基于SNP标记桃抗蚜性状的基因定位

doi:10.3864/j.issn.0578-1752.2017.23.014

摘要/Abstract

摘要： 【目的】挖掘与桃抗蚜性状紧密连锁的SNP位点及候选基因，为桃抗性分子标记辅助选择育种奠定基础，并为进一步揭示桃抗蚜性状的遗传基础和分子机制提供依据。【方法】以来源于‘粉寿星’的抗蚜桃‘01-77-3’为母本，栽培品种‘中油桃13号’为父本，杂交获得F1代分离群体进行基因定位。以‘96-5-1’（抗蚜）为母本，‘10-7’（感蚜）为父本杂交获得F1分离群体作为验证定位位点准确性的材料。参考桃基因组序列（Prunus persica Genome v2.0.a1）开发基于Sanger测序的SNP标记，在亲本（‘01-77-3’‘中油桃13号’）及各4个子代中PCR扩增后进行Sanger测序，获得候选SNP后，扩大群体验证，实现对抗性基因的初步定位。对亲本（‘01-77-3’‘中油桃13号’‘96-5-1’‘10-7’）进行覆盖度约为70×的全基因组深度测序，并基于重测序数据，在初定位区间内开发基于Sanger测序与HRM分析的SNP标记，筛选多态性标记，对‘01-77-3’×‘中油桃13号’杂交后代141株实生苗进行基因分型，以获得与桃抗蚜型基因紧密连锁的分子标记。通过双亲（‘96-5-1’‘10-7’）表型与基因型一致，在精细定位区间内开发与桃抗蚜性状紧密连锁的InDel位点，以验证InDel标记与抗蚜表型是否连锁。最后，参考桃基因组分析定位区间的候选基因。【结果】通过人工接种观察蚜虫对桃F1后代单株新梢危害表明，抗蚜与感蚜比例接近1﹕1（P值为0.556；χ²为0.348），符合孟德尔遗传规律。基于Sanger测序结果，初步将抗蚜基因定位在桃基因组Pp01_38011783与Pp01_47231340之间，物理距离约为9.22 Mb。亲本全基因组深度测序分别产生Clean data 17.109 Gb，平均覆盖度为75.19×，在Pp01初定位区间内开发基于HRM与Sanger测序的SNP标记共29对，筛选后得到11对紧密连锁的标记。基因分型结果表明，与桃抗蚜基因紧密连锁的标记位于桃基因组Pp01的45.66 Mb和46.12 Mb处，Pp01_45.66处等位基因为T和C，Pp01_46.12处等位基因为G和C，遗传距离分别为1.4 cM、2.1 cM；与抗蚜基因完全连锁的标记SNP_Pp01_45712702，位于桃基因组Pp01_45.71处，等位基因为G和T。基于亲本（‘96-5-1’‘10-7’）重测序数据，在精细定位区间内开发紧密连锁的InDel标记KYYZ_Pp01_45799758，位于Pp01_45.79处。对验证群体92个单株进行PCR扩增，经聚丙烯酰胺凝胶电泳检测表明，仅1个单株基因型与表型不符，分子鉴定准确率为98.91%。【结论】利用亲本深度测序与SNP标记技术相结合的方法，精细定位了控制桃抗蚜性状的基因，将抗蚜基因缩小到物理区间460 Kb内，共包含56个转录本，包括52个候选基因。

关键词: 桃, SNP标记, 抗蚜, 基因定位

Abstract: 【Objective】 The objective of this study is to identify the SNP loci tightly linked to peach( Prunus persica (L.) Batsch ) aphid(Myzus persicae (Sulzer)) resistance traits, revealing its genetic basis and laying a foundation for the marker assistant selection in resistance breeding of peach.【Method】In this study, the population used for the mapping study consisted of141 individuals which were obtained from a cross between female parent (‘01-77-3’ ) and male parent (‘CN13’). Referencing the peach genome and using Sanger sequencing, single nucleotide polymorphism (SNP) markers were developed in female and male parents and 8 progenies to obtain markers linked to the target loci which were tested on the whole population. Subsequently, using whole genome re-sequencing data of two parents, SNPs for fine mapping were selected based on the genotype of two parents, andemployed to conduct genotyping to obtain the SNP marker linked to resistance traits. Ultimately, the fine mapping region was validated by using an InDel marker to verify the genotype of F₁ population generated from ‘96-5-1’ × ‘10-7’. 【Result】 As a result of phenotype identification of 141 progenies, the segregation ratio of resistance to aphid to susceptible ones showed 1﹕1 (P: 0.556; χ²: 0.348). Using Sanger sequencing we mapped the resistant gene to an approximate 9.92 Mb physical distance between two SNP markers, Pp01_38011783 and Pp01_47231340 on Pp01. For fine mapping, a total of 17.109 Gb clean data was generated from genome re-sequencing and the average coverage depth is 75.19×. 11 of 29 pairs of primers which were designed based on genome re-sequencing data were effective and linked to target trait. As a result of genotyping, we obtained two SNP makers tightly linked to desired trait, SNP_Pp01_45665389 and SNP_Pp01_46120950, with genetic distance of 1.4 cM and 2.1 cM, respectively. The target locus was between these two markers, an approximate physical distance of 460 Kb, and the gene was co-segregating with another marker SNP_Pp01_45712702. With fine gene mapping region, an InDel marker, KYYZ_Pp01_45799758, was designed and used to verify the phenotype of 92 individuals generated from an F₁ segregation population of ‘96-5-1’ ×‘10-7’ with 98.91% accuracy.【Conclusion】The SNP loci and candidate genes related closely with aphid-resistant gene of peach were identified in this study. The resistant gene had been mapped to an approximate 460 kb physical distance between two SNP markers, SNP_Pp01_45665389 and SNP_Pp01_46120950 at the bottom of Pp01 which contains 56 transcripts (52 candidate genes).

