNP at 24 838 146 bp on chromosome 3 was found to be the most significant one with high resistance correlation to Alternaria brown spot disease, and the genotype at this location could be effectively used to distinguish the resistant varieties. The candidate genes responding to Alternaria brown spot disease in mandarin were discussed, i.e., Ciclev10019874, Ciclev10018604, Ciclev10023485, Ciclev10024586, and Ciclev10023486.

Key words: citrus mandarin, Alternaria brown spot, molecular marker, genome-wide association study, fixation index

杨胜男, 程莉, 谈月霞, 朱延松, 江东. 宽皮柑橘褐斑病抗性的全基因组关联分析[J]. 中国农业科学, 2023, 56(18): 3642-3654.

YANG ShengNan, CHENG Li, TAN YueXia, ZHU YanSong, JIANG Dong. Genome Wide Association Study for Resistance to Citrus Brown Spot Disease[J]. Scientia Agricultura Sinica, 2023, 56(18): 3642-3654.

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参考文献 41

[1]	GAI Y P, MA H J, CHEN Y N, LI L, CAO Y Z, WANG M S, SUN X P, JIAO C, RIELY B K, LI H Y. Chromosome-scale genome sequence of Alternaria alternata causing Alternaria brown spot of Citrus. Molecular Plant-Microbe Interactions, 2021, 34(7): 726-732. doi: 10.1094/MPMI-10-20-0278-SC
[2]	LLORENS E, FERNÁNDEZ-CRESPO E, VICEDO B, LAPEÑA L, GARCÍA-AGUSTÍN P. Enhancement of the citrus immune system provides effective resistance against Alternaria brown spot disease. Journal of Plant Physiology, 2013, 170(2): 146-154. doi: 10.1016/j.jplph.2012.09.018
[3]	陈昌胜, 黄峰, 程兰, 冯春刚, 黄涛江, 李红叶. 红橘褐斑病病原鉴定. 植物病理学报, 2011, 41(5): 449-455.
	CHEN C S, HUANG F, CHENG L, FENG C G, HUANG T J, LI H Y. Identification of the pathogenic fungus causing brown spot on tangerine (Citrus reticulata CV. Hongjv. Acta Phytopathologica Sinica, 2011, 41(5): 449-455. (in Chinese)
[4]	张玉洁, 杨续旺, 张志信, 李红超, 田洪, 张铁. 柑橘褐斑病的病原分离和药物筛选. 北方园艺, 2010(14): 169-171.
	ZHANG Y J, YANG X W, ZHANG Z X, LI H C, TIAN H, ZHANG T. Isolation of Citrus black patch pathogen and comparison of the sensitivities to eleven kind fungicides. Northern Horticulture, 2010(14): 169-171. (in Chinese)
[5]	REIS R F, DE ALMEIDA T F, STUCHI E S, DE GOES A. Susceptibility of citrus species to Alternaria alternata, the causal agent of the Alternaria brown spot. Scientia Horticulturae, 2007, 113(4): 336-342. doi: 10.1016/j.scienta.2007.04.005
[6]	SOLEL Z, KIMCHI M. Susceptibility and resistance of Citrus genotypes to Alternaria alternata pv. citri. Journal of Phytopathology, 1997, 145(8/9): 389-391. doi: 10.1111/jph.1997.145.issue-8-9
[7]	符雨诗, 罗君琴, 徐建国, 李红叶. 不同柑橘品种对链格孢褐斑病的感病性离体评价. 浙江农业学报, 2016, 28(1): 84-89.
	FU Y S, LUO J Q, XU J G, LI H Y. In vitro susceptibility evaluation of citrus cultivars to Alternaria alternata pv.citri. Acta Agriculturae Zhejiangensis, 2016, 28(1): 84-89. (in Chinese)
[8]	DALKILIC Z, TIMMER L W, GMITTER F G. Linkage of an Alternaria disease resistance gene in mandarin hybrids with RAPD fragments. Journal of the American Society for Horticultural Science, 2005, 130(2): 191-195. doi: 10.21273/JASHS.130.2.191
