老芒麦种质资源遗传多样性的SRAP分析

引用本文

顾晓燕, 郭志慧, 张新全, 周永红, 白史且, 张昌兵, 蒋忠荣, 刘新, 周朝杰, 马啸. 老芒麦种质资源遗传多样性的SRAP分析. 23(1): 205-216
GU Xiao-yan, GUO Zhi-hui, ZHANG Xin-quan, ZHOU Yong-hong, BAI Shi-qie, ZHANG Chang-bing, JIANG Zong-rong, LIU Xin, ZHOU Chao-jie, MA Xiao. Genetic diversity ofElymus sibiricus germplasm resources revealed by SRAP markers . Acta Prataculturae Sinica, 23(1): 205-216 复制到剪切板

Permissions

本文属于开放获取期刊。

老芒麦种质资源遗传多样性的SRAP分析

顾晓燕¹, 郭志慧¹, 张新全¹, 周永红², 白史且³, 张昌兵³, 蒋忠荣⁴, 刘新¹, 周朝杰¹, 马啸^1*

1.四川农业大学动物科技学院草业科学系,四川雅安 625014

2.四川农业大学小麦研究所,四川成都,611130

3.四川省草原科学研究院,四川成都 611731

4.四川省甘孜藏族自治州畜牧科学研究所,四川康定 626000

*通讯作者。E-mail:maroar@126.com

作者简介:顾晓燕(1989-),女,甘肃兰州人,在读硕士。E-mail:mobei_ttxs@126.com

基金:国家科技支撑计划(2011BAD17B03,2012BAD13B06),四川省科技支撑计划(2011NZ0098-11)和国家自然科学基金(31101763)资助;

摘要

利用SRAP标记,对来自亚洲的84份老芒麦种质的遗传多样性和遗传关系进行了分析。23个引物组合共产生337条扩增带,其中203条为多态性带,多态性比率为60.24%。各种质间遗传相似系数的变幅为0.783~0.965,平均值为0.865。来自于青藏高原和蒙古的种质间的平均遗传相似性(GS)值最小(0.830),而来自于俄罗斯和蒙古的种质间的平均GS值最大(0.897)。对84份种质的聚类分析表明,供试种质可以划分成2大类,而且聚类结果与原始相似性矩阵间具有很高的吻合度(r=0.88)。同时,主向量分析(PCoA)也得到了与聚类分析类似的结果。方差分析(AMOVA)表明在总的遗传变异中有79.62%发生在地理类群内,有20.38%发生在类群间(Φ_ST=0.204),类群间和类群内的变异均为极显著(P<0.0001)。基于各地理类群间Φ_ST值进行的聚类分析也表明青藏高原类群明显区别于其他地理类群。这种聚类模式可能依赖于种质地理来源赋予其的特殊生态地理适应性。本研究结果对于今后老芒麦种质的利用和品种选育提供了有益信息。

关键词: 老芒麦; 种质资源; 遗传多样性; SRAP

中图分类号:S543.03 文献标志码:A 文章编号:1004-5759(2014)01-0205-12

Genetic diversity ofElymus sibiricus germplasm resources revealed by SRAP markers

GU Xiao-yan¹, GUO Zhi-hui¹, ZHANG Xin-quan¹, ZHOU Yong-hong², BAI Shi-qie³, ZHANG Chang-bing³, JIANG Zong-rong⁴, LIU Xin¹, ZHOU Chao-jie¹, MA Xiao^1*

1.Animal Science and Technology College, Sichuan Agricultural University, Ya#cod#x02019;an 625014, China

2.Institute of Triticeae, Sichuan Agricultural University, Chengdu 611130, China

3.Sichuan Academy of Grassland Science, Chengdu 611731, China

4.Institute of Animal Husbandry of Ganzi Autonomous Prefecture of Sichuan Province, Kangding 626000, China

Fund:

Abstract

SRAP markers were used to analyze genetic diversity and genetic relationship among eighty-fourElymus sibiricus accessions from Asia. A set of 23 primer combinations yielded 337 bands, of which 203 were polymorphic (60.24%). Genetic similarity values (GS) among the accessions ranged between 0.783 and 0.965 with a mean of 0.865. On the average, Mongolian and Russian accessions were the most similar while, Mongolian and Qinghai-Tibet Plateau accessions were the most distant ones. Cluster analysis grouped the 84 accessions into two clusters, which has quite a high fit (r=0.88) to the original similarity matrix. Results of cluster analysis which was supported strongly by the principal coordinate analysis. The molecular variance analysis (AMOVA) showed that the proportion of variation explained by within geographic group and between geographic groups diversity was 0.7962 and 0.2038, respectively. Similar results were obtained when genetic diversity was estimated using the Shannon’s index of diversity. Based on pairwiseΦ_ST between geographic groups, cluster analysis showed a clear demarcation between accessions from Qinghai-Tibet Plateau and the others as separate groups. The clustering pattern was probably dependent on geographic origin and ecological adaptability of the accessions. The results of present study can be useful in collecting germplasm and the establishment of core collections ofE. sibiricus. The results in this study will provide useful information for the use ofE. sibiricus germplasm and variety breeding.

Keyword: Elymus sibiricus; germplasm resources; genetic diversity; SRAP

Show Figures

引言

研究和了解植物种质间的遗传变异,对于其在育种上的有效利用非常有益。根据遗传变异的信息,可以确定具有优异农艺性状的亲本组合^{[ 1]},拓宽植物品种的遗传基础,防止其育种进程中的渐进性遗传衰退。另外,遗传多样性还可以为物种保护计划的制定和实施提供参考和借鉴。

老芒麦( Elymus sibiricus),别名西伯利亚野麦草,是披碱草属的模式种,为多年生、疏丛型、自花授粉的异源四倍体禾草,具有StStHH的染色体组构成^{[ 2]}。它是欧亚大陆的广布种,在北美的阿拉斯加和加拿大北部也有少量分布^{[ 3]}。老芒麦一般生长于湿润的草地、河滩、灌木丛中或森林边缘地带。在中亚及中国新疆、内蒙古和青藏高原地区,老芒麦作为一种重要的牧草在草地畜牧业中发挥了巨大作用^{[ 4, 5, 6]}。

目前对老芒麦种质的遗传多样性研究很少。袁庆华等^{[ 7]}发现21份老芒麦种质在13个农艺和形态学性状上存在高度变异。但形态性状具有不少缺点,如数目有限、易受环境影响和非均匀分布等。DNA分子标记的发展,极大地增加了研究遗传多样性的方法,它可以揭示整个基因组上可能存在的大量变异^{[ 8]}。序列相关扩增多态性(sequence related amplified polymorphisms,SRAPs)是一种简单的分子标记系统,已经在植物分子作图和基因定位上被证明其有效性^{[ 9]}。该技术是基于正向和反向引物对模板DNA的PCR(polymerase chain reaction,聚合酶链式反应)扩增,引物具有3个明显不同的序列框:5'端存在的10~11个随机碱基序列,紧跟着CCGG(正向引物)或AATT(反向引物)的碱基序列,最后是位于3'端的3个选择性碱基。从技术的可操作性来看,每个引物5'端的10个随机碱基序列可以促进任意序列的扩增,即类似于RAPD(random amplified polymorphic DNA,随机扩增多态性DNA标记)标记的结果^{[ 10]}。正向引物的CCGG 序列可以优先与具有丰富GC碱基的外显子序列退火结合,而反向引物的AATT 序列可优先与具有丰富AT碱基的内含子和启动子序列退火结合^{[ 9]}。引物3'端的3个选择性碱基可以对模板DNA进行选择,与在AFLP(amplified fragment length polymorphism,扩增片段长度多态性)选择性扩增中使用的引物的3'端很相似^{[ 11]}。基于这种独特的引物设计,SRAP标记具有相对于其他标记更好的重复性、稳定性和简易性。SRAP标记已经应用于芸苔属( Brassica)^{[ 9]}、南瓜属( Cucurbita)^{[ 12]}、野牛草( Buchloe dactyloides)^{[ 13]}、紫花苜蓿( Medicago sativa)^{[ 14]}、黑麦草( Lolium)^{[ 15]}和芍药属( Paeonia)^{[ 16]}。Budak等^{[ 17]}发现在揭示遗传关系相近的野牛草品种间的遗传多样性时SRAP标记比SSR(simple sequence repeats,简单重复序列)、ISSR(inter-simple sequence repeat,简单序列重复区间扩增多态性)和 RAPD标记更加有效。

