苹果部分种质资源分子身份证的构建

doi:10.3864/j.issn.0578-1752.2015.19.011

摘要/Abstract

摘要： 【目的】以国家果树种质兴城梨、苹果圃保存的131份苹果地方品种、育成品种及其野生近缘种为试材，利用TP-M13-SSR标记构建苹果种质分子身份证。【方法】基于TP-M13-SSR指纹图谱，筛选可以将苹果种质区分的引物组合，并对其等位基因进行编码建立种质分子身份证。【结果】（1）从131份材料中随机选取两份材料，对第一次PCR条件进行优化和引物筛选，从32对合成引物中筛选出16对稳定性高和重复性好的TP-M13-SSR引物用于131份苹果属植物指纹图谱构建。（2）16对SSR引物在供试种质间共检测出等位基因326个，每对引物平均检测到等位基因数为20.3个。CH05d04对种质扩增的等位基因数最多为49个，位点期望杂合度最高为0.878；其次是CH01f07a为48个。利用PopGen32软件计算引物的多态性信息含量，16对引物的平均多态性信息含量为0.7558。16对SSR引物可区分供试苹果种质资源数量从11份到71份不等，平均每对SSR引物可区分49份苹果种质，区分率为8.09%—52.21%。其中对苹果种质区分率最高的是CH01f07a，最低的为BGT23b。（3）根据引物扩增的多态性信息含量和对苹果种质的区分率，将两者均较高的引物CH05d04、CH01f07a、CH03d07、CH04e03、CH04h02和CH04g07两两组合，CH04h02和CH01f07a引物组合分辨率最高，可以区分120份苹果种质。继续增加组合中引物数量，在增加到3对引物时，即可将全部苹果种质区分开来。（4）把可以将全部供试苹果种质资源材料全部区分的3对核心引物CH04h02、CH05d04和CH01f07a获得等位基因按照从大到小的顺序排列，并用阿拉伯数字从01开始赋值；将每份材料在3个位点获得的等位基因按照赋值数字编码获得每份供试材料独有的字符串，利用条码技术将每对引物的分子身份证转化成可被机器快速扫描的条码分子身份证。【结论】依据引物扩增的多态性信息含量和对苹果种质的区分率，筛选核心引物组合，区分全部供试苹果地方品种、育成品种及其野生近缘种质资源，并基于指纹图谱构建其可被机器快速识别的分子身份证，使每份种质具有可辨的分子身份证，达到利用最少、最特异引物区分最多苹果种质的目的。

关键词: 苹果, 种质资源, TP-M13-SSR, 分子身份证

Abstract: 【Objective】 A total of 131 apple germplasms including landraces, bred cultivars and related species selected from the National Repository of Pear and Apple Germplasm Resources in Xingcheng, China were studied with tailed primer M13 microsatellite markers (TP-M13-SSR). An analysis was made to establish the molecular ID of 131 apple germplasms.【Method】Based on genetic fingerprints, germplasms were distinguished with selected SSR markers, alleles that were amplified by each marker were coded, then was combined as a molecular ID.【Result】Two accessions selected from a total of 131 accessions were used for optimization of the first PCR detecting conditions and SSR primer screening. 16 pairs of TP-M13-SSR primers with high stability and good repeatability were used to establish the fingerprints of 131 accessions of Malus Mill.. By using 16 selected SSR markers, 326 polymorphic sites were detected with a mean value of 20.3. The amplification of CH05d04 for all germplasms obtained 49 alleles with the highest expected heterozygosity 0.878, and the amplification of CH01f07a for all germplasms obtained 48 alleles. The polymorphism information content was calculated by PopGen32 with 0.7558 on average. 16 SSR markers could distinguish 11 accessions at least, 71 accessions at most, and 49 accessions on average. The identified rate was 8.09%-52.21%. The identified rate of CH01f07a was the highest, and the identified rate of BGT23b was the lowest. Based on the PIC of amplification and identified rate, each parir of two SSR primers was combined together to identify all the accessions. The combination of CH04h02 and CH01f07a could distinguish 120 accessions at most. More SSR primers were combined together. Finally three SSR primers could distinguish all the accessions. All the alleles of the three core SSR markers were sequenced from small to large, and the assignment was from number 01. A character string was constituted by combining all the codes of the three primers for every accession. By using barcode technology molecular ID can be transferred into a barcode ID that can be quickly scanned by machine.【Conclusion】Based on the PIC of amplification and identified rate, core SSR primers can be screened out to distinguish all the landraces, bred cultivars, and related species. And by constructing fingerprints, every apple germplasm obtains its differentiable molecular ID that can be recognized by the machine. The purpose was to distinguish the most apple germplasms by using the least and the most specific primers.

