基于HRM获得与桃Tssd紧密连锁的SNP标记

doi:10.3864/j.issn.0578-1752.2017.08.014

Abstract

Abstract: 【Objective】 SNP marker is characterized as highly distribution, high resolution and co-domination in plant organism, which is considered as a promising molecular marker. The achievements of peach whole genome sequencing generated massive amounts of SNPs. How to establish a sensitive and effective method for identifying genotyping of different SNP genotypes is a base of further research on gene mapping, cultivar identification and linkage map construction. 【Method】 The aim of this study was to determine if HRM can detect the genotyping of different SNP genotypes and identify a SNP marker tightly linked to PpTssd gene. The segregation population of semi-dwarf progeny was selected with parents 97-32-46 (ST) × 03-94-2 (Tssd). According to gene mapping, different types of SNP markers within mapping region were developed, and the HRM analysis was employed to conduct SNP genotyping and the SNP marker linked to desired traits were generated.【Result】As the key factors for genotyping, the proper concentrations of temperate DNA and Mg²⁺ were established. In the 15 µL PCR reaction system, genotyping could not be complete when template DNA was less than 5 ng and Mg²⁺ was less than 1.6 µmol·L^-1. The primers were designed based on the phenotype and genotype, spanning each desired SNP to amplify DNA fragments shorter than 150 bp. HRM analysis could discriminate four types of SNPs (A/T, A/G, A/C, and C/G) occurred via single nucleotide mutation and the result was validated by Sanger sequence. HRM analysis divided temperature-sensitive semi-dwarf and standard type individuals into two groups. A SNP tightly linked to Tssd gene was identified in 96 individuals consisting of 48 Tssd and 48 ST, respectively. The HRM technique distinguished the Tssd and ST into two groups except for one individual with null amplification. Ultimately, the homozygous A/A and heterozygous A/T were identified, and generated a SNP tightly linked to Tssd gene with 2.11cM with two recombinants.【Conclusion】SNP genotyping of different SNPs were established based on HRM analysis. Although HRM could not distinguish two types of homozygote, HRM analysis still can be a effective method for SNP genotyping and can be used for gene mapping, genetic diversity and cultivar identification in peach based on this study.

Key words: peach, high resolution melting (HRM), SNP genotyping, temperature-sensitive semi-dwarf type

LU ZhenHua, NIU Liang, ZHANG NanNan, CUI GuoChao, PAN Lei, ZENG WenFang, WANG ZhiQiang. SNP Marker Tightly Linked to Tssd for Peach Using High Resolution Melting Analysis[J].Scientia Agricultura Sinica, 2017, 50(8): 1505-1513.

References

[1] Semagn K, Babu R, Hearne S, Olsen M. Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Molecular Breeding, 2014, 33(1): 1-14.

[2] Schlotterer C. The evolution of molecular markers-just a matter of fashion? Nature Reviews Genetics, 2004, 5: 63-69.

[3] Micheletti D, Troggio M, Zharkikh A, Costa F, Malnoy M, Velasco R, Salvi S. Genetic diversity of the genus Malus and implications for linkage mapping with SNPs. Tree Genetics & Genomes, 2011, 7(4): 857-868.

[4] CAO K, ZHENG Z J, WANG L R, LIU X, ZHU G R, FANG W C, CHENG S F, ZENG P, CHEN C W, WANG X W, XIE M, ZHONG X, WANG X L, ZHAO P, BIAN C, ZHU Y L, ZHANG J H, MA G S, CHEN C X, LI Y J, HAO F G, LI Y, HUANG G D, LI Y X, LI H Y, GUO J, XU X, WANG J. Comparative population genomics reveals the domestication history of the peach, Prunus persica, and human influences on perennial fruit crops. Genome Biology, 2014, 15: 415.

[5] Abe A, Kosugi S, Yoshida K, Natsume S, Takagi H, Kanzaki H, Matsumura K, Yoshida K, Mitsuoka C, Tamiru M, Innan H, Cano L, Kamoun S, Terauchi R. Genome sequencing reveals agronomically important loci in rice using MutMap. Nature Biotechnology, 2012, 30(2): 174-179.

