PCR-SSCP技术与比较基因组学结合定位棉花Li1基因

Abstract
Figure/Table
References
Related Citation (15)

Download: PDF (3371 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract Polymerase chain reaction-single strand conformation ploymorphism (PCR-SSCP), as an important technique in detecting minor differences between genomic DNA bases, has the characteristics of simplicity, rapidity and sensitivity. So far, a large number of genome sequences provide lots of DNA sequences for the development of novel genetic markers through comparative genomics and the mapping of target genes. Hence, the possibility was investigated on the application of PCR-SSCP technique combined with comparative genomics to develop novel molecular markers in cotton (Gossypium spp.) genetic mapping in the study, taking Ligon lintless-1 (Li1) mutant gene mapping as an example and the published sequences of Arabidopsis and cotton genome D as references. The optimization of SSCP conditions for samples of large fragment PCR products (＞400 bp) were carried out in the experiment. The results showed that the optimal experiment conditions were as follows: voltage of 150 V, gel concentration of 10%, electrophoresis temperature of 4 ℃, and 5∶1 of loading dye to PCR product. According to the linear segments of Arabidopsis and cotton, we selected 102 cotton EST sequences to develop the SSCP primers. The optimized PCR-SSCP system was utilized to construct a genetic map consisting of 28 markers and spanning 149.6 cM . This genetic map contained 4 genes with functions closely related to Li1 loci. Additionally, using genome D sequences, 185 SSCP primers were developed and there were 86 primers showing polymorphism between the two parents (Li1 and H7124). In this experiment, Li1 gene was firstly mapped in a genetic map consisting of 17 SSCP markers and the Li1 loci, which spanned 40.3 cM. The Li1 gene was flanked by W058 and P095, which were the nearest markers, and the distance from Li1 loci was 0.6 and 0.3 cM respectively. Using the published Li1 genetic map to analyze the relationship between D genome sequences and two genetic maps constructed in this experiment, the study suggested that there were good homologous between these genetic maps. The results presented here could offer a useful foundation for future research work on fine mapping and cloning of Li1 gene. In addition, with the development of the third generation genome sequencing technology, constantly enrichment of the GenBank and the publishment of cDNA sequences, the optimization of SSCP technology plays an important role in cotton genetic linkage map construction, map-based cloning, molecular marker-assisted breeding and other aspects.

Key words： Cotton Molecular markers PCR-SSCP Ligon lintless-1( Li1) Comparative genomics Genetic mapping

Received: 17 September 2015 Published: 24 February 2016

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	YANG Fen
	CAO Ti-Fen
	DAI Hua-Qin
	JIANG Yu-Rong

Cite this article:

YANG Fen,CAO Ti-Fen,DAI Hua-Qin, et al. Mapping of Li1 Gene Based on the Combination of PCR-SSCP Technique and Comparative Genomics in Cotton (Gossypium spp.)[J]. , 2016, 24(4): 613-624.

URL:

