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桤木属(Alnus Mill.)为非豆科固氮树种,根系富含根瘤,可改良土壤,是重要的先锋造林与生态功能树种。桤木属是现存桦木科植物中最原始的属,也是北半球新生代植物区系的重要植物类群,主要分布在欧亚和北美,拉丁美洲与非洲有少量分布[1-2]。四川省及邻近地区是桤木属的重要分布区,原生分布有桤木(Alnus cremastogyne, 又名四川桤木)、川滇桤木(A. ferdinandi-coburgii)、毛桤木(A. lanata)、尼泊尔桤木(A. nepalensis),可能是桤木属植物起源与分化的中心[1],其中桤木是我国最重要的一个特有种,也是研究最广泛的一个种,生长迅速、适应性强、童期短且结实量大,目前其适生栽培区域已扩大至长江中下游地区,是中国长江流域退耕还林工程、生态建设工程和混交造林的重要树种。
SSR(Simple sequence repeat,简单序列重复)分子标记具有分布广泛、多态性丰富、稳定、共显性等特点,自从被开发以来,在物种遗传改良上获得了广泛的应用。在桤木属植物SSR研究方面,Zhuk等最早开发出了桤木SSR引物[3]。随后,Lance等通过筛选海岸桤木(A. maritima)基因组文库获得了19条桤木SSR引物 [4]。使用Lance开发的引物,Jones等人系统研究了美国濒危物种海岸桤木的遗传多样性与群体结构等,为其提供了的濒危保护理论基础[5,6]。随后SSR技术逐渐的应用到其他桤木属树种中,这些树种的群体遗传变异基础与进化史也逐渐被深入揭示[7-10]。目前国内桤木遗传改良研究主要集中于育苗、栽培等常规育种方面,在群体遗传变异研究上,主要通过表型鉴定方法进行[11-14],采用分子标记手段的研究较少,仅有卓仁英等建立了RAPD体系[15]。在SSR分子标记方面,也仅有饶龙兵等基于桤木、欧洲桤木(A. glutinosa)、硬桤木(A. firma)转录组数据开发了适用于桤木属的SSR标记[16]。总之桤木群体遗传变异缺乏分子水平上的数据支撑,影响了其保护与进一步的推广利用。
此外,SSR分子标记从来源上说包括Genomic-SSR与EST-SSR,分别来源于基因组数据与表达序列标签(Express sequence tags,EST)数据。本研究分析了2种来源的桤木SSR标记的差异,以期推动其在国内桤木遗传变异研究上的应用。
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采样群体为天然次生林,共包括13个群体(表1)。群体范围包括成都平原区、盆周山地区、盆地丘陵区、川西高山峡谷区和川西南山地区。群体取样单株之间相距至少50m,采集桤木新鲜叶片,硅胶干燥保存带回实验室。
表 1 桤木群体基本信息
Table 1. Location and number of trees sampled in 13 Alnus Cremastogyne populations
群体编号
ID地点
Location群体数目
Size经度
Longitude (E)纬度
Latitude (N)海拔范围
Elevation range/mAA 美姑 8 103.08 28.30 1609–2352 AC 峨边 13 103.25 29.28 828–1180 B 泸定 12 102.13 29.77 1303–2343 F 冕宁 16 102.10 29.18 1884–1908 I 平武 11 104.50 32.30 845–1440 J 青川 12 105.14 32.41 552–1496 K 剑阁 13 105.45 32.16 569–808 N 巴中 24 106.63 31.68 368–591 Q 宣汉 10 107.62 31.55 482–998 S 金堂 12 103.80 30.74 740–960 T 邛崃 9 103.16 30.32 746–1118 U 沐川 16 103.71 29.15 500–530 V 峨眉山 8 103.33 29.59 650–1273 -
使用天根植物基因组提取试剂盒DNA(DP305)提取基因组DNA。
SSR扩增所用引物来源于2部分:(1)Genomic-SSR,公开发表文献中其它桤木属树种相关研究中使用的SSR引物[3-4, 17-18],10对引物;(2)EST-SSR[19],6对引物。具体信息见表2。
表 2 桤木10个Genomic-SSR与6个EST-SSR标记遗传参数