[6] Shirasawa K, Fukuoka H, Matsunaga H, Kobayashi Y, Kobayashi I, Hirakawa H, Isobe S, Tabata S. Genome- wide association studies using single nucleotide polymorphism markers developed by re-Sequencing of the genomes of cultivated tomato. DNA Research, 2013, 20: 593-603.

[7] Dardick C, Callahan A, Horn R, Ruiz K B, Zhebentyayeva T, Hollender C, Whitaker M, Abbott A, Scorza R. PpeTAC1 promotes the horizontal growth of branches in peach trees and is a member of a functionally conserved gene family found in diverse plants species. The Plant Journal, 2013, 75: 618-630.

[8] Takaji H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, Uemura A, Utsushi H, Tamiru M, Takuno S, Innan H, Cano L M, Kamoun S, Terauchi R. QTL-seq: rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. The Plant Journal, 2013, 74: 174-183.

[9] Hanke M, Wink M. Direct DNA sequencing of PCR-amplified vector inserts following enzymatic degradation of primer and dNTPs. Biotechniques, 1994, 17: 858-860.

[10] TILL B J, REYNOLDS S H, GREENE E A, CODOMO C A, ENNS L C, JOHNSON J E, BURTNER C, ODDEN A R, YOUNG K, TAYLOR N E, Henikoff J G, Comai L, Henikoff S. Large-scale discovery of induced point mutations with high-throughput Tilling. Genome Research, 2003, 13: 524-530.

[11] Glava? D, Dean M. Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations. Human Mutation, 2005, 2(5): 404-414.

[12] GUNDERSON K L, STEEMERS F J, LEE G, MENDOZA L G, CHEE M S. A genome-wide scalable SNP genotyping assay using microarray technology. Nature Genetics, 2005, 37: 549-554.

[13] FULLWOOD M J, WEI C L, LIU E T, RUAN Y. Next-generation DNA sequencing of paired-end tags (PET) for transcriptome and genome analyses. Genome Research, 2009, 19: 521-532.

[14] GUNDRY C N, VANDERSTEEN J G, REED G H, PRYOR R J, CHEN J, WITTWER C T. Amplicon melting analysis with labeled primers: A closed-tube method for differentiating homozygotes and heterozygotes. Clinical Chemistry, 2003, 49: 396-406.

[15] GARRITANO S, GEMIGNANI F, VOEGELE C, NGUYEN- DUMONT T, LE CALVEZ-KELM F, DE SILVA D, LESUEUR F, LANDI S, TAVTIGIAN S V. Determining the effectiveness of high resolution melting analysis for SNP genotyping and mutation scanning at the TP53 locus. BMC Genetics, 2009, 10: 5. doi: 10.1186/1471- 2156-10-5.

[16] CHANGÉ D, GASIC K, CROWHURSTT R N, HAN Y P, BASSETT H C, BOWATTE D R, LAWRENCE T J, RIKKERINK E H A, GARDINER S E, KORBAN S S. Development of a set of SNP markers present in expressed genes of the apple. Genomics, 2008, 92: 353-358.

[17] Botticella E, Sestili F, Hernandez-Lopez A, Phillips A, Lafiandra D. High resolution melting analysis for the detection of EMS induced mutations in wheat SbeIIa genes. BMC Plant Biology, 2011, 11: 156.

[18] 李炜, 田义轲, 王彩虹, 白牡丹, 侯董亮. 通过HRM技术筛查与梨矮生性状决定位点PcDw紧密连锁的SNP标记. 园艺学报, 2015, 42(2): 214-220.

LI W, TIAN Y K, WANG C H, BAI M D, HOU D L. Screening of SNP markers tightly linked to PcDw locus determining Pear dwarf trait using HRM technology. Acta Horticulturae Sinica, 2015, 42(2): 214-220. (in Chinese)

[19] LI J S, WANG X M, DONG R X, YANG Y, ZHOU J, YU C L, CHENG Y, YAN C Q, CHEN J P. Evaluation of high-resolution melting for gene mapping in rice. Plant Molecular Biology Reporter, 2011, 29: 979-985.

[20] DARDICK C, CALLAHAN A, HORN R, RUIZ K B, ZHEBENTYAYEVA T, HOLLENDER C, WHITAKER M, ABBOTT A, SCORZA R. PpeTAC1 promotes the horizontal growth of branches in peach trees and is a member of a functionally conserved gene family found in diverse plants species. The Plant Journal, 2013, 75: 618-630.

