葡萄无核性状的SSR新分子标记开发及应用

doi:10.3864/j.issn.0578-1752.2018.13.017

摘要/Abstract

摘要： 【目的】开发葡萄无核性状的SSR新分子标记，对葡萄杂种后代进行早期无核性状鉴定，为分子标记辅助育种奠定基础。【方法】对母本‘红地球’与父本‘森田尼无核’及F₁代杂交群体的133份葡萄材料进行RAD-seq，将测序结果比对到参考基因组上；运用SAMtools软件生成CaSFS软件所需的pileup和glf文件，过滤获得亲本之间的有效SNP集；采用窗口滑动的方法，确定每个窗口的基因型，选择以每15个SNP为一个窗口，每次滑动一个SNP，得到每个个体的基因型并生成bin图；对生成的bin图采用拟测交的方式利用Joinmap软件进行连锁分析，构建‘红地球’与‘森田尼无核’遗传连锁图谱；用WindowQTLCartographer2.0软件进行QTL定位，在定位区间通过Perl程序语言找到符合SSR特征序列的35个SSR分子标记，用Primer premier5.0软件设计35个SSR分子标记引物对，通过HRM技术筛选亲本之间在无核性状上存在差异的SSR分子标记，并在131株F₁代杂交群体及65个葡萄品种的自然群体中检测无核分型正确率、无核检测率、无核保持率。【结果】在‘红地球’与‘森田尼无核’构建的遗传连锁图谱上，将葡萄无核性状定位在chr18号染色体上，定位区间26 835 846—26 960 426，对无核的贡献率为77.9%，LOD阈值为26.3。亲本之间在无核性状上存在差异的SSR分子标记为VvSD10，其在111 bp等位点能对葡萄无核性状进行鉴定，利用分子标记VvSD10上的111 bp等位点通过HRM技术在葡萄F₁代杂交群体及自然群体中进行葡萄无核性状的鉴定。结果表明，分子标记VvSD10在F₁代杂交群体及自然群体中的无核分型正确率分别为97%、94%，其无核检测率均为56%，无核保持率分别为77%、85%。【结论】分子标记VvSD10在遗传群体及自然群体中的无核分型均能提供较高的准确信息，为无核葡萄的分子标记辅助育种奠定了基础。

关键词: 葡萄, 无核, QTL定位, SSR分子标记, 辅助育种

Abstract: 【Objective】The objective of this study is to develop a new SSR molecular marker for seedless traits in grape, and to identify the early seedless traits of grape hybrids, and to lay the foundation for molecular marker assisted selection breeding. 【Method】RAD-seq was carried out in 133 grape materials including the female parent ‘Red Globe’, the male parent ‘Centennial Seedless’ and their F₁ hybrids, and the sequencing results were compared to the reference genome. SAMtools software was used to generate CaSFS software required for pileup and GLF files, and the effective SNP sets between parents were obtained by filtration. A window sliding method was used to determine the genotype of each window, and selected one window per 15 SNP and slided one SNP at a time. The genotype of each individual was obtained and the bin generated. Linkage analysis was carried out based on Joinmap software for generated bin, and genetic linkage map was constructed between ‘Red Globe’ and ‘Centennial Seedless’. The QTL loci was mapped using WindowQTLCartographer2.0 software. 35 SSR molecular markers consistent with the SSR characteristic sequence were identified by Perl programming language in the QTL mapping interval, and then 35 pairs of SSR molecular marker primers were designed. The molecular marker was screened in the differences with the seedless traits between parents by HRM technology, and the accuracy of seedless typing, the rate of seedless detection and the rate of seedless holding were detected in 131 F₁ hybrid population and natural population including 65 grape varieties.【Result】On the genetic linkage map between ‘Red Globe’ and ‘Centennial Seedless’, the seedless traits of grape were mapped on chromosome 18, and the mapping interval was 26 835 846-26 960 426, and the contribution rate to seedless was 77.9%, and the LOD threshold was 26.3. The molecular marker VvSD10 was screened in the differences with the seedless traits between parents, and there was a 111 bp loci in seedless traits of grape. Identification of seedless traits in grape F₁ hybrid population and natural population by HRM technology using 111 bp loci on VvSD10 marker, the results indicated that in F1 hybrid population and natural population, the correct typing rate of molecular marker VvSD10 was 97% and 94%, respectively, and the rate of seedless detection of both populations was 56%, the rate of seedless holding was 77% and 85%, respectively. 【Conclusion】Seedless typing in grape by Molecular marker VvSD10 could provide high accurate information in both genetic and natural populations and laid the foundation for molecular marker assisted breeding program in seedless grape.

