Optimization of SSR-PCR reaction system and primer sceening in Malus crabapple

doi:10.3969/j.issn.1000-2006.201801006

HUANG Bin, NI Zhouxian, ZHOU Ting, ZHANG Wangxiang, XU Li'an

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2018, Vol. 42 ›› Issue (03) : 51-57.

PDF(1697361 KB)

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2018, Vol. 42 ›› Issue (03) : 51-57. DOI: 10.3969/j.issn.1000-2006.201801006

Optimization of SSR-PCR reaction system and primer sceening in Malus crabapple

HUANG Bin, NI Zhouxian, ZHOU Ting, ZHANG Wangxiang, XU Li'an^*

Author information +

History +

Abstract

Abstract: 【Objective】 To explore the optimal PCR conditions for SSR molecular markers of Malus crabapples, orthogonal design was used to optimize the SSR-PCR system, and SSR primers suitable for Malus crabapples were screened using the optimized system. 【Method】Six diploid Malus crabapple DNA templates were used as material. DNA template concentration, Taq enzyme concentration, primer concentration, Mg^{2 +} ion concentration, and dNTP concentration were optimized by the L₁₆(4⁵)orthogonal test and the optimum reaction system for SSR-PCR of crabapples was established. 【Results】 The results showed that 15 μL of optimized system was composed of DNA template 5 mg/L, Taq enzyme 1.25 U, primer 0.3 μmol/L, Mg²⁺ 2 mmol/L, dNTP 0.25 mmol/L and 1×Buffer 1.5 μL. Fifteen pairs of SSR primers from Malus with clear bands, abundant polymorphisms, and good repeatability were screened out using the optimization system. Its polymorphic information content was greater than 0.25. 【Conclusion】 Orthogonal test results achieved the purpose of optimization. The 15 polymorphic primers screened by the obtained system could be directly applied to the SSR molecular marker experiment for Malus crabapples, which provided an effective tool for identification, classification and genetic breeding of Malus crabapples.

Cite this article

EndNote

Ris (Procite)

Bibtex

Download Citations

HUANG Bin, NI Zhouxian, ZHOU Ting, ZHANG Wangxiang, XU Li'an. Optimization of SSR-PCR reaction system and primer sceening in Malus crabapple[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2018, 42(03): 51-57 https://doi.org/10.3969/j.issn.1000-2006.201801006