[21] HOLLENDER C A, HADIARTOT, SRINIVASAN C, SCORZA R, DARDICK C. A brachytic dwar?sm trait (dw) in peach trees is caused by anonsense mutation within the gibberellic acid receptor PpeGID1c. New Phytologist, 2015, 210: 227-239.

[22] 王志强, 牛良, 刘淑娥, 宋银花, 宗学普. 半矮化油桃新种质—SD9238. 果树学报, 2004, 21(5): 503-504.

WANG Z Q, NIU L, LIU S E, SONG Y H, ZONG X P. ‘SD9238’, A New Semi-dwarf germplasm of nectarine, Journal of Fruit Science, 2004, 21(5): 503-504. (in Chinese)

[23] Lu Z H, Niu L, Chagné D, Cui G C, Pan L, Foster T, Zhang R P, Zeng W F, Wang Z Q. Fine mapping of the temperature-sensitive semi-dwarf, (Tssd) locus regulating the internode length in peach (Prunus persica). Molecular Breeding, 2016, 36(2): 1-11.

[24] Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar S K, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Ri A D, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-Ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald L M, Gutin N, Lanchbury J, Macalma T, Mitchell J T, Reid J, Wardell B, Kodira C, Chen Z T, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D, Schaeffer S, Krishnan V, Wu C J, Chu V T, King S T, Vick J, Tao Q Z, Mraz A, Stormo A, Stormo K, Bogden R, Ederle D, Stella A, Vecchietti A, Kater M M, Masiero S, Lasserre P, Lespinasse Y, Allan A C, Bus V, Chagné D, Crowhurst R N, Gleave A P, Lavezzo E, Fawcett J A, Proost S, Rouzé P, Sterck L, Toppo S, Lazzari B, Hellens R P, Durel C, Gutin A, Bumgarner R E, Gardiner S E, Skolnick M, Egholm M, Peer Y V, Salamini F, Viola R. The genome of the domesticated apple (Malus ×domestica Borkh.). Nature Genetics, 2010, 42 (10): 833-839.

[25] Shulaev V, Sargent D J, Crowhurst R N, Mockler T C, Folkerts O, Delcher A L, Jaiswal P, Mockaitis K, Liston A, Mane S P, Burns P, Davis T M, Slovin J P, Bassil N, Hellens R P, Evans C, Harkins T, Kodira C, Desany B, Crasta O R, Jensen R V, Allan A C, Michael T P, Setubal J C, Celton J M, Rees D J G, Williams K P, Holt S H, Rojas J J R, Chatterjee M, Liu B, Silva H, Meisel L, Adato A, Filichkin S A, Troggio M, Viola R, Ashman T L, Wang H, Dharmawardhana P, Elser J, Raja R, Priest H D, Bryant D W, Fox S E, Givan S A, Wilhelm L J, Naithani S, Christoffels A, Salama D Y, Carter J, Girona E L, Zdepski A, Wang W, Kerstetter R A, Schwab W, Korban S S, Davik J, Monfort A, Denoyes-Rothan B, Arus P, Mittler R, Flinn B, Aharoni A, Bennetzen J L, Salzberg S L, Dickerman A W, Velasco R, Borodovsky M, Veilleux R E, Folta K M. The genome of woodland strawberry (Fragaria vesca). Nature Genetics, 2011, 43 (2): 109-116.

[26] Wu J, Wang Z, Shi Z, Zhang S, Ming R, Zhu S, Khan M A, Tao S, Korban S S, Wang H, Chen N J, Nishio T, Xu X, Cong L, Qi K, Huang X, Wang Y, Zhao X, Wu J, Deng C, Gou C, Zhou W, Yin H, Qin G, Sha Y, Tao Y, Chen H, Yang Y, Song Y, Zhan D, Wang J, Li L, Dai M, Gu C, Wang Y, Shi D, Wang X, Zhang H, Zeng L, Zheng D, Wang C, Chen M, Wang G, Xie L, Sovero V, Sha S, Huang W, Zhang S, Zhang M, Sun J, Xu L, Li Y, Liu X, Li Q, Shen J, Wang J, Paull R E, Bennetzen J L, Wang J, Zhang S. The genome of the pear (Pyrus bretschneideri Rehd.). Genome Research, 2012, 23(2): 396-408.