比较具有不同地理分布的植物种质,是进化生物学和植物育种学的重要研究内容。因此,老芒麦地理分布的遗传变异研究,对于收集和管理其种质资源非常重要。然而,目前还未见对不同国家或大的生态区来源的老芒麦种质的遗传多样性水平研究的报道。本研究的目的在于:1)评价SRAP标记在老芒麦种质研究中的有效性;2) 确定来自于3个国家的84份老芒麦种质间的遗传关系;3)检测遗传多样性水平与地理来源的关系。这是首次关于利用SRAP标记检测老芒麦遗传多样性的研究报告。

1 材料与方法

1.1 植物材料

84份老芒麦供试种质分别从美国国家遗传资源库(NPGS,USDA)、四川农业大学小麦研究所和四川草原科学研究院获得( 表1)。种质的护照信息显示它们分别采集自俄罗斯(西伯利亚地区为主)、蒙古国、中国新疆、中国的青藏高原地区(川西北高原为主)。每份材料取10~20粒种子,置于有盖培养皿内的吸水滤纸上萌发。萌发后的种子转移至内含砂子-泥炭混合基质的盆钵内生长,盆钵置于温室内。植株生长至开花期进行形态学鉴定,标本凭证保存于四川农业大学草业科学系。

表1 老芒麦种质材料地理来源 Table 1 List of geographical origin of E. sibiricus germplasm material

序号 No.	材料编号 Accession code	地理来源 Geographical origin	序号 No.	材料编号 Accession code	地理来源 Geographical origin
1	W6 21536	蒙古 Mongolia	43	Y0737	新疆和静Hejing, Xinjiang
2	W6 21365	蒙古Mongolia	44	Y0488	新疆和静Hejing, Xinjiang
3	PI 639813	蒙古Mongolia	45	Y0473	新疆和静Hejing, Xinjiang
4	PI 639771	蒙古Mongolia	46	PI 595174	新疆Xinjiang
5	PI 634231	蒙古Mongolia	47	Y0760	新疆库车Kuche, Xinjiang
6	PI 634230	蒙古Mongolia	48	Y0909	新疆木垒Mulei, Xinjiang
7	PI 628726	蒙古Mongolia	49	PI 595169	新疆Xinjiang
8	PI 610886	蒙古Mongolia	50	Y1690	新疆清河Qinghe, Xinjiang
9	PI 610876	蒙古Mongolia	51	Y0822	新疆特克斯Tekesi, Xinjiang
10	PI 610866	蒙古Mongolia	52	Y0549	新疆温宿Wensu, Xinjiang
11	PI 610862	蒙古Mongolia	53	PI 628677	新疆焉耆Yanqi, Xinjiang
12	PI 610857	蒙古Mongolia	54	Y0860	新疆伊吾Yiwu, Xinjiang
13	PI 610850	蒙古Mongolia	55	Y0867	新疆伊吾Yiwu, Xinjiang
14	PI 610860	蒙古Mongolia	56	Y0811	新疆昭苏Zhaosu, Xinjiang
15	PI 639791	蒙古Mongolia	57	Y005	新疆乌鲁木齐Wulumuqi, Xinjiang
16	PI 639807	蒙古Mongolia	58	Y0486	新疆乌鲁木齐Wulumuqi, Xinjiang
17	PI 611013	俄罗斯西伯利亚阿尔泰区Altay, Siberia, Russia	59	PI 595162	新疆 Xinjiang
18	PI 610994	俄罗斯西伯利亚Siberia, Russia	60	PI 499458	甘肃山丹 Shandan, Gansu
19	PI 598795	俄罗斯西伯利亚阿尔泰山Altay Mountain, Siberia, Russia	61 62	PI 636676 ZY3117	甘肃夏河 Xiahe, Gansu 甘肃夏河 Xiahe, Gansu
20	PI 598800	俄罗斯西伯利亚阿尔泰区Altay, Siberia, Russia	63	Y2906	甘肃合作 Hezuo, Gansu
21	PI 598789	俄罗斯西伯利亚赤塔Chita, Siberia, Russia	64	204451	甘肃合作 Hezuo, Gansu
22	PI 598787	俄罗斯阿穆尔州Amurskaya Oblast, Russia	65	204441	青海青海湖Qinghai Lake, Qinghai
23	PI 598781	俄罗斯布利亚特Buryatiya, Russia	66	504463	青海青海湖Qinghai Lake, Qinghai
24	PI 598780	俄罗斯远东地区滨海边疆区The seaside border and east in Russia	67 68	204404 204251	西藏日喀则Rikaze, Xizang 西藏丁青县Dingqing, Xizang
25	PI 598479	俄罗斯阿尔泰区Altay, Russia	69	W6 23559	四川红原 Hongyuan, Sichuan
26	PI 598777	俄罗斯哈巴罗夫斯克区Khabarovsk, Russia	70	PI 639859	四川红原 Hongyuan, Sichuan
27	PI 406467	俄罗斯西伯利亚Siberia, Russia	71	ZY3040	四川红原 Hongyuan, Sichuan
28	PI 370673	俄罗斯克拉斯诺亚尔斯克边疆区Krasnoyarskiy Kray, Russia	72	川草2号 Chuancao No.2	四川若尔盖 Ruoergai, Sichuan
29	PI 345600	俄罗斯克拉斯诺亚尔斯克边疆区Krasnoyarskiy Kray, Russia	73	204081	四川红原龙日坝Longriba, Hongyuan, Sichuan
30	PI 345599	俄罗斯赤塔州Chita, Russia	74	204089	四川红原刷金寺Shuajingsi, Hongyuan, Sichuan
31	PI 326266	俄罗斯阿穆尔州Amurskaya Oblast, Russia	75	205185	四川红原刷金寺Shuajingsi, Hongyuan, Sichuan
32	PI 598782	俄罗斯布利亚特Buryatiya, Russia	76	205171	四川若尔盖包座Baozuo, Ruoergai, Sichuan
33	PI 610991	俄罗斯布利亚特Buryatiya, Russia	77	205172	四川若尔盖求吉Qiuji, Ruoergai, Sichuan
34	PI 326267	俄罗斯赤塔州Chita, Russia	78	205173	四川若尔盖巴西Baxi, Ruoergai, Sichuan
35	PI 598478	俄罗斯新西伯利亚New Siberia, Russia	79	205179	四川若尔盖巴西Baxi, Ruoergai, Sichuan
36	PI 557455	俄罗斯Russia	80	205226	四川阿坝麦尔玛乡Maierma, Aba, Sichuan