Table 2. Genetic diversity of 164 trees in A. cremastogyne revealed by 10 Genomic-SSRs and 6 EST-SSRs
Locus Repeat motif Size range Tm(oC) Na Ne Ho Ht Gst Nm Primer source Acg3 (CT)3CC(CT)2CC
(CT)13AT(CT)5210-262 55 18.00 5.156 0.963 0.859 0.035 6.893 L3.1[9] Acg7 (AG)11 265-293 56 13.00 3.897 0.939 0.791 0.028 8.679 Alma7[10] Acg8 (AG)12 177-217 56 16.00 4.093 0.829 0.804 0.016 15.375 Alma12[10] Acg11 (GT)12(GA)10 248-256 58 5.00 2.249 0.884 0.582 0.002 124.75 Alma20[10] Acg16 (AG)14 156-168 60 7.00 4.422 0.854 0.832 0.034 7.103 CAC-A105[17] Acg19 (TC)15 130-138 58 5.00 2.724 0.893 0.663 0.006 41.417 CAC-B113[17] Acg20 (GA)12 142-196 58 25.00 2.564 0.652 0.656 0.031 7.815 CAC-C118[17] Acg21 (TC)12 247-287 58 20.00 6.111 0.994 0.871 0.013 18.981 Ag01[18] Acg22 (TC)11 184-210 58 8.00 3.101 0.963 0.720 0.026 9.365 Ag05[18] Acg23 (TG)12 248-280 58 14.00 3.468 0.518 0.763 0.017 14.456 Ag09[18] 均值 13.10 3.779 0.849 0.754 0.021 11.655 Ace1 (TC)11n(TC)8ta
(TC)6220-248 60 13.00 1.768 0.560 0.467 0.020 12.250 FQ338662[19] Ace3 (CT)12(CA)6 210-240 60 11.00 3.778 0.939 0.794 0.042 5.702 FQ335170[19] Ace27 (CT)13(CA)7 155-185 58 15.00 7.113 0.988 0.884 0.002 124.750 FQ351578[19] Ace29 (TC)26 219-285 58 27.00 8.467 0.994 0.934 0.010 24.750 FQ344263[19] Ace35 (TC)20 194-200 58 4.00 1.670 0.713 0.468 0.087 2.624 FQ334282[19] Ace37 (GA)19 119-137 58 10.00 1.402 0.415 0.314 0.029 8.371 FQ351410[19] 均值 13.33 4.033 0.768 0.643 0.027 9.009 总均值 13.19 3.874 0.819 0.713 0.025 9.750 Na: 等位基因数; Ne: 有效等位基因数; Ho: 观察杂合度; Ht: 期望杂合度; Gst: 分化系数; Nm: 基因流
Na: number of alleles; Ne: number of effective alleles; Ho: observed heterozygosity; Ht: expected heterozygosity; Gst: differentiation coefficient; Nm: gene flowSSR上游引物添加FAM荧光标记。PCR扩增使用Takara Taq 聚合酶(Takara, Dalian, China) ,20 uL反应体系包括: 13.85 μL ddH2O, 2.0 μL 10 × buffer, 2.0 μL 2.0 mM dNTP, 0.5 μL of each primer (at 10 μM), 1 μL基因组DNA模版(30-50ng), and 0.75 U Taq DNA聚合酶。扩增程序如下:94 ℃预变性5 min;94 ℃变性20 s,退火温度退火20 s,72 ℃延伸40 s,25-30个循环;72 ℃延长5 min,4 ℃保存。退火温度根据文献或引物设计软件给出的数据。PCR产物进行毛细管荧光电泳分型,SSR片段长度由软件GeneMarker version 2.2.0 (SoftGenetics, USA)判读。