Key words: apple(Malus Mill.), germplasm resources, TP-M13-SSR, molecular ID

高源，刘凤之，王昆，王大江，龚欣，刘立军. 苹果部分种质资源分子身份证的构建[J]. 中国农业科学, 2015, 48(19): 3887-3898.

GAO Yuan, LIU Feng-zhi, WANG Kun, WANG Da-jiang, GONG Xin, LIU Li-jun. Establishment of Molecular ID for Some Apple Germplasm Resources[J]. Scientia Agricultura Sinica, 2015, 48(19): 3887-3898.

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参考文献

[1] 俞德俊. 中国果树分类学. 北京: 中国农业出版社, 1979: 2-81.

Yu D J. Taxonomy of China Fruits. Beijing: China Agriculture Press, 1979: 2-81. (in Chinese)

[2] 董玉琛, 刘旭. 中国作物及其野生近缘植物: 果树卷. 北京: 中国农业出版社, 2006: 55-84.

Dong Y C, Liu X. Crops and Their Wild Relatives in China: Vol. Fruit Crops. Beijing: China Agriculture Press, 2006: 55-84. (in Chinese)

[3] 李育农, 李晓林. 苹果属植物过氧化物酶同工酶酶谱的研究. 西南农业大学学报, 1995, 17(5): 371-377.

Li Y N, Li X L. Research on peroxidase isozyme in Malus Mill. Journal of Southwest Agricultural University, 1995, 17(5): 371-377. (in Chinese)

[4] 史燕山, 骆建霞. 苹果元帅系品种过氧化物酶同工酶酶谱的比较. 天津农学院学报, 1995, 1: 11-14.

Shi Y S, Luo J X. Comparison of peroxidese zymogrames of “Delicious” apple cultivers. Journal of Tianjin Agricultural College, 1995, 1: 11-14. (in Chinese)

[5] 王爱德, 李天忠, 许雪峰, 韩振海. 苹果品种的SSR分析. 园艺学报, 2005, 32(5): 875-877.

Wang A D, Li T Z, Xu X F, Han Z H. SSR analysis for apple cultivars. Acta Horticulturae Sinica, 2005, 32(5): 875-877. (in Chinese)

[6] 祝军, 王涛, 赵玉军, 张文, 李光晨. 应用AFLP分子标记鉴定苹果品种. 园艺学报, 2007, 27(2): 102-106.

Zhu J, Wang T, Zhao Y J, Zhang W, Li G C. Identification of apple varieties with AFLP molecular markers. Acta Horiculture Sinica, 2007, 27(2): 102-106. (in Chinese)

[7] 高源, 刘凤之, 曹玉芬, 王昆. 苹果属种质资源亲缘关系的SSR分析. 果树学报, 2007, 24(2): 129-134.

Gao Y, Liu F Z, Cao Y F, Wang K. SSR analysis of genetic relationship for apple germplasm resources. Journal of Fruit Science, 2007, 24(2): 129-134. (in Chinese)

[8] 高源, 王昆, 田路明, 曹玉芬, 刘凤之. 应用TP-M13-SSR技术鉴定苹果品种. 果树学报, 2010, 27(5): 833-837.

Gao Y, Wang K, Tian L M, Cao Y F, Liu F Z. Identification of apple cultivars by TP-M13-SSR technique. Journal of Fruit Science, 2010, 27(5): 833-837. (in Chinese)

[9] Scheulke M. An economic method for the fluorescent labeling of PCR fragments. Nature Biotechnology, 2000, 18: 233-234.

[10] 李会勇, 王天宇, 黎裕, 石云素, 宋艳春, 陆平. TP-M13自动荧光检测法在高粱SSR基因型鉴定中的应用. 植物遗传资源学报, 2005, 6(1): 68-70.

Li H Y, Wang T Y, Li Y, Shi Y S, Song Y C, Lu P. Application of the TP-M13 automated fluorescent-lablled system of SSR genotyping in sorghum. Journal of Plant Genetic Resources, 2005, 6(1): 68-70. (in Chinese)

[11] 王壮伟, 赵密珍, 袁骥, 吴伟民, 钱亚明. 38 个欧美草莓栽培品种SSR指纹图谱的构建. 果树学报, 2011, 28(6): 1032-1037.

Wang Z W, Zhao M Z, Yuan J, Wu W M, Qian Y M. Establishment of SSR fingerprinting database for 38 cultivars of strawberry (Fragaria ananassa) native to Europe and America. Journal of Fruit Science, 2011, 28(6): 1032-1037. (in Chinese)

[12] 徐雷锋, 葛亮, 袁素霞, 任君芳, 袁迎迎, 李雅男, 刘春, 明军. 利用荧光标记SSR构建百合种质资源分子身份证. 园艺学报, 2014, 41(10): 2055-2064.