表1 老芒麦种质材料地理来源 Table 1 List of geographical origin of E. sibiricus germplasm material

续表 1 Continued 1

1.2 DNA提取

每份种质采集5~10个单株的等量新鲜叶片,用液氮研磨成粉,保存于-80℃低温冰箱内。基因组DNA提取参照Doyle^{[ 18]}描述的CTAB(hexadecyl trimethyl ammonium bromide,十六烷基三甲基溴化铵)法进行。对比已知浓度的标准λDNA与样本DNA在0.8%(w/v)琼脂糖凝胶上的电泳图谱,利用Quantity One 软件(Bio-Rad,USA),计算出老芒麦各种质的DNA浓度。将所有样本的基因组DNA用0.1×TE缓冲液[1 mmol/L Tris-HCl,0.1 mmol/L EDTA(ethylene diamine tetraacetic acid,乙二胺四乙酸),pH 8.0]稀释至10 ng/μL,储存于-80℃冰箱内,供SRAP扩增使用。

1.3 SRAP 扩增

引物由上海生工生物工程技术公司合成( 表2)。PCR反应混合液(20 μL总体积)由0.2 mmol/L dNTPs, 0.3 μmol/L正向和反向引物,2.5 mmol/L Mg²⁺,10×Taq buffer,1 U Taq DNA 聚合酶(北京天根生物技术公司),40 ng 模板DNA组成。扩增在PTC-200 热循环仪内进行(MJ Research,Waltham MA, USA),采用以下PCR程序:94℃初变性5 min;94℃变性1 min,35℃退火1 min,72℃延伸2 min,共5个循环;在随后的30个循环中,退火温度增加至50℃;最后72℃延伸8 min。PCR扩增产物在2%琼脂糖凝胶(含有0.1 mg/mL的溴化乙锭)上用0.5×TBE(tris-borate-edta,三羟甲基氨基甲烷-硼酸-乙二胺四乙酸)缓冲液进行电泳分离,并用BIO-RAD自动凝胶成像系统照相保存。