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首先利用Excel2007整理整合GeneMarker输出的基因型分子量数据,随后数据输入基于R环境的Polysat 1.6软件[20],整合相关信息后,最后输出为GenoDive格式文件进行进一步分析。利用GenoDive 2.0b27[21]计算等位基因数(Na)、有效等位基因数(Ne)、观测杂合度(Ho)、总望杂合度(Ht)、固定系数(Gst,同Fst)等遗传参数,评价各群体的遗传多样性水平。基因流值(Nm)根据公式(1-GST)/(4GST)估算[22]。
利用GenoDive 2.0b27计算Nei’s(1978)遗传距离,运用NTSYS-pc 2.10s 软件生成UPGMA聚类图并计算各遗传距离的相关系数[23-24]。
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利用10个Genomic-SSR位点与6个EST-SSR个位点检测了13个桤木群体164个个体的遗传多样性参数,16个位点全部具有多态性。进一步分析结果表明(表2),在10个Genomic-SSR位点中,平均等位基因数为13.10,平均有效等位基因数为3.779,平均观察杂合度为0.849,平均期望杂合度为0.754;对EST-SSR来说,平均等位基因数为13.33,平均有效等位基因数为4.033,平均观察杂合度为0.768,平均期望杂合度为0.643。平均等位基因数EST-SSR位点高于Genomic-SSR位点,而对检测的杂合度来说,Genomic-SSR位点高于EST-SSR位点。就单个位点而言,揭示遗传多性度最高的为EST-SSR位点Ace29,其等位基因数达到27,有效等位基因数为8.467,观察杂合度为0.994。在10个基因组SSR位点中,等位基因数≥20的位点有2个,在10与20之间的位点有4个,10以下的位点有4个;而6个EST-SSR位点中,有1个超过20,在10(包括等于)与20之间的位点有4个,10以下的位点有1个。
针对13个桤木群体来说,除了平均有效等位基因数EST-SSR位点高于Genomic-SSR(4.251>3.941)之外,其余3个遗传参数(平均等位基因数、观察杂合度、期望杂合度)Genomic-SSR位点均高于EST-SSR。对于具体群体来说,Genomic-SSR与EST-SSR位点分析均显示F(冕宁)群体遗传多样性水平最高,但是Genomic-SSR位点显示I(平武、Ne值最小)、J(青川、Na与Ho值最小)、Q(宣汉、Ht值最小)群体遗传多样性水平最低,EST-SSR位点显示I(平武、Na、Ne、Ho值最小)、U(沐川、Ht值最小)群体遗传多样性水平最低。
表 4 基于Genomic-SSR、EST-SSR与Genomic-SSR+EST-SSR计算的Nei’s遗传距离相关性
Table 4. Correlation between genetic similarity coefficients based on Genomic-SSR, EST-SSR, and Genomic-SSR+EST-SSR
标记
MarkerGenomic-SSR遗传距离
Genetic distance of Genomic-SSREST-SSR遗传距离
Genetic distance of EST-SSRGenomic-SSR 1 EST-SSR 0.908** 1 Genomic-SSR+EST-SSR 0.975** 0.966** 注: **表示在 0.01水平相关显著。
Note: * * indicates significant correlation at 0.01 level. -
为确定两种标记对桤木群体遗传关系的鉴定准确度,本研究基于Nei’s遗传距离使用UPGMA方法对参试材料进行聚类分析,由图1(A、B)可知,Genomic-SSR与EST-SSR均将AA与F群体归为1类,进一步Genomic-SSR标记将其余群体归为3类:AC、B;I、S、T、V;J、Q、U、K、N,而EST-SSR划分的3类为:AC;B、T;I、J、K、N、Q、U、V、S。说明在大的区域分类上,Genomic-SSR与EST-SSR相一致,而在小的分类上有差异。
图 1 基于Nei’s遗传距离的桤木群体UPGMA聚类
