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基于SSR和SRAP标记的红花玉兰品种遗传关系分析及分子鉴定

张妹 何正权 马江 桑子阳 朱仲龙 张德春 马履一 陈发菊

张妹, 何正权, 马江, 桑子阳, 朱仲龙, 张德春, 马履一, 陈发菊. 基于SSR和SRAP标记的红花玉兰品种遗传关系分析及分子鉴定[J]. 北京林业大学学报, 2019, 41(9): 69-80. doi: 10.13332/j.1000-1522.20190204
引用本文: 张妹, 何正权, 马江, 桑子阳, 朱仲龙, 张德春, 马履一, 陈发菊. 基于SSR和SRAP标记的红花玉兰品种遗传关系分析及分子鉴定[J]. 北京林业大学学报, 2019, 41(9): 69-80. doi: 10.13332/j.1000-1522.20190204
Zhang Mei, He Zhengquan, Ma Jiang, Sang Ziyang, Zhu Zhonglong, Zhang Dechun, Ma Lüyi, Chen Faju. Genetic relationship analysis and molecular identification of Magnolia wufengensis cultivars based on SSR and SRAP markers[J]. Journal of Beijing Forestry University, 2019, 41(9): 69-80. doi: 10.13332/j.1000-1522.20190204
Citation: Zhang Mei, He Zhengquan, Ma Jiang, Sang Ziyang, Zhu Zhonglong, Zhang Dechun, Ma Lüyi, Chen Faju. Genetic relationship analysis and molecular identification of Magnolia wufengensis cultivars based on SSR and SRAP markers[J]. Journal of Beijing Forestry University, 2019, 41(9): 69-80. doi: 10.13332/j.1000-1522.20190204

基于SSR和SRAP标记的红花玉兰品种遗传关系分析及分子鉴定

doi: 10.13332/j.1000-1522.20190204
基金项目: 林业公益性行业科研专项(201504704)
详细信息
    作者简介:

    张妹。主要研究方向:生物化学与分子生物学。Email:335339931@qq.com 地址:443000 湖北省宜昌市西陵区大学路8号三峡大学生物与制药学院

    责任作者:

    陈发菊,博士,教授。主要研究方向:森林培育。Email:chenfj616@163.com 地址:同上

  • 中图分类号: S718.46;S718.49

Genetic relationship analysis and molecular identification of Magnolia wufengensis cultivars based on SSR and SRAP markers

  • 摘要: 目的为了解析红花玉兰各品种间的遗传多样性和遗传差异等问题,本研究采用SSR和SRAP分子标记方法对红花玉兰不同品种的遗传多样性水平和遗传分化程度进行评估,以建立其分子标记体系。方法以35个红花玉兰品种为实验材料,分别进行SSR和SRAP标记扩增,计算出SSR和SRAP分子标记的各遗传参数。利用NTSYS-pc2.1软件计算各品种间的遗传相似系数并用非加权法(UPGMA)进行聚类分析。通过R语言分析筛选出能够完全区分红花玉兰品种的引物对组合。结果经筛选共获得18对具有多态性且条带清晰的SSR引物,分别以35个红花玉兰品种基因组DNA为模板,共扩增出DNA条带128条,每对引物扩增条带在2 ~ 15之间,平均每对引物扩增7.1条,平均带型数为10.6,平均有效带型数为5.4,平均分辨能力(D)为0.72;不同引物的多态性位点百分比变化范围很大,在0.142 9 ~ 1.000 0之间,均值为0.730 2;Shannon’s指数平均为0.304 2,范围是0.086 4 ~ 0.433 7;平均期望杂合度为0.248 8,范围是0.059 2 ~ 0.282 3。利用筛选获得的11对SRAP引物对35个红花玉兰品种进行鉴定,共扩增156条DNA条带,引物扩增条带数在7 ~ 18之间,平均每对引物扩增14.2条,平均带型数为22.7,平均有效带型数为13.2,平均分辨能力(D)为0.93;11对引物的多态性位点百分比平均数为0.815 6,变异范围是0.657 1 ~ 1.000 0;平均Shannon’s指数为0.375 9,变异区间在0.287 2 ~ 0.455 3;平均期望杂合度为0.240 4,范围在0.180 2 ~ 0.311 6之间。结论红花玉兰具有较高的遗传多样性,经筛选和优化后的SSR和SRAP引物组合均能将现有红花玉兰品种完全区分开,实现了对红花玉兰品种的简便、快速、准确地鉴定,并为红花玉兰的保护和繁育,以及新品种的选育提供了重要的参考。