http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2016/V24/I4/613

Wang M, LI C Q, Wang Q L. Quantitative trait loci mapping and genetic dissection for lint percentage in upland cotton (Gossypium hirsutum)[J]. Journal of Genetics, 2014, 93(2): 371-378.
RoyChowdhury M, JIA Y L, Jackson A, et al. Analysis of rice blast resistance gene?Pi-zin rice germplasm using pathogenicity assays and DNA markers[J]. Euphytica, 2012,?184(1): 35-46.
Radix S, Hiroyuki S, Yuichi T. Phylogenetic study of Japanese Dickeya spp. and development of new rapid identi?cation methods using PCR–RFLP [J]. Journal of General Plant Pathology, 2014, 80(3): 237-254.
Chen M L, Wu J, Wang L F, Zhang X Y ,?et al. Development of mapped simple sequence repeat markers from common bean (Phaseolus vulgaris?L.) based on genome sequences of a Chinese landrace and diversity evaluation[J]. Molecular Breeding, 2014, 33(20): 489-496.
Ghaffari S ,?Hasnaoui N ,?Zinelabidine L H, et al. Genetic diversity and parentage of Tunisian wild and cultivated grapevines (Vitis vinifera?L.) as revealed by single nucleotide polymorphism (SNP) markers[J]. Tree Genetics and Genomes, 2014,?10(4): 1103-1112.
Han Z G, Wang C B, Song X L, et al. Characteristics, development and mapping of Gossypium hirsutum derived EST-SSRs in allotetraploid cotton[J]. Theoretical and Applied Genetics, 2006, 112(3): 430-439.
He S E, Zheng Y N, Chen A Q, et al. Converting restriction fragment length polymorphism to single‐strand conformation polymorphism markers and its application in the fine mapping of a trichome gene in cotton[J]. Plant Breeding, 2013, 132(3): 1-7.
谢晓兵, 于霁雯, 吴嫚, 等. 利用SSCP技术分析棉花纤维差异表达的基因[J]. 棉花学报, 2011a，23(04):306-310.( Xie X B, Yu J J, Wu M, et ai. Analysis of SSCP for Cotton Differentially Expressed Genes Related to Fiber Development[J]. Cotton Science, 2011(04):306-10.)
谢晓兵, 于霁雯, 吴嫚, 等. 棉花油份合成相关基因的SSCP标记开发[J]. 分子植物育种, 2011b, 9(3):336-342.(Xie X B, Yu J J, Wu M, et ai. Development of SSCP Makers for Genes Related to Oil Synthesis in Cotton[J]. Molecular Plant Breeding, 2011, 9(3):336-342.)
吴兆海, 许庆方. PCR-SSCP技术的研究与应用[J]. 畜牧与饲料科学, 2010, 31(8): 47-48.(Wu Z H, Xu Q F. Research and Application Development of PCR-SSCP Technology[J]. Animal Husbandry and Feed Science, 2010, 31(8): 47-48.)
Kuhn D N, Livingstone III D, Main D, Zheng P, et al. Motamayor J C.. Identification and mapping of conserved ortholog set (COS) II sequences of cacao and their conversion to SNP markers for marker-assisted selection in Theobroma cacao and comparative genomics studies[J]. Tree Genetics and Genomes, 2012, 8(1): 97-111.
Tang H B, Bowers J E, Wang X Y, et al. Synteny and Collinearity in Plant Genomes[J]. Science, 2008, 320(5875): 486-488 .
Rong J K , Bowers J E, Schulze S R. Comparative genomics of Gossypium and Arabidopsis:Unravling the consequences of both ancient and recent polyploidy[J]. Genome Research, 2005a, 15(9): 1198-1210.
Fukuoka H, Miyatake K, Nunome T, et al. Development of gene-based markers and construction of an integrated linkage map in eggplant by using Solanum orthologous (SOL) gene sets[J]. Theoretical and Applied Genetics, 2012, 125(1): 47-56.
Wang K B , Wang Z W , Li F G , et al. The draft genome of a diploid cotton Gossypium ramondii[J]. Nature Genetics, 2012, 44(10): 1098-1103.
Griffee F, Ligon L L. Occurrence of lintless cotton plants and the inheritance of the character ‘lintless’[J]. Agron J, 1929, 21: 711–717.
Kohel R J, Stelly D M, Yu J. Tests of six cotton (Gossypium hirsutum L.) mutants for association with aneuploids[J]. Journal of Heredity, 2002, 93(2): 130–132.