Figure 1. Unweighted pair-group method with arithmetic means (UPGMA) cluster analysis of 13 A. cremastogyne populations based on Nei’s genetic distance (A: Genomic-SSR; B: EST-SSR; C: Genomic-SSR+EST-SSR)
基于Genomic-SSR与EST-SSR总的16个标记的Nei’s遗传距离构建的UPGMA聚类树显示桤木群体可明显地分为4个类群(AA与F、B、I、其它)(图1 C),与Genomic-SSR标记结果更为相似,说明本研究中Genomic-SSR更能精确地鉴别出桤木群体遗传关系。
对 Genomic-SSR、EST-SSR以及 Genomic-SSR+EST-SSR计算出的遗传距离进行相关性分析,结果显示(表3):3部分相关系数呈极显著正相关(P<0.01),但是Genomic-SSR与Genomic-SSR+EST-SSR 相关系数稍高,即两种标记计算的综合相关系数与Genomic-SSR标记更为相似,表明本研究中Genomic-SSR能更准确地揭示不同桤木个体间的遗传关系,与上述聚类分析结果相一致。
表 3 基于10个Genomic-SSR与6个EST-SSR标记的桤木群体遗传多样性
Table 3. Genetic diversity of 13 A. cremastogyne populations revealed by 10 Genomic-SSRs and 6 EST-SSRs
群体 gSSR eSSR gSSR+eSSR Na Ne Ho Ht Na Ne Ho Ht Na Ne Ho Ht AA(美姑) 7.90 4.664 0.875 0.763 8.50 4.788 0.875 0.739 8.125 4.631 0.875 0.747 AC(峨边) 7.10 3.555 0.831 0.711 6.67 4.304 0.718 0.609 6.938 3.756 0.788 0.663 B(泸定) 7.70 4.584 0.875 0.782 7.00 4.084 0.792 0.667 7.438 4.366 0.844 0.736 F(冕宁) 9.60 4.920 0.913 0.785 9.83 5.328 0.854 0.743 9.688 4.961 0.891 0.762 I(平武) 7.10 3.318 0.827 0.707 5.33 3.401 0.682 0.560 6.438 3.295 0.773 0.646 J(青川) 6.10 3.711 0.808 0.694 5.50 3.740 0.833 0.619 5.875 3.669 0.818 0.660 K(剑阁) 6.60 3.522 0.846 0.694 7.00 4.499 0.756 0.605 6.75 3.806 0.813 0.656 N(巴中) 8.20 3.683 0.821 0.693 8.33 4.918 0.75 0.570 8.25 4.11 0.794 0.644 Q(宣汉) 6.60 3.578 0.840 0.692 6.50 4.109 0.767 0.578 6.562 3.691 0.813 0.641 S(金堂) 6.90 4.088 0.842 0.723 5.67 3.581 0.722 0.576 6.438 3.798 0.797 0.657 T(邛崃) 6.50 3.847 0.911 0.749 6.00 4.127 0.852 0.663 6.312 3.853 0.889 0.707 U(沐川) 7.90 4.055 0.838 0.718 7.83 4.457 0.719 0.552 7.875 4.072 0.793 0.648 V(峨眉山) 6.10 3.708 0.850 0.746 5.67 3.933 0.708 0.556 5.938 3.694 0.797 0.665 均值 7.254 3.941 0.852 0.727 6.910 4.251 0.771 0.618 7.125 3.977 0.822 0.679 Na: 等位基因数; Ne: 有效等位基因数; Ho: 观察杂合度; Ht: 期望杂合度.
Na: number of alleles; Ne: number of effective alleles; Ho: observe the heterozygosity; Ht: Expected heterozygosity.