     

  • 图  1  红花玉兰品种的地理位置

    Figure  1.  Geographic distribution of the analyzed M. wufengensis samples from Wufeng

    图  2  35个红花玉兰品种的聚类分析树状图

    Figure  2.  Cluster analysis tree diagram of 35 M. wufengensis cultivars

    图  3  35个红花玉兰品种的聚类分析树状图

    Figure  3.  Cluster analysis tree diagram of 35 M. wufengensis cultivars

    图  4  基于Mantel检验的SSR、SRAP数据和综合数据的相关性

    Figure  4.  Correlation of SSR, SRAP data and comprehensive data based on Mantel test

    表  1  35个红花玉兰品种采样信息表

    Table  1.   Details of sampling of the M. wufengensis investigated in this study

    序号 No.品种代号 Cultivar code品种名 Cultivar来源 Origin经度 Longitude纬度 Latitude海拔 Altitude/m
    1 HHYL1 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    2 HHYL2 娇月 Jiaoyue 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    3 HHYL3 娇逸 Jiaoyi 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    4 HHYL4 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    5 HHYL5 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    6 HHYL6 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    7 HHYL7 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    8 HHYL8 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    9 HHYL9 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    10 HHYL10 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    11 HHYL11 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    12 HHYL12 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    13 HHYL13 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    14 HHYL14 ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    15 HHYL15 ~ 五峰牛庄 Niuzhuang, Wufeng 110°21′55″E 30°13′58″N 1 800
    16 HHYL16 ~ 五峰独树坪 Dushuping, Wufeng 110°24′11″E 30°09′53″N 1 400
    17 LZP1 ~ 五峰栗子坪 Liziping, Wufeng 110°21′12″E 30°06′12″N 1 690
    18 LZP2 ~ 五峰栗子坪 Liziping, Wufeng 110°21′12″E 30°06′12″N 1 690
    19 LZP4 ~ 五峰栗子坪 Liziping, Wufeng 110°21′12″E 30°06′12″N 1 690
    20 LZP18 ~ 五峰栗子坪 Liziping, Wufeng 110°21′12″E 30°06′12″N 1 690
    21 16B ~ 五峰独树坪 Dushuping, Wufeng 110°24′11″E 30°09′53″N 1 620
    22 WT ~ 五峰湾潭 Wantan, Wufengan 110°25′50″E 30°02′53″N 1 400
    23 JH1 娇红一号 Jiaohong1 五峰罗筐岩 Luokuangyan, Wufeng 110°24′09″E 30°05′35″N 1 470
    24 JH2 娇红二号 Jiaohong2 五峰高峰 Gaofeng, Wufeng 110°44′53″E 30°15′27″N 1 800
    25 JY 娇玉 Jiaoyu 五峰栗子坪 Liziping, Wufeng 110°21′12″E 30°06′12″N 1 690
    26 JL 娇莲 Jiaolian 五峰栗子坪 Liziping, Wufeng 110°21′12″ E 30°06′12″N 1 690
    27 JD 娇丹 Jiaodan 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    28 CYH ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    29 JZX ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    30 LKY2 娇婉 Jiaowan 五峰罗筐岩 Luokuangyan, Wufeng 110°24′09″E 30°09′35″N 1 470
    31 DBH 娇韵 Jiaoyun 五峰栗子坪 Liziping, Wufeng 110°21′12″E 30°06′12″N 1 690
    32 SLX 娇荷 Jiaohe 五峰栗子坪 Liziping, Wufeng 110°21′12″E 30°06′12″N 1 690
    33 HH1 ~ 五峰后荒 Houhuang, Wufeng 110°38′58″E 30°17′43″N 1 800
    34 DSP 娇阳 Jiaoyang 五峰独树坪 Dushuping, Wufeng 110°24′11″E 30°09′53″N 1 620
    35 YTYT ~ 五峰黄粱坪 Huangliangping, Wufeng 110°37′47″E 30°07′20″N 1 400
    注:~代表新品种申请中。Note: ~ represents new cultivar in application.
    下载: 导出CSV