References

[1] ]ABE J, XU D, SUZUKI Y, et al. Soybean germplasm pools in Asia revealed by nuclear SSRs[J]. Theor Appl Genet, 2003, 106(3): 445-453. DOI:10.1007/s00122-002-1073-3.
[2] 徐立安, 李新军, 潘惠新, 等. 用SSR研究栲树群体遗传结构[J].植物学报, 2001, 43(4): 409-412. DOI:10.3321/j.issn:1672-9072.2001.04.015. XU L A, LI X J, PAN H X, et al. Study on population genetic structure in Castanopsis fargesii with microsatellite markers [J]. Acta Botanica Sinica, 2001, 43(4): 409-412.
[3] 沈敬理, 白天道, 陈亚斌, 等. 马尾松无性系种子园亲本指纹图谱构建 [J].分子植物育种, 2015, 13(3): 1-6. DOI:10.13271/j.mpb.013.000646. SHEN J L, BAI T D, CHEN Y B, et al. The fingerprints construction of clones in Pinus massoniana seed orchard [J]. Molecular Plant Beeding, 2015, 13(3): 1-6.
[4] RÖDER M S, KORZUN V, WENDEHAKE K, et al. A microsatellite map of wheat[J]. Genetics, 1998, 149(4): 2007-2023. DOI:10.1139/g98-009.
[5] MCCOUCH S R, TEYTELMAN L, XU Y, et al. Development and mapping of 2240 new SSR markers for rice(Oryza sativa L.)(supplement)[J]. DNA Res, 2002, 9(6): 257-279. DOI:10.1093/dnares/9.6.199.
[6] 张宁, 沈红香, 高遐虹, 等. 苹果属部分观赏品种与中国野生种的亲缘关系 [J].园艺学报, 2007.34(5): 1227-1234. DOI:10.3321/j.issn:0513-353x.2007.05.027. ZHANG N, SHEN H X, GAO X X, et al. Phylogenetic relationship between Malus and wild species of Malus in China [J]. Acta Horticulturae Sinica, 2007, 34(5): 1227-1234.
[7] 邢俊连. 皂葉属系统进化分析及SSR分子标记开发 [D]. 合肥:安徽农业大学,2016. XING J L. Phylogenetic analyses and SSR mining of genus Gleditisa [D]. Hefei: Anhui Agricultural University, 2016.
[8] ORAGUZIE N C, YAMAMOTO T, SOEJIMA J, et al. DNA fingerprinting of apple(Malus spp.)rootstocks using Simple Sequence Repeats[J]. Plant Breeding, 2005, 124(2): 197-202. DOI:10.1111/j.1439-0523.2004.01064.x.
[9] 王雷宏, 郑玉红, 汤庚国. 基于SSR标记的8个山荆子居群遗传多样性和遗传关系分析[J]. 植物资源与环境学报, 2012, 21(1): 42-46. DOI:10.3969/j.issn.1674-7895.2012.01.006. WANG L H, ZHENG Y H, TANG G G. Analyses of genetic diversity and genetic relationship of eight populations of Malus baccata based on SSR markers[J]. Journal of Plant Resources and Environment, 2012, 21(1): 42-46.
[10] 孙萍, 宗宇, 刘晶,等. 基于SSR标记的清凉峰地区三叶海棠遗传多样性研究 [J].果树学报, 2013, 30(1): 8-15. SUN P, ZONG Y, LIU J, et al. Study on genetic diversity of Malus sieboldii in Qingliangfeng Region based on SSR markers [J]. Journal of Fruit Science, 2013, 30(1): 8-15.
[11] 高源.苹果属种质资源亲缘关系和遗传多样性的SSR分析 [D]. 北京:中国农业科学院, 2007. GAO Y. SSR analysis of Phyogenetic Relationship and Genetic Diversity for Malus Mill. Germplasm Resources [D]. Beijing: Chinese Academy of Agricultural Science, 2007.
[12] 唐建民, 周世良, 成明昊,等.用RAPD和SSR分子标记鉴定小金海棠F1代杂种实生苗的研究 [J]. 农业生物技术科学, 2006, 36-40. DOI:10.3969/j.issn.1000-6850.2006.02.011. TANG J M, ZHOU S L, CHEN M H, et al. Identification of the F1 Hybrids of Malus xiaojinensis Using RAPD and SSR molecular markers [J]. Chinese Agricultural Science Bulletin, 2006, 36-40.
[13] STEWART CN J, VIA LE. A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications[J]. BioTechniques, 1993, 14(5): 748-750.
[14] COTA-SÁNCHEZ J H, REMARCHUK K, UBAYASENA K. Ready-to-use DNA extracted with a CTAB method adapted for herbarium specimens and mucilaginous plant tissue[J]. Plant Molecular Biology Reporter, 2006, 24(2): 161-167. DOI:10.1007/bf02914055.
[15] LISTON A, RIESEBERG L H, ADAMS R P, et al. A method for collecting dried plant specimens for DNA and isozyme analyses, and the results of a field test in Xinjiang, China[J]. Annals of the Missouri Botanical Garden, 1990, 77(4): 859. DOI:10.2307/2399681.
[16] 邓振伟, 于萍, 陈玲. SPSS软件在正交实验设计、结果分析中的应用[J]. 电脑学习, 2009(10): 15-16.DOI:10.3969/j.issn.2095-2163.2009.05.007. DENG Z W, YU P, CHEN L. Application of SPSS software in orthogonal design and result analysis [J]. Computer Study, 2009(10): 15-16.
[17] BOTSTEIN D, WHITE RL, SKOLNICK M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. Am J Hum Genet, 1980, 32(3): 314-331.
[18] 苏辉, 李志刚, 宋书宏. 正交设计优化大豆SSR-PCR反应体系及引物筛选 [J].华北农学报, 2009, 24(2): 99-102. DOI:10.7668/hbnxb.2009.02.021. SU H, LI Z G, SONG S H. Optimization of SSR-PCR system on soybean by orthogonal design and SSR primer selection [J]. Acta Agriculturae Boreali-Sinica, 2009, 24(2): 99-102.