[9]	CUENCA J, ALEZA P, VICENT A, BRUNEL D, OLLITRAULT P, NAVARRO L. Genetically based location from triploid populations and gene ontology of a 3.3-mb genome region linked to Alternaria brown spot resistance in citrus reveal clusters of resistance genes. PLoS ONE, 2013, 8(10): e76755. doi: 10.1371/journal.pone.0076755
[10]	CUENCA J, ALEZA P, GARCIA-LOR A, OLLITRAULT P, NAVARRO L. Fine mapping for identification of Citrus Alternaria brown spot candidate resistance genes and development of new SNP markers for marker-assisted selection. Frontiers in Plant Science, 2016, 7: 1948.
[11]	ARLOTTA C, CIACCIULLI A, STRANO M C, CAFARO V, SALONIA F, CARUSO P, LICCIARDELLO C, RUSSO G, SMITH M W, CUENCA J, ALEZA P, CARUSO M. Disease resistant Citrus breeding using newly developed high resolution melting and CAPS protocols for Alternaria brown spot marker assisted selection. Agronomy, 2020, 10(9): 1368. doi: 10.3390/agronomy10091368
[12]	唐科志, 周常勇. 红橘响应褐斑病菌侵染的转录组学分析. 中国农业科学, 2020, 53(22): 4584-4600. doi: 10.3864/j.issn.0578-1752.2020.22.006. doi: 10.3864/j.issn.0578-1752.2020.22.006
	TANG K Z, ZHOU C Y. Transcriptome analysis of Citrus reticulata blanco, cv.Hongjv infected with Alternaria alternata tangerine pathotype. Scientia Agricultura Sinica, 2020, 53(22): 4584-4600. doi: 10.3864/j.issn.0578-1752.2020.22.006. (in Chinese) doi: 10.3864/j.issn.0578-1752.2020.22.006
[13]	DEMIRJIAN C, VAILLEAU F, BERTHOMÉ R, ROUX F. Genome-wide association studies in plant pathosystems: Success or failure? Trends in Plant Science, 2023, 28(4): 471-485. doi: 10.1016/j.tplants.2022.11.006
[14]	LI Y L, RUPERAO P, BATLEY J, EDWARDS D, DAVIDSON J, HOBSON K, SUTTON T. Genome analysis identified novel candidate genes for Ascochyta blight resistance in chickpea using whole genome re-sequencing data. Frontiers in Plant Science, 2017, 8: 359.
[15]	刘荣萍, 胡军华, 姚廷山, 王雪莲, 左佩佩, 王延杰, 李鸿筠. 柑橘褐斑病室内快速评价方法的研究. 果树学报, 2013, 30(5): 889-892.
	LIU R P, HU J H, YAO T S, WANG X L, ZUO P P, WANG Y J, LI H Y. A rapid laboratory evaluation method of citrus brown spot caused by Alternaria alternate. Journal of Fruit Science, 2013, 30(5): 889-892. (in Chinese)
[16]	王小柯, 江东, 孙珍珠. 利用GBS技术研究240份宽皮柑橘的系统演化. 中国农业科学, 2017, 50(9): 1666-1673. doi: 10.3864/j.issn.0578-1752.2017.09.012. doi: 10.3864/j.issn.0578-1752.2017.09.012
	WANG X K, JIANG D, SUN Z Z. Study on phylogeny of 240 mandarin accessions with genotyping-by-sequencing technology. Scientia Agricultura Sinica, 2017, 50(9): 1666-1673. doi: 10.3864/j.issn.0578-1752.2017.09.012. (in Chinese) doi: 10.3864/j.issn.0578-1752.2017.09.012
[17]	WRIGHT S. The genetical structure of populations. Annals of Eugenics, 1951, 15(4): 323-354. doi: 10.1111/j.1469-1809.1949.tb02451.x pmid: 24540312
[18]	PEEVER T L, OLSEN L, IBAÑEZ A, TIMMER L W. Genetic differentiation and host specificity among populations of Alternaria spp.causing brown spot of grapefruit and tangerine × grapefruit hybrids in Florida. Phytopathology, 2000, 90(4): 407-414. doi: 10.1094/PHYTO.2000.90.4.407