    表  2  实验所用SSR引物信息

    Table  2.   Information of SSR primers used in the experiment

    引物
    Primer
    正向引物(5′—3′)
    Forward sequence (5′−3′)
    反向引物(5′—3′)
    Reverse sequence (5′−3′)
    重复单元
    Repeat motif
    参考文献
    Reference
    MC3 CCGCAGCAAACCCTACGC GCACACGCCAACAATGGAAAG (CT)16 [16]
    MC8 GAGTTCCGTGAGTCCCA CATAATAGAAGTCATAAATCCC (TC)15 [16]
    MC11 TCAACAGCACAAACCGAC AGAACAACAGAACCAGGG (CT)4(CT)3(CT)(CT)2 [16]
    MC35 TACATCTAAAGTCCCCACAT CTCATTTCCAGCCCATAC (CT)3(CT)4(CT)3 [16]
    MMA51 CGATGCAGCCTAAAAAGAGC CGATCATCTCTCCCGTCACT (GA)10 [17]
    SGA5 GAGATGAGTCACCGCCTGTT ATTCAGTTGCACGGCTCTCT (AG)15 [17]
    SGA15 CTGACGTAACCCGACCTGAT CCTGACTTGATCCCACCACT (GA)14 [17]
    Ksep11 GACGGGGTCCCTCCACTA TCACACCAACCAATCAGC (CT)10 [18]
    Ksep13 TAGATGTTGGACAGTTTGC CATTGATTTTGATTTGGTG (GA)11(G)2(GA)8 [18]
    L78 GAGTAAGAAATGAAGACGCTCG AGTAACAAGCCAATCAGGAGG (AG)13 [19]
    M10D6 CGACGACGAAACTACTAACA TTAAGTTGAGGTGGAATGAC (CT)11 [20]
    M17D3 AAAATTACCATAGAAGAACA TTAACAGAAACAAGCACTTA (CT)19 [20]
    LT078 ACTGGGCCGTTTATACTTTT TGACCTTTCCCTTATTCTCA (AACA)5 [4]
    LT083 ATTACGCAGCTTCCCTTAC GGAGTTCTGGTATGGTTGAG (ACAACC)4 [4]
    LT092 GGGGTTTTGCTTAATGTGA CATTCCCTACCTCCTTCTCT (GGAGCC)4 [4]
    LT115 CTCTCATTCCGACCTTCATA ACTTTTCCTGCAACTACTGC (TCA)9 [4]
    LT149 GTGAAGACCAAGGAAAATGA GGAAAAGAGAGGAGGAATGT (CTT)6 [4]
    LT164 AGCTGCCAAGACCTACAAC CCTCTTCAGGTTCCACATTA (CTT)7 [4]
    下载: 导出CSV

    表  3  基于SSR标记的红花玉兰品种遗传多样性分析

    Table  3.   Genetic diversity of M. wufengensis cultivars analyzed by SSR

    引物
    Primer
    样本量
    Number of samples
    期望杂合度
    Expected heterozygosity
    信息指数
    Shannon index
    多态性位点百分比
    Percentage of polymorphic loci
    Kesp11 35 0.171 4 0.278 3 0.657 1
    Kesp13 35 0.279 5 0.433 7 0.914 3
    L78 35 0.123 7 0.194 5 0.428 6
    LT078 35 0.261 4 0.417 7 1.000 0
    LT083 35 1.229 5 0.273 9 0.714 3
    LT092 35 0.272 5 0.425 8 0.942 9
    LT115 35 0.188 5 0.325 2 0.942 9
    LT149 35 0.162 1 0.290 3 0.914 3
    LT164 35 0.225 9 0.367 8 0.971 4
    M10D6 35 0.059 2 0.086 4 0.142 9
    M17D3 35 0.180 0 0.264 6 0.457 1
    MC3 35 0.147 2 0.255 5 0.800 0
    MC8 35 0.238 9 0.377 7 0.857 1
    MC11 35 0.139 1 0.245 5 0.771 4
    MC35 35 0.082 8 0.120 9 0.200 0
    MMA51 35 0.282 3 0.431 6 0.857 1
    SGA5 35 0.240 5 0.366 9 0.714 3
    SGA15 35 0.193 6 0.319 1 0.857 1
    均值 Mean 35 0.248 8 0.304 2 0.730 2
    下载: 导出CSV