表2 SRAP正向和反向引物序列 Table 2 Sequences of forward and reverse SRAP primers

1.4 数据分析

只记录清晰可辨的SRAP扩增条带,排除弱带和弥散带。对所有材料,有带(即具有相同分子量)记作1,无带记作0,形成原始二元数据矩阵。

用NTSYS-pc2.11x软件分析二元矩阵,计算各种质间的Nei和Li^{[ 19]}遗传相似系数,利用SAHN模块,基于UPGMA法进行聚类分析,构建聚类树形图。利用NTSYS-pc2.11x软件基于聚类树矩阵计算其协表征矩阵(cophenetic matrix),再利用Mantel检验,计算遗传相似性(GS)矩阵与协表征矩阵间的相关系数,从而反映聚类结果与GS矩阵的符合度^{[ 20]}。利用WINBOOT 软件^{[ 21]}基于bootstrap重抽样法,对聚类树形图节点分支的可靠性进行了检测^{[ 22]},设定重复抽样次数为1000。除聚类分析外,利用NTSYS-pc2.11x软件,基于GS矩阵还进行了主向量分析(PCoA,principal coordinates analysis),特征向量以二维形式显示。PCoA分析可以直观地观察各种质间的相关程度,这是因为二维图上各点之间的距离,反映了各点所代表的各种质之间的遗传相似性。

对每对引物扩增的条带而言,下列参数即:总扩增带数(total number of bands, TNB)、多态性带数(number of polymorphic bands,NPB)、多态性条带百分比 (percentage of polymorphic bands,PP)、多态性信息量(polymorphic information content,PIC)[也可称作杂合度(heterozygosity,H)]、条带信息指数(band informativeness,BI)、分辨力(resolving power,RP)和标记指数(marker index,MI)按下文所述进行计算。 PP=NPBs/TNB, PIC_i=2 f_i(1- f_i),式中, PIC_i 是第 i个标记的 PIC值, f_i是扩增带等位基因(即记为“1”的条带)的频率,而(1- f_i)是无效带等位基因(即记为“0”的条带)的频率^{[ 23]}。对每个引物组合而言,计算PIC应该求平均值[或称之为平均杂合度(average heterozygosity, H_av),即 PIC_av=H_av=∑ PIC_i/N,式中, N是各引物对产生的多态性带数(NPB); BI_i=1-(2×|0.5- f_i|),式中, BI_i是第 i个标记的 BI值, f_i是各种质具有的扩增带(即记为“1”的条带)的频率,而 RP=∑ BI_i。对每对SRAP引物而言,计算 BI时应该求平均值,即 BI_av=∑ BI_i/N,式中, N是各引物对产生的多态性带数(NPB)。对每个引物组合而言,标记指数(MI)可以按下述公式计算: MI=NPB×PI $\begin{matrix} {C_{av}}^{[24]} \end{matrix}$ ,式中, NPB是每对SRAP引物组合产生的多态性条带数。

为了解老芒麦种质不同地理类群间和类群内的SRAP变异,利用ARLEQUIN 3.1软件进行了分子变异方差分析(AMOVA)^{[ 25]},计算类群间的遗传分化系数 Φ_ST。同时利用AMOVA分析,还可以得到不同地理类群间的遗传距离(称之为 Φ统计量)^{[ 26]}。方差成分及类群间遗传距离的显著性检测采用9999次随机置换进行。

2 结果与分析

2.1 SRAP分析

从140个引物组合中,筛选出23个可以产生清晰可辨条带的引物组合。这23个引物组合对84份老芒麦种质共产生337条扩增带,分子量范围为75~1580 bp( 图1)。其中,203条(60.24%)为多态性带( 表3)。单个引物组合产生的多态性带为2~19条。平均每个引物组合产生14.7条带,其中多态性带为8.8条。单个引物的PIC值在0.151~0.438之间变化,平均值为0.284( 表3)。引物组合的平均条带信息指数值为0.408,变幅为0.230(Em9+Me15)~0.696(Em12+Me16);23个引物组合的标记分辨力(RP)的变幅为1.119(Em12+Me5)~9.333(Em1+Me10),平均值为3.631。具有较高的分辨力和标记指数的引物组合一般同时具有较高的多态性,能够辨别更多的种质。

表3 23对SRAP引物对84份老芒麦种质产生的总扩增带数(TNB)、多态性带数(NPB)、多态性条带百分比(PP)、多态性信息量(PIC)、条带信息指数(BI)、条带分辨力(RP)和标记指数(MI) Table 3 Total number of bands (TNB), number of polymorphic bands (NPB), percent polymorphism (PP), polymorphic information content (PIC), band informativeness (BI), resolving power (RP) and marker index (MI) of the 23 primer combinations used to generate SRAP markers in 84 E. sibiricus accessions