[19]	BHATTARAI G, SHI A N, MOU B Q, CORRELL J C. Resequencing worldwide spinach germplasm for identification of field resistance QTLs to downy mildew and assessment of genomic selection methods. Horticulture Research, 2022, 9: uhac205. doi: 10.1093/hr/uhac205
[20]	ALAVILLI H, LEE J J, YOU C R, POLI Y, KIM H J, JAIN A, SONG K. GWAS reveals a novel candidate gene CmoAP2/ERF in Pumpkin (Cucurbita moschata) involved in resistance to powdery mildew. International Journal of Molecular Sciences, 2022, 23(12): 6524. doi: 10.3390/ijms23126524
[21]	PHUKE R M, HE X Y, JULIANA P, KABIR M R, ROY K K, MARZA F, ROY C, SINGH G P, CHAWADE A, JOSHI A K, SINGH P K. Identification of genomic regions and sources for wheat blast resistance through GWAS in Indian wheat genotypes. Genes, 2022, 13(4): 596. doi: 10.3390/genes13040596
[22]	TERAKAMI S, ADACHI Y, IKETANI H, SATO Y, SAWAMURA Y, TAKADA N, NISHITANI C, YAMAMOTO T. Genetic mapping of genes for susceptibility to black spot disease in Japanese pears. Genome, 2007, 50(8): 735-741. pmid: 17893733
[23]	MORIYA S, TERAKAMI S, OKADA K, SHIMIZU T, ADACHI Y, KATAYOSE Y, FUJISAWA H, WU J, KANAMORI H, YAMAMOTO T, ABE K. Identification of candidate genes responsible for the susceptibility of apple (Malus × domestica Borkh.) to Alternaria blotch. BMC Plant Biology, 2019, 19(1): 1-13. doi: 10.1186/s12870-018-1600-2
[24]	DALIO R J D, MAGALHÃES D M, RODRIGUES C M, ARENA G D, OLIVEIRA T S, SOUZA-NETO R R, PICCHI S C, MARTINS P M M, SANTOS P J C, MAXIMO H J, PACHECO I S, DE SOUZA A A, MACHADO M A. PAMPs, PRRs, effectors and R-genes associated with citrus-pathogen interactions. Annals of Botany, 2017, 119(5): 749-774. doi: 10.1093/aob/mcw238 pmid: 28065920
[25]	KOURELIS J, VAN DER HOORN R A L. Defended to the nines: 25 years of resistance gene cloning identifies nine mechanisms for R protein function. The Plant Cell, 2018, 30(2): 285-299. doi: 10.1105/tpc.17.00579 pmid: 29382771
[26]	WANG J, TAN S J, ZHANG L, LI P, TIAN D C. Co-variation among major classes of LRR-encoding genes in two pairs of plant species. Journal of Molecular Evolution, 2011, 72(5/6): 498-509. doi: 10.1007/s00239-011-9448-1
[27]	SCHWESSINGER B, ZIPFEL C. News from the frontline: recent insights into PAMP-triggered immunity in plants. Current Opinion in Plant Biology, 2008, 11(4): 389-395. doi: 10.1016/j.pbi.2008.06.001 pmid: 18602859
[28]	CHAPARRO-GARCIA A, WILKINSON R C, GIMENEZ-IBANEZ S, FINDLAY K, COFFEY M D, ZIPFEL C, RATHJEN J P, KAMOUN S, SCHORNACK S. The receptor-like kinase SERK3/ BAK1 is required for basal resistance against the late blight pathogen Phytophthora infestans in Nicotiana benthamiana. PLoS ONE, 2011, 6(1): e16608. doi: 10.1371/journal.pone.0016608
[29]	DANNA C H, MILLET Y A, KOLLER T, HAN S W, BENT A F, RONALD P C, AUSUBEL F M. The Arabidopsis flagellin receptor FLS 2 mediates the perception of Xanthomonas Ax21 secreted peptides. Proceedings of the National Academy of Sciences of the United States of America, 2011, 108(22): 9286-9291.