Xu L F, Ge L, Yuan S X, Ren J F, Yuan Y Y, Li Y N, Liu C, Ming J. Using the fluorescent labeled SSR markers to establish molecular identity of Lily germplasms. Acta Horiculture Sinica, 2014, 41(10): 2055-2064. (in Chinese)

[13] 陈昌文, 曹珂, 王力荣, 朱更瑞, 方伟超. 中国桃主要品种资源及其野生近缘种的分子身份证构建. 中国农业科学, 2011, 44(10): 2081-2093.

Chen C W, Cao K, Wang L R, Zhu G R, Fang W C. Molecular ID establishment of main China peach varieties and peach related species. Scientia Agricultura Sinica, 2011, 44(10): 2081-2093. (in Chinese)

[14] 高运来, 朱荣胜, 刘春燕, 李文福, 蒋洪蔚, 李灿东, 姚丙晨, 胡国华, 陈庆山. 黑龙江部分大豆品种分子ID 的构建. 作物学报, 2009, 35(2): 211-218.

Gao Y L, Zhu R S, Liu C Y, Li W F, Jiang H W, Li C D, Yao B C, Hu G H, Chen Q S. Establishment of molecular ID in soybean varieties in Heilongjiang, China. Acta Agronomica Sinica, 2009, 35(2): 211-218. (in Chinese)

[15] 杨阳, 刘振, 赵洋, 梁国强. 湖南省主要茶树品种分子指纹图谱的构建. 茶叶科学, 2010, 30(5): 367-373.

Yang Y, Liu Z, Zhao Y, Liang G Q. Construction of DNA fingerprints for tea cultivars originated from Hunan province. Journal of Tea Science, 2010, 30(5): 367-373. (in Chinese)

[16] 刘新龙, 马丽, 陈学宽, 应雄美, 蔡青, 刘家勇, 吴才文. 云南甘蔗自育品种DNA指纹身份证构建. 作物学报, 2010, 36(2): 202-210.

Liu X L, Ma L, Chen X K, Ying X M, Cai Q, Liu J Y, Wu C W. Establishment of DNA fingerprint ID in sugarcane cultivars in Yunnan. Acta Agronomica Sinica, 2010, 36(2): 202-210. (in Chinese)

[17] Pan Y B. Databasing molecular identities of sugarcane (Saccharum spp.) clones constructed with microsatellite (SSR) DNA markers. American Journal of Plant Sciences, 2010, 1(2): 87-94.

[18] 王晓飞, 陈建华, 栾明宝, 祁建民, 许英, 孙志民. 苎麻种质资源分子身份证构建的初步研究. 植物遗传资源学报, 2010, 11(6): 802-805.

Wang X F, Chen J H, Luan M B, Qi J M, Xu Y, Sun Z M. Establishment of molecular identification in ramie germplasms. Journal of Plant Genetic Resources (in Chinese), 2010, 11(6): 802-805.

[19] 王黎明, 焦少杰, 姜艳喜, 严洪冬, 苏德峰, 孙广全. 142份甜高粱品种的分子身份证构建. 作物学报, 2011, 37(11): 1975-1983.

Wang L M, Jiao S J, Jiang Y X, Yan H D, Su D F, Sun G Q. Establishment of molecular identity in 142 sweet sorghum varieties. Acta Agronomica Sinica, 2011, 37(11): 1975-1983. (in Chinese)

[20] 颜静宛, 田大刚, 许彦, 王锋. 杂交稻主要亲本的SSR分子身份证数据库的构建. 福建农业学报, 2011, 26(2): 148-152.

Yan J W, Tian D G, Xu Y, Wang F. Constructing SSR molecular database for identity system of hybrid rice parents. Fujian Journal of Agricultural Sciences, 2011, 26(2): 148-152. (in Chinese)

[21] Ohtsubo K, Nakamura S. Cultivar identification of rice (Oryza sativa L.) by polymerase chain reaction method and its application to processed rice products. Journal of Agricultural and Food Chemistry, 2007, 55(4): 1501-1509.

[22] 邱杨, 李锡香, 李清霞, 陈亦辰, 沈镝, 王海平, 宋江萍. 利用SSR标记构建萝卜种质资源分子身份证. 植物遗传资源学报, 2014, 15(3): 648-654.

Qiu Y, Li X X, Li Q X, Chen Y C, Shen D, Wang H P, Song J P. Establishment of the molecular identification for radish germplasm using SSR markers. Journal of Plant Genetic Resources, 2014, 15(3): 648-654. (in Chinese)

[23] 刘倩, 戴志刚, 陈基权, 温岚, 龚友才, 粟建光. 应用SRAP分子标记构建红麻种质资源分子身份证. 中国农业科学, 2013, 46(10): 1974-1983.