    表  4  SSR引物对鉴别能力

    Table  4.   Discriminating power of SSR primer pairs

    引物对
    Primer pair
    带型数
    Number of banding pattern
    X2DDL有效带型数
    Number of effective banding pattern
    X
    Kesp11 9 28.00 0.823 5 0.800 0 5.000 0 105
    Kesp13 12 28.43 0.873 9 0.849 0 6.621 6 75
    L78 7 56.00 0.647 1 0.628 6 2.692 3 210
    LT078 19 44.80 0.905 9 0.880 0 8.333 3 56
    LT083 6 55.69 0.584 9 0.568 2 2.315 7 247
    LT092 16 54.14 0.865 5 0.840 8 6.282 1 80
    LT115 5 79.71 0.354 6 0.344 5 1.525 5 384
    LT149 5 94.57 0.267 2 0.259 6 1.350 6 436
    LT164 13 58.97 0.816 8 0.793 5 4.841 9 109
    M10D6 4 41.23 0.468 9 0.455 5 1.836 6 316
    M17D3 5 8.29 0.774 8 0.752 7 4.042 9 134
    MC11 4 28.89 0.559 7 0.543 7 2.191 4 262
    MC3 20 22.71 0.944 5 0.917 6 12.128 7 33
    MC35 3 16.69 0.522 7 0.507 8 2.031 5 284
    MC8 15 41.71 0.879 0 0.853 9 6.843 6 72
    MMA51 23 16.91 0.963 0 0.935 5 15.506 3 22
    SGA15 8 18.26 0.833 6 0.809 8 5.257 5 99
    SGA5 17 42.23 0.895 8 0.870 2 7.704 4 62
    均值 Mean 10.61 40.96 0.721 1 0.700 6 5.361 4 166
    注:D. 引物分辨能力;DL. 引物极限分辨能力;X. 单对无法区分的样品对数;X2,各引物带型的分布情况。表8同此。Notes: D, discriminating power of primer; DL, promer limitation of discriminating power; X, number of sample pairs can’t be distinguished by a single primer pair; X2 indicates the distribution of each primer belt type. The same as Tab.8.
    下载: 导出CSV

    表  5  SSR引物对组合优化分析

    Table  5.   Majorization of SSR primer pair combination

    序号
    No.
    引物组合
    Prime pair combination
    多态性条带数
    Number of polymorphic bands
    DDL无法区分的样品对数
    Indistinguishable cultivar pairs
    1 LT078 + MC3 + MMA51 35 1 0.971 4 0
    2 LT078 + MC3 + MC8 34 1 0.971 4 0
    3 LT164 + MC3 + MMA51 33 1 0.971 4 0
    4 MC3 + SGA15 + SGA5 33 1 0.971 4 0
    5 LT092 + MC3 + MMA51 32 1 0.971 4 0
    6 LT092 + MC3 + MC8 31 1 0.971 4 0
    7 LT092 + MC3 + SGA5 31 1 0.971 4 0
    8 Kesp11 + LT092 + MC3 30 1 0.971 4 0
    9 LT078 + MC8 + SGA15 30 1 0.971 4 0
    10 MC3 + MC8 + MMA51 30 1 0.971 4 0
    11 MC3 + MMA51 + SGA5 30 1 0.971 4 0
    下载: 导出CSV