[30]	TAKAI R, ISOGAI A, TAKAYAMA S, CHE F S. Analysis of flagellin perception mediated by flg22 receptor OsFLS2 in rice. Molecular Plant-Microbe Interactions, 2008, 21(12): 1635-1642. doi: 10.1094/MPMI-21-12-1635 pmid: 18986259
[31]	ENCISO-RODRÍGUEZ F E, GONZÁLEZ C, RODRÍGUEZ E A, LÓPEZ C E, LANDSMAN D, BARRERO L S, MARIÑO-RAMÍREZ L. Identification of immunity related genes to study the Physalis peruviana: Fusarium oxysporum pathosystem. PLoS ONE, 2013, 8(7): e68500. doi: 10.1371/journal.pone.0068500
[32]	LI J Y, WANG X D, ZHANG L R, MENG Q F, ZHANG N, YANG W X, LIU D Q. A wheat NBS-LRR gene TaRGA19 participates in Lr19-mediated resistance to Puccinia triticina. Plant Physiology and Biochemistry, 2017, 119: 1-8. doi: 10.1016/j.plaphy.2017.08.009
[33]	LV L L, LIU Y S, BAI S H, TURAKULOV K S, DONG C H, ZHANG Y G. A TIR-NBS-LRR gene MdTNL1 regulates resistance to Glomerella leaf spot in apple. International Journal of Molecular Sciences, 2022, 23(11): 6323. doi: 10.3390/ijms23116323
[34]	GAO Y L, WANG B W, XU Z L, LI M Y, SONG Z B, LI W Z, LI Y P. Tobacco serine/threonine protein kinase gene NrSTK enhances black shank resistance. Genetics and Molecular Research, 2015, 14(4): 16415-16424. doi: 10.4238/2015.December.9.11 pmid: 26662438
[35]	马璐琳, 段青, 杜文文, 张艺萍, 崔光芬, 贾文杰, 吴学尉, 王祥宁, 王继华. 泸定百合丝氨酸/苏氨酸蛋白激酶基因(LsS/TK)的克隆与表达分析. 分子植物育种, 2020, 18(18): 5925-5932.
	MA L L, DUAN Q, DU W W, ZHANG Y P, CUI G F, JIA W J, WU X W, WANG X N, WANG J H. Cloning and expression analysis of a serine/threonine protein kinase gene (LsS/TK) in Lilium sargentiae. Molecular Plant Breeding, 2020, 18(18): 5925-5932. (in Chinese)
[36]	BRUEGGEMAN R, DRADER T, KLEINHOFS A. The barley serine/threonine kinase gene Rpg1 providing resistance to stem rust belongs to a gene family with five other members encoding kinase domains. Theoretical and Applied Genetics, 2006, 113(6): 1147-1158. doi: 10.1007/s00122-006-0374-3 pmid: 16896706
[37]	CHAKRABORTY S, NGUYEN B, WASTI S D, XU G Z. Plant leucine-rich repeat receptor kinase (LRR-RK): Structure, ligand perception, and activation mechanism. Molecules, 2019, 24(17): 3081. doi: 10.3390/molecules24173081
[38]	CHEN T S. Identification and characterization of the LRR repeats in plant LRR-RLKs. BMC Molecular and Cell Biology, 2021, 22(1): 9. doi: 10.1186/s12860-021-00344-y pmid: 33509084
[39]	MATSUSHIMA N, MIYASHITA H. Leucine-rich repeat (LRR) domains containing intervening motifs in plants. Biomolecules, 2012, 2(2): 288-311. doi: 10.3390/biom2020288 pmid: 24970139
[40]	DÍAZ L, DEL RÍO J A, PÉREZ-GILABERT M, ORTUÑO A. Involvement of an extracellular fungus laccase in the flavonoid metabolism in Citrus fruits inoculated with Alternaria alternata. Plant Physiology and Biochemistry, 2015, 89: 11-17. doi: 10.1016/j.plaphy.2015.02.006
[41]	ORTUÑO A, NEMSA I, ALVAREZ N, LACASA A, PORRAS I, GARCIA LIDÓN A, DEL RÍO J A. Correlation of ethylene synthesis in Citrus fruits and their susceptibility to Alternaria alternata pv. citri. Physiological and Molecular Plant Pathology, 2008, 72(4/5/6): 162-166. doi: 10.1016/j.pmpp.2008.08.003