Liu Q, Dai Z G, Chen J Q, Wen L, Gong Y C, Su J G.. Establishment of molecular identity for kenaf germplasm using SRAP marker. Scientia Agricultura Sinica, 2013, 46(10): 1974-1983. (in Chinese)

[24] 艾呈祥, 张力思, 魏海蓉, 苑克俊, 金松南, 刘庆忠. 甜樱桃品种SSR指纹图谱数据库的建立. 中国农学通报, 2007, 23(5): 55-58.

Ai C X, Zhang L S, Wei H R, Yuan K J, Jin S N, Liu Q Z. Construction of molecular fingerprinting database for sweet cherry using SSR markers. Chinese Agricultural Science Bulletin, 2007, 23(5): 55-58. (in Chinese)

[25] 杜晶晶, 刘国银, 魏军亚, 刘德兵, 杨小振. 基于SSR标记构建葡萄种质资源分子身份证. 植物研究, 2013, 33(2): 232 -237.

Du J J, Liu G Y, Wei J Y, Liu D B, Yang X Z. Establishment of molecular ID for grape germplasm based on SSR markers. Bulletin of Botanical Research, 2013, 33(2): 232 -237. (in Chinese)

[26] 张靖国, 田瑞, 陈启亮, 杨晓平, 胡红菊. 基于SSR标记的梨栽培品种分子身份证的构建. 华中农业大学学报, 2014, 33(1): 12-17.

Zhang J G, Tian R, Chen Q L, Yang X P, Hu H J. Establishment of molecular ID for pear cultivars based on SSR markers. Journal of Huazhong Agricultural University, 2014, 33(1): 12-17. (in Chinese)

[27] Moriya S, Iwanami H, Okada K, Yamamoto T, Abe K. A practical method for apple cultivar identification and parent-offspring analysis using simple sequence repeat markers. Euphytica, 2011, 177(1): 135-150.

[28] 王立新, 张小军, 史星云, 高华, 赵政阳. 苹果栽培品种SSR指纹图谱的构建. 果树学报, 2012, 29(6): 971-977.

Wang L X, Zhang X J, Shi X Y, Gao H, Zhao Z Y. Establishment of SSR fingerprinting database on major apple (Malus ×Domestica) cultivars. Journal of Fruit Science, 2012, 29(6): 971-977. (in Chinese)

[29] 高源, 王昆, 刘凤之, 聂继云, 王大江, 龚欣, 刘立军. 适宜加工用苹果品种 TP-M13-SSR 指纹图谱构建及遗传关系分析. 园艺学报, 2014, 41(5): 946-956.

Gao Y, Wang K, Liu F Z, Nie J Y, Wang D J, Gong X, Liu L J. Establishment of TP-M13-SSR fingerprints and analysis of genetic relationship for 44 processing apple cultivars. Acta Horiculture Sinica (in Chinese), 2014, 41(5): 946-956.

[30] Liebhard R, Gianfrancechi L, Koller B, Ryder C D, Tarchini R, Vandeweg E, Gessler C. Development and characterization of 140 new microsatellites in apple (Malus×domestica Borkh.). Molecular Breeding, 2002, 10: 217-241.

[31] Yamamoto T, Kimura T, Sawamura Y, Manabe T, Kotobuki K, Hayashi T, Ban Y, Matsuta N. Simple sequence repeats for genetic analysis in pear. Euphytica, 2002, 124: 129-137.

[32] Yamamoto T, Kimura T, Shoda M, Ban Y, Hayashi T, Matsuta N. Development of microsatellite markers in the Japanese pear (Pyrus pyrifolia Nakai). Molecular Ecology Notes, 2002, 2: 14-16.

[33] 郝晨阳, 王兰芬, 贾继增, 董玉琛, 张学勇. SSR荧光标记和银染技术的比较分析. 作物学报, 2005, 31(2): 144-149.

Hao C Y, Wang L F, Jia J Z, Dong Y C, Zhang X Y. Comparison of fluorescence and silver-staining detection systems of microsatellite markers. Acta Agronomica Sinica, 2005, 31(2): 144-149. (in Chinese)

[34] 李莉, 杨剑波, Mackill D J, Colowit P M. 水稻SSR不同检测和分析方法的比较研究. 中国水稻科学, 2000, 14(3): 185-188.

Li L, Yang J B, Mackill D J, Colowit P M. Comparison of different detection methods for rice SSR analysis. Chinese Journal of Rice Science, 2000, 14(3): 185-188. (in Chinese)