    表  6  实验所用SRAP引物信息

    Table  6.   Information of SRAP primers used in the experiment

    引物 Primer正向引物(5 ′—3′) Forward sequence (5′−3′)引物 Primer反向引物(5′—3′) Reverse sequence (5′−3′)
    me1 TGAGTCCAAACCGGATA em1 GACTGCGTACGAATTAAT
    me2 TGAGTCCAAACCGGAGC em2 GACTGCGTACGAATTTGC
    me3 TGAGTCCAAACCGGAAT em3 GACTGCGTACGAATTGAC
    me4 TGAGTCCAAACCGGACC em4 GACTGCGTACGAATTTGA
    me5 TGAGTCCAAACCGGAAG em5 GACTGCGTACGAATTAAC
    me6 TGAGTCCAAACCGGGAT em6 GACTGCGTACGAATTGCA
    me7 TGAGTCCAAACCGGTGC em7 GACTGCGTACGAATTTAG
    me8 GTACATAGAACCGGAGT em8 GACTGCGTACGAATTATT
    me9 AGCGAGCAAGCCGGTGG em9 GACTGCGTACGAATTATG
    me10 TGGGGACAACCCGGCTT em10 GACTGCGTACGAATTCTG
    me11 TTCAGGGTGGCCGGATG em11 AGGCGGTTGTCAATTGAC
    me12 GACCAGTAAACCGGATG em12 TGTGGTCCGCAAATTTAG
    下载: 导出CSV

    表  7  基于SRAP标记的红花玉兰品种遗传多样性分析

    Table  7.   Genetic diversity of M. wufengensis cultivars analyzed by SRAP

    引物
    Primer
    样本量
    Number of samples
    期望杂合度
    Expected heterozygosity
    信息指数
    Shannon index
    多态性位点百分比
    Percentage of polymorphic loci
    me1-em1 35 0.223 5 0.366 9 0.885 7
    me3-em1 35 0.284 2 0.448 2 1.000 0
    me4-em5 35 0.274 2 0.424 4 0.885 7
    me5-em1 35 0.207 8 0.319 3 0.657 1
    me5-em5 35 0.265 5 0.411 2 0.828 6
    me5-em6 35 0.311 6 0.456 2 0.800 0
    me8-em5 35 0.198 0 0.308 1 0.685 7
    me9-em7 35 0.219 5 0.353 7 0.857 1
    me9-em9 35 0.296 4 0.455 3 0.885 7
    me10-em10 35 0.180 2 0.287 2 0.685 7
    me11-em7 35 0.183 1 0.304 4 0.800 0
    均值 Mean 35 0.240 4 0.375 9 0.815 6
    下载: 导出CSV

    表  8  SRAP引物对鉴别能力

    Table  8.   Discriminating power of SRAP primer pairs

    引物对
    Primer pair
    带型数
    Number of banding pattern
    X2DDL有效带型数
    Number of effective banding pattern
    X
    me1-em1 29 8.91 0.984 9 0.956 7 23.113 2 9
    me10-em10 17 51.94 0.879 0 0.853 9 6.843 6 72
    me11-em7 26 26.66 0.959 7 0.932 2 14.759 0 24
    me3-em1 23 15.60 0.964 7 0.937 1 15.909 1 21
    me4-em5 28 9.00 0.983 2 0.955 1 22.272 7 10
    me5-em1 22 72.49 0.885 7 0.860 4 7.163 7 68
    me5-em5 19 31.77 0.926 1 0.899 6 9.959 3 44
    me5-em6 25 25.71 0.958 0 0.930 6 14.411 8 25
    me8-em5 24 65.80 0.905 9 0.880 0 8.333 3 56
    me9-em7 25 14.29 0.971 4 0.943 7 17.753 6 17
    me9-em9 12 50.37 0.820 2 0.796 7 4.919 7 107
    均值 Mean 22.72 33.87 0.930 8 0.904 2 13.221 7 41
    下载: 导出CSV

    表  9  SRAP标记引物对组合优化

    Table  9.   Majorization of SRAP primer pair combination

    序号
    No.
    引物组合
    Primer pair combination
    多态性条带数
    Number of polymorphic band
    DDL无法区分的样品对数
    Indistinguishable cultivar pair
    1me1-em1 + me10-em103010.971 40
    2me1-em1 + me3-em13010.971 40
    3me1-em1 + me9-em73310.971 40
    4me1-em1 + me9-em93510.971 40
    5me10-em10 + me11-em73010.971 40
    6me10-em10 + me5-em62710.971 40
    7me10-em10 + me9-em72710.971 40
    8me11-em7 + me4-em53610.971 40
    9me3-em1 + me8-em52610.971 40
    10me4-em5 + me8-em53210.971 40
    11me4-em5 + me9-em73310.971 40
    下载: 导出CSV
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出版历程
  • 收稿日期:  2019-04-29
  • 修回日期:  2019-06-27
  • 网络出版日期:  2019-07-10
  • 刊出日期:  2019-09-01

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