2.2 种质间的遗传相似性

84份种质间的遗传相似性(GS)系数变幅为0.783(204251与PI610886)~0.965(205119与205151),平均值为0.865。图2描述了3486个GS值的频数分布。前2个最高的GS值频数分别分布在0.886~0.891和0.880~0.885。约95%的GS值分布在0.810~0.921之间,只有0.8%的GS值高于0.93,同时也只有0.1%的GS值低于0.80。这说明供试材料间的遗传关系较近。

	Figure Option View Download New Window
	图2 基于203条多态带计算出的84份老芒麦种质间的遗传相似系数的分布Fig.2 The distribution of genetic similarities among 84 E. sibiricus accessions based on SRAP data

2.3 聚类系统树和主向量分析(PCA)对种质的划分

基于SRAP标记的UPGMA系统树( 图3), 揭示了84份老芒麦种质间的遗传关系。系统树可以在不同的GS水平上将供试种质划归为不同的组群。在GS=0.84的水平上,供试种质可以被划分成2个明显不同的组群。第Ⅰ组群包括来自于俄罗斯(20份)、蒙古(16份)和中国新疆(23份)的种质。第Ⅱ组群包括全部来自于青藏高原的25份种质。第Ⅱ组群又可划分为3个亚组:亚组1包括甘肃种质3份、青海种质2份,西藏种质1份;亚组2包括13份四川种质和2份甘肃种质;亚组3包括3份四川种质和1份西藏种质。协表征矩阵与GS矩阵间的相关系数达0.88,说明聚类结果与原始GS矩阵能较好地吻合。基于Bootstrap分析所得的对系统树分支节点置信度的支持值(bootstrap value)被标注在图3中聚类图上。共有19个分支节点具有高于50%的置信度。第Ⅰ和第Ⅱ组分支节点被91%和100%的bootstrap值支持,表明了青藏高原来源的种质类群(Ⅱ)与其他地理来源的种质类群(Ⅰ)具有明显差异。

	Figure Option View Download New Window
	图3 基于SRAP数据利用UPGMA法获得的老芒麦种质的聚类树形图Fig.3 Dendrogram of E. sibiricus accessions based on SRAP data using UPGMA method

基于84份种质GS矩阵的主向量分析(PcoA)的结果表明,第1和第2主向量分别可以解释16.7%和5.8%的遗传变异。依据图4上各种质间的空间关系,可以直观地看出84份材料被明显划分成两大组,其中青藏高原组又可划分成3个亚组。这与UPGMA聚类结果保持一致。

	Figure Option View Download New Window
	图4 基于SRAP数据描述84份老芒麦种质间遗传关系的主向量分析Fig.4 Two-dimensional principal coordinate analysis depicting genetic relationships among 84 E. sibiricus accessions based on SRAP data

2.4 老芒麦地理类群的遗传结构分析

根据采集地及其生态条件的差异,可以将供试种质划分为四大类群,即青藏高原、新疆、蒙古和俄罗斯类群。基于扩增带(记为1的带)和无效带(记为0的带)的频率,对群体遗传结构进行了分析。AMOVA分析结果显示:在总的遗传变异中有79.62%发生在类群内,有20.38%发生在类群间( Φ_ST=0.204),群体间和群体内的变异均为极显著( P<0.0001)( 表4)。

表4 4个老芒麦地理类群的分子方差分析(AMOVA) Table 4 Analysis of molecular variance (AMOVA) of four germplasm groups of E. sibiricus

2.5 老芒麦地理类群的聚类分析

基于AMOVA分析,可以得到类群间两两比较的 Φ_ST 值,它可以代表标准化的类群间遗传距离(pairwise Φ_ST distance)( 表5)。青藏高原类群与其他类群间的遗传距离均比较大,而剩余3个类群间的遗传距离比较小。类群间成对 Φ_ST的变幅为0.0371(蒙古类群与俄罗斯类群间)~0.3547(蒙古类群与青藏高原类群间)。为更好地了解各类群间的遗传关系,利用表5中的 Φ_ST距离,进行UPGMA聚类分析( 图5)。4个地理群被明显划分成两支,一支为青藏高原类群,另一支包括蒙古、俄罗斯和新疆类群。青藏高原类群明显区别于其他3个类群,这与对84份种质的UPGMA聚类结果一致( 图3)。

表5 4个地理类群间的成对 Φ_ST遗传距离 Table 5 Pairwise Φ_ST distances among four ecogeographic groups