Key words: Prunus persica, SNP markers, aphid-resistant, gene mapping

张南南，鲁振华，崔国朝，潘磊，曾文芳，牛良，王志强. 基于SNP标记桃抗蚜性状的基因定位[J]. 中国农业科学, 2017, 50(23): 4613-4621.

ZHANG NanNan, LU ZhenHua, CUI GuoChao, PAN Lei, ZENG WenFang, NIU Liang, WANG ZhiQiang. Gene Mapping of Aphid-Resistant for Peach Using SNP Markers[J]. Scientia Agricultura Sinica, 2017, 50(23): 4613-4621.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.23.014

https://www.chinaagrisci.com/CN/Y2017/V50/I23/4613

参考文献

[1] 牛良, 鲁振华, 曾文芳, 崔国朝, 潘磊, 徐强, 李国怀, 王志强. ‘粉寿星’对桃绿蚜抗性的遗传分析. 果树学报, 2016, 33(5): 578-584.

NIU L, LU Z H, ZENG W F, CUI G C, PAN L, XU Q, LI G H, WANG Z Q. Inheritance analysis of resistance to green peach aphid (Myzus persicae (Sulzer)) for peach cultivar ‘Fen Shouxing’ (Prunus persica var. densa). Journal of Fruit Science, 2016, 33(5): 578-584. (in Chinese)

[2] SMITH C M, CHUANG W B. Plant resistance to aphid feeding: Behavioral, physiological, genetic and molecular cues regulate aphid host selection and feeding. Pest Management Science, 2014, 70(40): 528-540.

[3] BLACKMAN R L, EASTOP V F. Aphids on the world's crops. John Wiley & Sons Ltd, 2000.: an identification and information guide

[4] DEVONSHIRE A L, FIELD L M, FOSTER S P. The evolution of insecticide resistance in the peach-potato aphid, Myzus persicae. Philosophical Transactions of the Royal Society B: Biological Sciences, 1998, 353(1376): 1677-1684.

[5] FUENTES-CONTRERAS E, FIGUEROA C C, REYES M.Genetic diversity and insecticide resistance of Myzus persicae (Hemiptera: Aphididae) populations from tobacco in Chile: evidence for the existence of a single predominant clone. Bulletin of Entomological Research, 2004, 94(1): 11-18.

[6] FOSTER J C, RIDEOUT W. Storm enhanced density: Magnetic conjugacy effects.Annales Geophysicae, 2007, 25(8): 1791-1799.

[7] MASSONIÉ G, MAISON P, MONET R. Résistance au puceron vert du pêcher Myzus persicae Sulzer (Homoptera: Aphididae) chez Prunus persicaL. Batsch. et d’autres espèces de prurrus. Agronomie, 1982, 2(1): 63-70.

[8] MONET R, MASSONIÉ G. Déterminisme génétique de la résistance au puceron vert (Myzus persicae) chez le pêcher Résultats complémentaires. Agronomie, 1994, 14(3): 177-182.

[9] SAUGE M H, KERVELLA J, RAHBÉ Y. Probing behaviour of the green peach aphid Myzus persicae on resistant Prunus genotypes. Entomologia Experimentalis et Applicata, 1998, 89(3): 223-232.

[10] SAUGE M H, LACROZE J P, POËSSEL J L. Induced resistance by Myzus persicae in the peach cultivar ‘Rubira’. Entomologia Experimentalis et Applicata, 2002, 102(1): 29-37.

[11] SAUGE M H, MUS F, LACROZE J P. Genotypic variation in induced resistance and induced susceptibility in the peach-Myzus persicae aphid system. Oikos, 2006, 113(2): 305-313.

[12] PASCAL T, PFEIFFER F, KERVELLA J, LACROZE J P, Sauge M H, Weber W E. Inheritance of green peach aphid resistance in the peach cultivar ‘Rubira’. Plant Breeding, 2002, 121(5): 459-461.

[13] LU Z, NIU L, CHAGNÉ D. Fine mapping of the temperature- sensitive semi-dwarf (Tssd) locus regulating the inter-node length in peach (Prunus persica). Molecular Breeding, 2016, 36(2): 1-11.