Genetic Differences Revealed by Genomic-SSR and EST-SSR Markers in Alnus cremastogyne
More Information-
摘要: 桤木(Alnus cremastogyne)为非豆科固氮树种,也是我国最重要的一个桤木属特有种,具有重要的生态功能。本文分析了桤木基因组SSR与EST-SSR两种来源标记的遗传差异,结果显示EST-SSR的平均等位基因数与平均有效等位基因数高于Genomic-SSR,而对平均观察杂合度与平均期望杂合度来说,Genomic-SSR高于EST-SSR,聚类分析显示Genomic-SSR与EST-SSR在小的分群上有差异,说明Genomic-SSR与EST-SSR标记在解析遗传多样性与遗传关系方面有一定差异,综合两种标记可以获得更加客观的结果。Abstract: As a non-leguminous and nitrogen-fixing tree species, Alnus cremastogyne is also the most important endemic species of Alnus in China, which has important ecological functions. In this paper, the genetic differences of Genomic-SSR and EST-SSR markers in Alnus cremastogyne genome were analyzed. The results showed that the average number of alleles and the average number of effective alleles with EST-SSR were higher than those of genomic-SSR, while the average observed heterozygosity and average expected heterozygosity were higher than those of EST-SSR. Cluster analysis showed that there were differences between genomic-SSR and EST-SSR in small groups, which indicated that there were some differences between Genomic-SSR and EST-SSR in analyzing genetic diversity and genetic relationship to a certain extent, and more objective results could be obtained by combining the two marker methods.
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Key words:
- Alnus cremastogyne;
- SSR;
- Genetic diversity;
- Polyploid;
- GenoDive
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表 1 桤木群体基本信息
Tab. 1 Location and number of trees sampled in 13 Alnus Cremastogyne populations
群体编号
ID地点
Location群体数目
Size经度
Longitude (E)纬度
Latitude (N)海拔范围
Elevation range/mAA 美姑 8 103.08 28.30 1609–2352 AC 峨边 13 103.25 29.28 828–1180 B 泸定 12 102.13 29.77 1303–2343 F 冕宁 16 102.10 29.18 1884–1908 I 平武 11 104.50 32.30 845–1440 J 青川 12 105.14 32.41 552–1496 K 剑阁 13 105.45 32.16 569–808 N 巴中 24 106.63 31.68 368–591 Q 宣汉 10 107.62 31.55 482–998 S 金堂 12 103.80 30.74 740–960 T 邛崃 9 103.16 30.32 746–1118 U 沐川 16 103.71 29.15 500–530 V 峨眉山 8 103.33 29.59 650–1273 表 2 桤木10个Genomic-SSR与6个EST-SSR标记遗传参数
Tab. 2 Genetic diversity of 164 trees in A. cremastogyne revealed by 10 Genomic-SSRs and 6 EST-SSRs
Locus Repeat motif Size range Tm(oC) Na Ne Ho Ht Gst Nm Primer source Acg3 (CT)3CC(CT)2CC
(CT)13AT(CT)5210-262 55 18.00 5.156 0.963 0.859 0.035 6.893 L3.1[9] Acg7 (AG)11 265-293 56 13.00 3.897 0.939 0.791 0.028 8.679 Alma7[10] Acg8 (AG)12 177-217 56 16.00 4.093 0.829 0.804 0.016 15.375 Alma12[10] Acg11 (GT)12(GA)10 248-256 58 5.00 2.249 0.884 0.582 0.002 124.75 Alma20[10] Acg16 (AG)14 156-168 60 7.00 4.422 0.854 0.832 0.034 7.103 CAC-A105[17] Acg19 (TC)15 130-138 58 5.00 2.724 0.893 0.663 0.006 41.417 CAC-B113[17] Acg20 (GA)12 142-196 58 25.00 2.564 0.652 0.656 0.031 7.815 CAC-C118[17] Acg21 (TC)12 247-287 58 20.00 6.111 0.994 0.871 0.013 18.981 Ag01[18] Acg22 (TC)11 184-210 58 8.00 3.101 0.963 0.720 0.026 9.365 Ag05[18] Acg23 (TG)12 248-280 58 14.00 3.468 0.518 0.763 0.017 14.456 Ag09[18] 均值 13.10 3.779 0.849 0.754 0.021 11.655 Ace1 (TC)11n(TC)8ta