Karaca M, Saha S, Jenkins J N, et al. Simple sequence repeat (SSR) markers linked to the Ligon lintless (Li1) mutant in cotton[J]. Journal of Heredity. 2002, 93(3): 221-224.
Rong J K, Pierce G J, Waghmare V N, et al. Genetic mapping and comparative analysis of seven mutants related to seed fiber development in cotton[J]. Theoretical and Applied Genetics, 2005b, 111(6): 1137-1146.
Paterson A H A, Brubaker C L, Wendel J F. A rapid method for extraction of cotton (Grossypium spp)genomic DNA suitable for RFLP or PCR analysis[J]. Plant Molecular Biology Reporter, 1993, 11(2): 122-127.
宋国立, 崔荣霞, 王坤波, 等. 改良CTAB法快速提取棉花DNA[J]. 棉花学报, 1998, 10(5): 273-275.(Song G L, Cui R X, Wang K B, et al. A Rapid Improved CTAB Method for Extraction of Cotton Genomic DNA[J]. Cotton Science, 1998, 10(5): 273-275.)
Rong J K, Alex F F, Waghmare V N, et al. Meta-analysis of Polyploid Cotton QTL Shows Unequal Contributions of Subgenomes to a Complex Network of Genes and Gene Clusters Implicated in Lint Fiber Development[J]. Genetis, 2007, 176(4): 2577-2588.
李绍华, 熊远著, 郑嵘, 等. 猪MSTN基因多态性及其SNPs的研究[J]. 遗传学报, 2002, 29(4): 326-331.(LI S H, Xiong Y Z, Zheng R, et al. Polymorphism of Porcine Myostatin Gene[J]. Acta Genetica Sinica, 2002, 29(4): 326-331.)
Callon Cécile, Delbès Céline, Duthoit Frédérique, et al. Application of SSCP-PCR fingerprinting to profile the yeast community in raw milk Salers cheeses [J]. Systematic and Applied Microbiology, 2006, 29(2): 172-180.
Shintani S, Nakahara Y, Waghmare V N, et al. et al. Inactivation of the p14ARF, p15INK4B and p16INK4A genes is a frequent event in human oral squamous cell carcinomas [J]. Oral Oncology, 2001, 37(6): 498-504.
李媛媛. SSCP技术在植物中的应用[J]. 北方园艺, 2009, (5): 122-124.(Li Y Y. Single-Strand Conformation Polymorphism and Its Application in Plant Research[J]. Northern Horticulture, 2009(5): 122-124.)
朱琴, 汪桂玲. 中国明对虾（Penaeus chinensis kishinouye）Sox基因PCR-SSCP分析条件优化的研究[J]. 现代渔业, 2006, 21(12): 7-9.(Zhu Q, Wang G L. A Study on Optimization of the Conditions Affecting SSCP Analysis of Sox Gene in Penaeus chinensis Kishinouye[J]. Modern Fisheries Information, 2006, 21(12): 7-9.)
Orita M, Suzuki Y, Sekiya T, et al. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction[J]. Genomics, 1989, 5(4): 874-879.
Hayashi K, Yandell D W. How sensitive is PCR-SSCP[J]. Human Mutation,1993, 2(5): 338-346.
姜运良, 李宁, 赵兴波, . 影响PCR-SSCP的因素分析[J]. 农业生物技术学报, 2000, 8(3): 245-247.(Jiang Y L, Li N, Zhao X B, et al. Analysis on the Factors Affecting PCR-SSCP[J]. Joumal of Agriculture Biotechnology, 2008, 8(3): 245-247.)
Ravnik, Glawac M, Glacvc D. Sensivity of single strand conformation polymorphism and heteroduplex method for mutation detection in the cystic frbrosis gene [J]. Human Molecular Genetics, 1994, 3 (5): 801-807.
Kodama Y, Ha L T, Watanabe K. Sulfurospirillum cavolei sp. nov. a facultatively anaerobic sulfur-reducing bacterium isolated from an underground crude oil storage cavity[J]. International Journal of Systematic and Evolutionary Microbiology, 2007, 57(4): 827?831.
余桂红, 唐克轩, 马鸿翔, 等. 小麦SSCP分子标记体系的优化[J]. 核农学报, 2007, 21(4): 333-338.(Yu G H, Tang K X, Ma H X, et al. Optimization of the Conditions Affecting SSCP Technique in Wheat[J]. Journal ofNuclear Agricultural Sciences, 2007, 21(4): 333-338.)
Gilbert M K, Turley R B, Kim H J, et al. Transcript profiling by microarray and marker analysis of the short cotton (Gossypium hirsutum L.) fiber mutant Ligon lintless-1 (Li1)[J]. BMC Genomics, 2013, 14(403): 1-17.
Rong J K, Abbey C, Bowers J E., et al. A 3347-Locus Genetic Recombination Map of Sequence-Tagged Sites Reveals Features of Genome Organization, Transmission and Evolution of Cotton (Gossypium)[J]. Genetics, 2004, 166(1): 389-417.