[19] CHEN S, ZHENG X, CAO H, et al. A simple and efficient method for extraction of Taq DNA polymerase[J]. Electronic Journal of Biotechnology, 2015, 18(5): 355-358. DOI:10.1016/j.ejbt.2015.08.001.
[20] TANWEER F A, RAFII M Y, SIJAM K, et al. Identification of suitable segregating SSR markers for blast resistance in rice using inheritance and disease reaction analysis in backcross families[J]. Australasian Plant Pathology, 2015, 44(6): 619-627. DOI:10.1007/s13313-015-0380-5.
[21] 王薪淇,姜龙,宋轶群,等. 糯玉米SSR-PCR暗影体系优化及多态性引物筛选 [J]. 吉林农业大学学报, 2016, 38(4): 398-404. DOI: 10.13327/j.jjlau.2016.2610. WANG X Q, JIANG L, SONG Y T, et al. Optimization of SSR-PCR system of waxy maize and selection of polymorphic primers [J]. Journal of Jilin Agricultural University, 2016, 38(4): 398-404.
[22] 贾波, 徐海滨, 徐阳,等. 鹅掌楸Genomic-SSR反应体系优化 [J]. 林业科学研究, 2013, 26(4): 506-510. DOI:10.3969/j.issn.1001-1498.2013.04.018. JIA B, XU H B, XU Y, et al. Optimization of genomic-SSR reaction system in Liriodendron [J]. Forest Research, 2013, 26(4): 506-510.
[23] 张美玲, 李巧明. 濒危植物望天树SSR-PCR反应体系的优化 [J].云南大学学报(自然科学版), 2011, 33(S2): 425-432. ZHANG M L, LI Q M. Optimization of SSR-PCR reaction system on an endangerd plant Parashorea chinensis [J]. Journal of Yunnan University, 2011, 33(S2): 425-432.
[24] 麦静, 杨志玲, 杨旭, 等. 濒危植物厚朴SSR引物筛选及反应体系优化 [J]. 生态与农村环境学报, 2015, 31(4): 600-607. DOI:10.11934/j.issn.1673-4831.2015.04.025 MAI J, YANG Z L, YANG X, et al. Screening of primers for SSR markers and optimization of reaction system for endangered plant Magnolia officinalis [J]. Journal of Ecology and Rural Environment, 2015, 31(4): 600-607.
[25] 郭鑫, 倪州献, 徐立安, 等. 桉树SSR-PCR 体系优化及引物筛选应用 [J]. 分子植物育种, 2017, 15(2): 626-632. DOI: 10.13271/j.mpb.015.000626. GUO X, NI Z X, X L A, et al. Optimization of SSR-PCR reaction system and primer screening of Eucalyptus [J]. Molecular Plant Breeding, 2017, 15(2): 626-632.
[26] 李爽. 苹果SSR标记体系的优化及遗传连锁图谱构建初探 [D]. 杨凌:西北农林科技大学,2011. LI S. Optimizing of SSR analysis sytem and genetic mapping of apple [D]. Yangling: Northwest A & F University, 2011.
[27] 姚利华. 苹果属EST-SSR标记开发及其在梨属上的转移性 [D]. 杭州: 浙江大学, 2008. YAO L H. Development of EST-SSR Markers from Malus and their transferability to Pyrus [D]. Hangzhou: ZheJiang University, 2008.
[28] 韩志校, 毕振良, 陆凤勤, 等. 利用苹果引物对不同山楂品种的SSR分析 [J]. 河北林果研究, 2014, 29(2): 165-168. DOI:10.13320/j.cnki.hjfor.2014.0036. HAN Z X, BI Z L, LU F Q, et al. Analysis of the relationship of different hawthorn cultivars by apple SSR primers [J]. Hebei Journal of Foresty and Orchard Research, 2014, 29(2): 165-168.
[29] 王彩虹, 田义轲, 赵静. 来自苹果的SSRs在蔷薇科植物资源上的通用性分析 [J]. 园艺学报, 2005, 32(3): 500-502. Doi:10.3321/j.issn:0513-353X.2005.03.025. WANG C H, TIAN Y K, ZHAO J. General application analysis of SSRs derived from apple on other species in Rosaceae [J]. Acta Horticultural Sinica, 2005, 32(3): 500-502.
[30] YAMAMOTO T, KIMURA T, SAWAMURA Y, et al. SSRs isolated from apple can identify polymorphism and genetic diversity in pear[J]. TAG Theoretical and Applied Genetics, 2001, 102(6): 865-870. DOI:10.1007/s001220000524.
[31] 张国盛, 张玮, 李小龙, 等. 北沙柳SSR-PCR反应体系优化及引物筛选 [J]. 内蒙古农业大学学报(自然科学版), 2013, 34(4): 59-63. ZHANG G S, ZHANG W, LI X L, et al. Optimization of the SSR-PCR system and selection of primers in Salix psammophila [J]. Journal of Inner Mongolia Agricultural University, 2013, 34(4): 59-63.
[32] 王萍, 母洪娜, 耿兴敏, 等. 利用正交试验设计优化桂花的 SSR-PCR 体系[J]. 江苏农业科学, 2017, 45(2): 44-46. DOI:10.15889/j.issn.1002-1302.2017.02.011. WANG P, MU H N, DI X M, et al. Optimization of Osmanthus fragrans SSR-PCR system by orthogonal design[J]. Jiangsu Agricultural Sciences, 2017, 45(2): 44-46.

PDF(1697361 KB)

Accesses

Citation

Detail

Sections

Recommended

The full text is translated into English by AI, aiming to facilitate reading and comprehension. The core content is subject to the explanation in Chinese.

www.nldxb.njfu.edu.cn

Edited ＆ Published by Editorial Department of Nanjing Forestry University(Natural Sciences Edition)
Address： No.159 Longpan Road,Nanjing 210037 Jiangsu,P.R.China
E-mail ：xuebao@njfu.edu.cn , xuebao@njfu.com.cn
Telephone +86-25-85428247,85427076
Distributed by China International Book Trading Corporation P.O. Box 399, Beijing ,P.R. China

〈

〉

Please choose a citation manager

Content to export