Key words: Vitis vinifera L., seedless, QTL mapping, SSR molecular marker, assisted breeding

马亚茹，冯建灿，刘崇怀，樊秀彩，孙海生，姜建福，张颖. 葡萄无核性状的SSR新分子标记开发及应用[J]. 中国农业科学, 2018, 51(13): 2622-2630.

MA YaRu, FENG JianCan, LIU ChongHuai, FAN XiuCai, SUN HaiSheng, JIANG JianFu, ZHANG Ying. Development and Application of SSR New Molecular Marker for Seedless Traits in Grape[J]. Scientia Agricultura Sinica, 2018, 51(13): 2622-2630.

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.0578-1752.2018.13.017

https://www.chinaagrisci.com/CN/Y2018/V51/I13/2622

参考文献

[1] MENCARELLI F, ANDREA B, GIANCARLO D. Grape: Post- harvest Operations[D]. Italy: University of Viterbo, 2005.

[2] 崔梦杰, 王晨, 张文颖, 汤崴, 朱旭东, 李晓鹏, 房经贵. 无核葡萄研究进展. 植物生理学报, 2017, 53(3): 317-330.

CUI M J, WANG C, ZHANG W Y, TANG W, ZHU X D, LI X P, FANG J G. Research advance of seedlessness in grape. Plant Physiology Journal, 2017, 53(3): 317-330. (in Chinese)

[3] 孙红, 李晓萍. 美国新注册的葡萄品种. 落叶果树, 2001, 33(5): 57-58.

SUN H, LI X P. Newly registered grape varieties in the United States. Deciduous Fruits, 2001, 33(5): 57-58. (in Chinese)

[4] 蒲胜海, 张计峰, 丁峰, 马雪琴. 新疆葡萄产业发展现状及研究动态. 北方园艺, 2013(13): 200-203.

PU S H, ZHANG J F, DING F, MA X Q. Development status and research trends of grape industry in Xinjiang. Northern Horticulture, 2013(13): 200-203. (in Chinese)

[5] 陶建敏. 葡萄无核育种技术研究及种质创新[D]. 南京: 南京农业大学, 2007.

TAO J M. Study on the technology in breeding of seedless grape and innovation of germplasm [D]. Nanjing: Nanjing Agricultural University, 2007. (in Chinese)

[6] BOUQUET A, DANGLOT Y. Inheritance of seedlessness in grapevine (Vitis vinifera L.). Vitis, 1996, 35(1): 35-42.

[7] WEI X, SYKES S R, CLINGELEFFER P R. An investigation to estimate genetic parameters in CSIRO's table grape breeding program. Euphytica, 2002, 128(3): 343-351.

[8] DOLIGEZ A, BOUQUET A, DANGLOT Y, LAHOGUE F, RIAZ S, MEREDITH C P, EDWARDS K J, THIS P. Genetic mapping of grapevine (Vitis vinifera L.) applied to the detection of QTLs for seedlessness and berry weight. Theoretical & Applied Genetics, 2002, 105(5): 780-795.

[9] EMANUELLI F, BATTILANA J, COSTANTINI L, LE CUNFF L, BOURSIQUOT J M, THIS P, GRANDO M S. A candidate gene association study on muscat flavor in grapevine (Vitis vinifera L.). Bmc Plant Biology, 2010, 10(1): 241.

[10] COSTANTINI L, BATTILANA J, LAMAJ F, FANIZZA G, GRANDO M S. Berry and phenology-related traits in grapevine (Vitis vinifera L.): From quantitative trait loci to underlying genes. BMC Plant Biology, 2008, 8(1): 38.