[14] 鲁振华, 牛良, 张南南, 崔国朝, 潘磊, 曾文芳, 王志强. 基于HRM获得与桃Tssd紧密连锁的SNP标记. 中国农业科学, 2017, 50(8): 1505-1513.

LU Z H, NIU L, ZHANG N N, CUI G C, PAN L, ZENG W F, WANG Z Q. SNP marker tightly linked to Tssd for peach using high resolution melting analysis. Scientia Agricultura Sinica, 2017, 50(8): 1505-1513. (in Chinese)

[15] BROOKES A J. The essence of SNPs. Gene, 1999, 234(2): 177-186.

[16] MURANTY H, JORGE V, BASTIEN C.Potential for marker-assisted selection for forest tree breeding: lessons from 20 years of MAS in crops. Tree Genetics & Genomes, 2014, 10(6): 1491-1510.

[17] DIRLEWANGER E, COSSON P, BOUDEHRI K. Development of a second-generation genetic linkage map for peach [Prunus persica (L.) Batsch] and characterization of morphological traits affecting flower and fruit. Tree Genetics & Genomes, 2006, 3(1): 1-13.

[18] PICAÑOL R, EDUARDO I, ARANZANA M J. Combining linkage and association mapping to search for markers linked to the flat fruit character in peach. Euphytica, 2013, 190(2): 279-288.

[19] MARTÍNEZ GARCÍA J S. Fracaso escolar, PISA y la difícil ESO. RASE: Revista de la Asociación de Sociología de la Educación, 2013, 2(1): 56-85.

[20] HAN Y, CHAGNÉ D, GASIC K. BAC-end sequence-based SNPs and Bin mapping for rapid integration of physical and genetic maps in apple. Genomics, 2009, 93(3): 282-288.

[21] VERDE I, BASSIL N, SCALABRIN S. Development and evaluation of a 9K SNP array for peach by internationally coordinated SNP detection and validation in breeding germplasm. PLoS One, 2012, 7(4): e35668.

[22] 许建兰, 张斌斌, 马瑞娟, 俞明亮, 沈志军, 周懋. 春季异常气候对桃树蚜虫发生及坐果率的影响. 江西农业学报, 2014, 26(11): 21-23, 28.

XU J L, ZHANG B B, MA R J, YU M L, SHEN Z J, ZHOU M. Effects of Spring abnormal climate on aphid occurrence and fruit setting rate of peach. Acta Agriculturae Jiangxi, 2014, 26(11): 21-23, 28. (in Chinese)

[23] 乔岩, 董杰, 王品舒, 岳瑾, 张保常, 张金良, 袁志强, 杨建国. 三种生物源农药对桃树蚜虫的防治效果研究. 生物技术进展, 2015, 5(6): 468-470.

QIAO Y, DONG J, WANG P S, YUE J, ZHANG B C, ZHANG J , YUAN Z Q, YANG J G. Control effects of three biogenic pesticides on controlling peach aphids. Current Biotechnology, 2015, 5(6): 468-470. (in Chinese)

[24] MENG J Y, ZHANG C Y, CHEN X J. Differential protein expression in the susceptible and resistant Myzus persicae (Sulzer) to imidacloprid. Pesticide Biochemistry and Physiology, 2014, 115: 1-8.

[25] CRAVEDI P, CERVATO P. Resistance to insecticides of the green peach aphid and integrated fruit production guidelines. IOBC wprs Bulletin, 1997, 20: 75-78.

[26] PAUQUET J, BURGET E, HAGEN L. Map-based cloning of the Vat gene from melon conferring resistance to both aphid colonization and aphid transmission of several viruses//Proceedings of the 8th EUCARPIA Meeting on Cucurbit Genetics and Breeding: Cucurbitaceae, 2004: 325-329.

[27] KALOSHIAN I, YAGHOOBI J, LIHARSKA T. Genetic and physical localization of the root-knot nematode resistance locus Mi in tomato. Molecular and General Genetics, 1998, 257(3): 376-385.

[28] MILLIGAN S B, BODEAU J, YAGHOOBI J. The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes. The Plant Cell, 1998, 10(8): 1307-1319.

[29] ALSTON F H, BRIGGS J B. Resistance to Sappaphis devecta (Wlk.) in apple. Euphytica, 1968, 17(3): 468-472.

[30] MONET R, GUYE A, MASSONIE G. Breeding for resistance to green aphid Myzus persicae Sulzer in the peach. IV International Peach Symposium 465, 1997: 171-176.

[31] LAMBERT P, PASCAL T. Mapping Rm2 gene conferring resistance to the green peach aphid (Myzus persicae Sulzer) in the peach cultivar ‘Rubira®’. Tree Genetics & Genomes, 2011, 7(5): 1057-1068.

[32] PASCAL T, ABERLENC R, CONFOLENT C. Mapping of new resistance (Vr2, Rm1) and ornamental (Di2, pl) Mendelian trait loci in peach. Euphytica, 2017, 213(6): 132.

[33] FURBANK R T. Plant phenomics: from gene to form and function. Functional Plant Biology, 2009, 36(10): 5-6.