(TC)6220-248 60 13.00 1.768 0.560 0.467 0.020 12.250 FQ338662[19] Ace3 (CT)12(CA)6 210-240 60 11.00 3.778 0.939 0.794 0.042 5.702 FQ335170[19] Ace27 (CT)13(CA)7 155-185 58 15.00 7.113 0.988 0.884 0.002 124.750 FQ351578[19] Ace29 (TC)26 219-285 58 27.00 8.467 0.994 0.934 0.010 24.750 FQ344263[19] Ace35 (TC)20 194-200 58 4.00 1.670 0.713 0.468 0.087 2.624 FQ334282[19] Ace37 (GA)19 119-137 58 10.00 1.402 0.415 0.314 0.029 8.371 FQ351410[19] 均值 13.33 4.033 0.768 0.643 0.027 9.009 总均值 13.19 3.874 0.819 0.713 0.025 9.750 Na: 等位基因数; Ne: 有效等位基因数; Ho: 观察杂合度; Ht: 期望杂合度; Gst: 分化系数; Nm: 基因流
Na: number of alleles; Ne: number of effective alleles; Ho: observed heterozygosity; Ht: expected heterozygosity; Gst: differentiation coefficient; Nm: gene flow表 4 基于Genomic-SSR、EST-SSR与Genomic-SSR+EST-SSR计算的Nei’s遗传距离相关性
Tab. 4 Correlation between genetic similarity coefficients based on Genomic-SSR, EST-SSR, and Genomic-SSR+EST-SSR
标记
MarkerGenomic-SSR遗传距离
Genetic distance of Genomic-SSREST-SSR遗传距离
Genetic distance of EST-SSRGenomic-SSR 1 EST-SSR 0.908** 1 Genomic-SSR+EST-SSR 0.975** 0.966** 注: **表示在 0.01水平相关显著。
Note: * * indicates significant correlation at 0.01 level.表 3 基于10个Genomic-SSR与6个EST-SSR标记的桤木群体遗传多样性
Tab. 3 Genetic diversity of 13 A. cremastogyne populations revealed by 10 Genomic-SSRs and 6 EST-SSRs
群体 gSSR eSSR gSSR+eSSR Na Ne Ho Ht Na Ne Ho Ht Na Ne Ho Ht AA(美姑) 7.90 4.664 0.875 0.763 8.50 4.788 0.875 0.739 8.125 4.631 0.875 0.747 AC(峨边) 7.10 3.555 0.831 0.711 6.67 4.304 0.718 0.609 6.938 3.756 0.788 0.663 B(泸定) 7.70 4.584 0.875 0.782 7.00 4.084 0.792 0.667 7.438 4.366 0.844 0.736 F(冕宁) 9.60 4.920 0.913 0.785 9.83 5.328 0.854 0.743 9.688 4.961 0.891 0.762 I(平武) 7.10 3.318 0.827 0.707 5.33 3.401 0.682 0.560 6.438 3.295 0.773 0.646 J(青川) 6.10 3.711 0.808 0.694 5.50 3.740 0.833 0.619 5.875 3.669 0.818 0.660 K(剑阁) 6.60 3.522 0.846 0.694 7.00 4.499 0.756 0.605 6.75 3.806 0.813 0.656 N(巴中) 8.20 3.683 0.821 0.693 8.33 4.918 0.75 0.570 8.25 4.11 0.794 0.644 Q(宣汉) 6.60 3.578 0.840 0.692 6.50 4.109 0.767 0.578 6.562 3.691 0.813 0.641 S(金堂) 6.90 4.088 0.842 0.723 5.67 3.581 0.722 0.576 6.438 3.798 0.797 0.657 T(邛崃) 6.50 3.847 0.911 0.749 6.00 4.127 0.852 0.663 6.312 3.853 0.889 0.707 U(沐川) 7.90 4.055 0.838 0.718 7.83 4.457 0.719 0.552 7.875 4.072 0.793 0.648 V(峨眉山) 6.10 3.708 0.850 0.746 5.67 3.933 0.708 0.556 5.938 3.694 0.797 0.665 均值 7.254 3.941 0.852 0.727 6.910 4.251 0.771 0.618 7.125 3.977 0.822 0.679 Na: 等位基因数; Ne: 有效等位基因数; Ho: 观察杂合度; Ht: 期望杂合度.
Na: number of alleles; Ne: number of effective alleles; Ho: observe the heterozygosity; Ht: Expected heterozygosity. -
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