[11] MEJIA N, GEBAUER M, MUNOZ L, HEWSTONE N, MUNOZ C, HINRICHSEN P. Identification of QTLs for seedlessness, berry size, and ripening date in a seedless x seedless table grape progeny. American Journal of Enology & Viticulture, 2007, 58(4): 499-507.

[12] FISCHER B M, SALAKHUTDINOV I, AKKURT M, EIBACH R, EDWARDS K J, TOPFER R, ZYPRIAN E M. Quantitative trait locus analysis of fungal disease resistance factors on a molecular map of grapevine. Theoretical & Applied Genetics, 2004, 108(3): 501-515.

[13] HANANIA U, VELCHEVA M, OR E, FLAISHMAN M, SAHAR N, Perl A. Silencing of chaperonin 21, that was differentially expressed in inflorescence of seedless and seeded grapes, promoted seed abortion in tobacco and tomato fruits.Transgenic Research,2007, 16: 515-525.

[14] HANANIA U, VELCHEVA M, SAHAR N, FLAISHMAN M, OR E, DEGANI O, PERL A. The ubiquitin extension protein S27a is differentially expressed in developing flower organs of Thompson seedless versus Thompson seeded grape isogenic clones. Plant Cell Reports, 2009, 28(7): 1033-1042.

[15] CABEZAS J A, CERVERA M T, RUIZGARCIA L, CARRENO J, MARTINEZ-ZAPATER J M. A genetic analysis of seed and berry weight in grapevine. Genome, 2006, 49(12): 1572-1585.

[16] MEJIA N, SOTO B, GUERRERO M, CASANUEVAL X, HOUEL C, MICCONO M A, RAMOS R, CUNFF L L, BOURSIQUOT J M, HINRICHSEN P, ADAM-BLONDON A F. Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine. BMC Plant Biology, 2011, 11(1): 57.

[17] BOSS P K, SENSI E, HUA C, DAVISE C, THOMAS M R. Cloning and characterisation of grapevine (Vitis vinifera L.) MADS-box genes expressed during inflorescence and berry development. Plant Science, 2002, 162(6): 887-895.

[18] DIAZ-RIQUELME J, LIJAVETZKY D, MARTINEZ-ZAPATER J M, CARMONA M J. Genome-wide analysis of MIKCC-type MADS box genes in grapevine. Plant Physiology, 2009, 149(1): 354-369.

[19] PARENICOVA L, FOLTER S D, KIEFFER M, HORNER D S, FAVALLI C, BUSSCHER J, COOK H E, INGRAM R M, KATER M M, DAVIES B, ANGENENT G C, COLOMBO L. Molecular and phylogenetic analyses of the complete MADS-box transcription factor family in Arabidopsis: New openings to the MADS world. Plant Cell, 2003, 15: 1538-1551.

[20] IMMINK R G, TONACO I A, FOLTER S D, SHCHSNNIKOVA A, DIJK A D V, BUSSCHER-LANGE B, BORST J W, ANGENENT G C. SEPALLATA3: The ‘glue’ for MADS box transcription factor complex formation. Genome Biology, 2009, 10(2): R24.

[21] 谭昌渊, 罗九甫, 任兆瑞. 高分辨率熔解曲线分析—分子诊断的新技术. 生命科学研究, 2009, 13(3): 268-271.

TAN C Y, LUO J F, REN Z R. High Resolution Melting-New method for molecular diagnosis. Life Science Research, 2009, 13(3): 268-271. (in Chinese)

[22] SOUZA R A, FALAAO J P. A novel high-resolution melting analysis- based method for Yersinia enterocolitica, genotyping. Journal of Microbiological Methods, 2014, 106(3): 129-134.

[23] 舒庆尧, 张华丽, 黄建中. 一种用于水稻光敏雄性核不育基因lncR分型的引物及其应用. CN 103146696 A[P]. 2013.