引物名称 Primer name	序列 Primer sequence	扩增长度 Length (bp)
SNP08-spare-F	AGGATTGTGAGGTGACGCTT	747
SNP08-spare-R	GCAACATATCCAAATGCGCC	747

基因名称 Gene name	引物名称 Primer name	序列 Primer sequence
β-Actin	F	CCCCATCGTTACCGTCCAG
β-Actin	R	CGCCTTGCCAGTTGAATATCC
Ciclev10018604	F	GGGAGCTTGTGGCACTGTAT
Ciclev10018604	R	TCCTTCACCACGCAACTTGA
Ciclev10019874	F	GAATCCCTTCTCTCCGGCTG
Ciclev10019874	R	GTGGTGGAGTGATGAGGTGG
Ciclev10023485	F	GAGTCACCTGACAAGAGAATGC
Ciclev10023485	R	ACTTGCAGCAACATCATCCAAG
Ciclev10024586	F	GCATAGGCCTTGCCAATGTT
Ciclev10024586	R	CGATAATCAACGACACGGGC
Ciclev10023486	F	AAACAGCATTCCAAGCGTGC
Ciclev10023486	R	AATCCCATCCGCATAGGTCG

来源 Source	编号 Code	染色体 Chromosome	位置 Location (bp)	P值/F_st值 P value/ F_stvalue
GWAS筛选 Screening of GWAS	SNP1	3	24838146	7.742227
GWAS筛选 Screening of GWAS	SNP2	3	29033460	6.01147
GWAS筛选 Screening of GWAS	SNP3	2	9623685	5.404526
GWAS筛选 Screening of GWAS	SNP4	3	10912656	4.899702
GWAS筛选 Screening of GWAS	SNP5	3	29379604	4.524974
GWAS筛选 Screening of GWAS	SNP6	3	29379461	4.504927
F_st筛选 Screening of F_st	SNP7	5	9384031	0.427099
F_st筛选 Screening of F_st	SNP8	3	21846432	0.407686
F_st筛选 Screening of F_st	SNP9	3	30824581	0.402405
F_st筛选 Screening of F_st	SNP10	2	32716438	0.400017
F_st筛选 Screening of F_st	SNP11	3	11889486	0.395677
F_st筛选 Screening of F_st	SNP12	3	30230610	0.391282
F_st筛选 Screening of F_st	SNP13	3	30132014	0.381140
F_st筛选 Screening of F_st	SNP14	3	33601151	0.380600

基因 Gene	注释 Annotation	连锁SNP编号 Chain SNP code
Ciclev10019874	丝氨酸/苏氨酸-蛋白激酶wag1 Serine/threonine-protein kinase wag1	SNP2
Ciclev10023485	非特异性丝氨酸/苏氨酸蛋白激酶；苏氨酸特异性蛋白激酶 Non-specific serine/threonine protein kinase; Threonine-specific protein kinase	SNP14
Ciclev10018604	蛋白激酶结构域；富含亮氨酸重复序列N端结构域（LRRNT_2）；富含亮氨酸重复序列（LRR_8） Protein kinase domain; Leucine rich repeat N-terminal domain (LRRNT_2); Leucine rich repeat (LRR_8)	SNP2
Ciclev10024586	富含亮氨酸重复序列（LRR_1）；富含亮氨酸重复序列N端结构域（LRRNT_2） Leucine Rich Repeat (LRR_1); Leucine rich repeat N-terminal domain (LRRNT_2)	SNP14
Ciclev10023486	富含亮氨酸重复序列的蛋白 Leucine-rich repeat-containing protein	SNP8