[27] Verde I, Abbott A G, Scalabrin S, Jung S, Shu S Q, Marroni F, Zhebentyayeva T, Dettori M T, Grimwood J, Cattonaro F, Zuccolo A, Rossini L, Jenkins J, Vendramin E, Meisel L A, Decroocq V, Sosinski B, Prochnik S, Mitros T, Policriti A, Cipriani G, Dondini L, Ficklin S, Goodstein D M, Xuan P F, Fabbro C D, Aramini V, Copetti D, Gonzalez S, Horner D S, Falchi R, Lucas S, Mica E, Maldonado J, Lazzari B, Bielenberg D, Pirona R, Miculan M, Barakat A, Testolin R, Stella A, Tartarini S, Tonutti P, Arús P, Orellana A, Wells C, Main D, Vizzotto G, Silva H, Salamini F, Schmutz J, Morgante M, Rokhsar D S. The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution. Nature Genetics, 2013, 45(5): 487-494.

[28] Doyle J J, Doyle J L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin, 1987, 19: 11-15

[29] Batley J, Edwards D. Mining for SNPs and SSRs using SNPServer, dbSNP and SSR taxonomy tree., Methods in Molecular Biology, 2009, 537: 303-321.

[30] BALL A, STAPLEY J, DAWSON D, BIRKHEAD T R, BURKE T, SLATE J. A comparison of SNPs and microsatellites as linkage mapping markers: lessons from the zebra finch (Taeniopygia guttata). BMC Genomics, 2010, 11(1): 218.

[31] Yu H, Xie W, Wang J, Xing Y, Xu C, Li X, Xiao J, Zhang Q. Gains in QTL detection using an ultra-high density snp map based on population sequencing relative to traditional RFLP/SSR markers. PLoS ONE, 2011, 6(3): e17595.

[32] Verde I o, Bassil N, Scalabrin S, Gilmore B, Lawley C T, Gasic K, Micheletti D, Rosyara U R, Cattonaro F, Vendramin E, Main D, Aramini V, Blas A L, Mockler T C, Bryant D W, Wilhelm L, Troggio M, Sosinski B, Aranzana M J, Arús P, Iezzoni A, Morgante M, Peace C. 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.

[33] Aranzana M J, Illa E, Howad W, Arús P. A first insight into peach [Prunus persica (L.) Batsch] SNP variability. Tree Genetics & Genomes, 2012, 8: 1359-1369.

[34] Mao F, Leung W Y, Xin X. Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnology, 2007, 7: 76-91.

[35] 吴波, 杨润婷, 朱世平, 钟云, 姜波, 曾继吴, 钟广炎. 宽皮柑橘单核苷酸多态性的高分辨率熔解曲线分型. 园艺学报, 2012, 39(4): 777-782.

WU B, YANG R T, ZHU S P, ZHONG Y, JIANG B, ZENG J W, ZHONG G Y. Genotyping single nucleotide polymorphisms in mandarin cultivars using high resolution melting analysis. Acta Horticulturae Sinica, 2012, 39(4): 777-782. (in Chinese)

[36] 赵均良, 张少红, 刘斌. 应用高分辨率熔解曲线技术分析水稻分子标记基因型. 中国农业科学, 2011, 44(18): 3701-3708.

ZHAO J L, ZHANG S H, LIU B. Application of high-resolution melting curve analysis for molecular marker genotyping in rice. Scientia Agricultura Sinica, 2011, 44(18): 3701-3708. (in Chinese)

[37] YAMAMOTO T, SHIMADA T, IMAI T, YAEGAKI H, HAJI T, MATSUTA N, YAMAGUCHI M, HAYASHI T. Characterization of morphological traits based on a genetic linkage map in peach. Breeding Science, 2001, 51: 271-278.

[38] VERDE I, QUARTA R, CEDROLA C, DETTORI M T. QTL analysis of agronomic traits in a BC₁ Peach population. Acta Horticulturae, 2002, 592: 291-297.

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