SHU Q Y, ZHANG H L, HUANG J Z. Primer and application for lncR typing of photoperiod sensitive genic male sterile gene in rice. CN 103146696 A[P]. 2013. (in Chinese)

[24] 尤崇革, 李玉民, 马克君, 施鑫鹤, 郜莉娜, 李菲菲. 基于高分辨熔解技术进行单核苷酸多态性基因分型的方法学评价. 中华临床医师杂志(电子版), 2012(7): 1786-1790.

YOU C G, LI Y M, MA K J, SHI X H, GAO L N, LI F F. Evaluation on the methodology of SNP genotyping based on high resolution melting. Chinese Clinicians, 2012(7): 1786-1790. (in Chinese)

[25] NOMOTO K, TSUTA K, TAKANO T, FUKUI T, YOKOZAWA K, SAKAMOTO H, YOSHIDA T, MAESHIMA A M, SHIBATA T, FURUTA K, OHE Y, MATSUNO Y. Detection of EGFR mutations in archivedcytologic specimens of non-small cell lung cancer using high-resolution melting analysis. American Journal of Clinical Pathology, 2006, 126(4): 608-615.

[26] LOCHLAINN S Ó, AMOAH S, GRAHAM N, ALAMER K, RIOS J J, KURUP S, STOUTE A, HAMMOND J P, OSTERGAARD L, KING G J, WHITE P J, BROADLEY M R. High resolution melt (HRM) analysis is an efficient tool to genotype EMS mutants in complex crop genomes. Plant Methods, 2011, 7(1): 1-9.

[27] MAAT W, VELDEN P A V D, OUTLUITING C, PLUG M, DIRKS A, JAGER M, GRUIS N A. Epigenetic inactivation of RASSF1a in uveal melanoma. Investigative Ophthalmology & Visual Science, 2007, 48(2): 486-490.

[28] 邓建强, 李文慧, 李亮, 张文彬, 龙仁, 蔡继峰. 应用HRM技术进行法医昆虫种属鉴定的初步研究. 中国热带医学, 2014, 14(1): 9-11.

DENG J Q, LI W H, LI L, ZHANG W B, LONG R, CAI J F. Preliminary study of species identification by HRM in forensic entomology. China Tropical Medicine, 2014, 14(1): 9-11. (in Chinese)

[29] 曹春鸽, 孙海燕, 周芳芳, 王诗铭, 陈红岩, 卢大儒. 应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型. 遗传, 2013, 35(7): 923-930.

CAO C G, SUN H Y, ZHOU F F, WANG S M, CHEN H Y, LU D R. Duplex genotyping of CYP2C19*2 and CYP2C19*3 by high-resolution melting curve analysis. Hereditas, 2013, 35(7): 923-930. (in Chinese)

[30] 嘎利兵嘎. 高分辨率熔解曲线(HRM)技术在动物病毒基因分型中的研究及应用[D]. 北京: 中国农业大学, 2015.

GA L B G. Research and application of high resolution melting curve analysis in the genotyping and detection of animal viruses [D]. Beijing: China Agricultural University, 2015. (in Chinese)

[31] SONG Q J, JIA G F, ZHU Y L, GRANT D, NELSON R T, HWANG E Y, HYTEN D, CREGAN P B. Abundance of SSR motifs and development of candidate polymorphic SSR markers (BARCSOYSSR_1.0) in soybean. Crop Science, 2010, 50(5): 1950-1960.

[32] KUBOTA S, LIU Q, KESSUWAN K, OKAMOTO N, SAKAMOTO T, NAKAMURA Y, SHIGENOBU Y, SUGAYA T, SANO M, UJI S, NOMURA K, OZAKI A. High-throughput simple sequence repeat (SSR) markers development for the kelp grouper (Epinephelus bruneus), and cross-species amplifications for Epinephelinae species. Advances in Bioscience and Biotechnology, 2014, 5(2): 117-130.

[33] KARAAGAC E, VARGAS A M, ANDRES M T D, CARRENO I, IBANEZ J, CARRENO J, MARTINE-ZAPATER J M, CABEZAS J A. Marker assisted selection for seedlessness in table grape breeding. Tree Genetics & Genomes, 2012, 8(5): 1003-1015.