	Figure Option View Download New Window
	图5 基于AMOVA获得的遗传距离对4个老芒麦地理类群的UPGMA聚类Fig.5 UPGMA clustering of four geographic groups of E. sibiricus using genetic distances generated from AMOVA

3 讨论

3.1 SRAP标记的多态性和变异

形态-农艺性状和生化标记(同工酶和种子贮藏蛋白)是评价植物种内遗传多样性的常用手段^{[ 7, 27, 28, 29, 30]}。由于不受环境影响、不受限于植物的发育阶段,分子标记目前已成为遗传多样性评价的强有力的主流研究手段。虽然目前有关利用分子标记研究老芒麦种质遗传变异的报道很少,但RAPD、SSR、AFLP、PCR-RFLP等DNA分子标记已广泛用作披碱草属植物的群体遗传结构和种间系统进化研究^{[ 31, 32, 33, 34]}。SRAP标记综合RAPD标记的简便性和AFLP标记的高多态性和稳定性于一身,其上下游通用引物可以搭配使用,极大地提高了引物的利用效率,是一种进行遗传多样性评价、品种鉴定和系统进化研究的有效工具^{[ 9, 13]}。本研究得到的老芒麦种质的SRAP多态性比率(PP)为60.24%,低于已报道的禾本科植物野牛草(95%)^{[ 13]}和鸭茅( Dactylis glomerata)(84.4%)^{[ 35]},而且也低于对青藏高原地区老芒麦SRAP研究的结果(86.48%)^{[ 36]}。本研究利用23个引物组合共扩增出337条带,其中多态性带203条,平均每个引物组合扩增多态性带8.8条,说明SRAP的扩增产率较高,非常适合对大规模种质的遗传变异检测。由于SRAP可以采用更加灵敏的聚丙烯酰胺凝胶银染或荧光标记来检测扩增带,而本实验采用的是较为简便的琼脂糖检测法,这可能是本研究所得的多态性比率较低的部分原因。

84份老芒麦种质间的平均遗传相似系数(GS)为0.865,表明供试材料间具有较近的遗传关系,这很可能与老芒麦的繁育方式相关。一般来说,相对于异花授粉和常异花授粉植物而言,自花授粉植物具有较高的群体间遗传变异和较低的群体内遗传变异^{[ 37]}。由于老芒麦存在较高的异交率,并非严格的自花授粉植物,属于常异花授粉植物,所以每份种质都可以看作是一个内部存在较高变异的小群体,而本实验采用的是混合单株提取DNA法(bulked DNA),可能会掩盖种质内的遗传变异,降低决定种质间遗传关系的多态性带的数目;加上种质间在繁殖后代过程中存在一定的基因流(花粉流)^{[ 38]},这两种因素可能会在一定程度上降低种质间的遗传差异,导致种质间较高平均GS值的出现。根据地理来源的不同,将供试种质划分为四大地理类群。地理隔离、生态隔离和繁殖隔离会极大地影响植物的种内遗传多样性^{[ 39]}。所以青藏高原类群与其他3个类群种质间平均GS值均较小,其原因可能是青藏高原与其他三地区的生态地理环境存在的巨大差异所致。

本研究运用AMOVA分析的结果表明:老芒麦地理类群内的遗传变异占总变异的79.62%,类群间的遗传变异占总变异的20.38%,基于Shannon指数的分析结果与AMOVA分析结果非常接近。另外在4个地理类群中,青藏高原类群具有最高的Shannon多样性(H_o=0.2398)。这表明进行老芒麦遗传资源的挖掘利用时,应更多地重视本地优异资源特别是青藏高原地区资源的收集、保护及隔离繁种。

3.2 供试种质间及其地理类群间的遗传关系

根据表现所有供试种质间遗传关系的聚类树形图和主向量空间分布图来看,各种质的聚类分布与地理来源有较强的关系^{[ 40]}。种质的地理来源代表了不同的宏观生态环境,而这些不同的生态环境条件是造成种质间的遗传变异的主要因素^{[ 41]}。本研究供试种质被分成两大类的原因,可能主要依赖于青藏高原种质的特有生态地理适应性。由于青藏高原的高海拔及它被喀喇昆仑山、阿尔金山和祁连山等著名高山与其他种质的来源地区隔离开来^{[ 42]},造成其植物种质可能具有特定的生态地理适应性。需要注意的是,来自于新疆、蒙古和俄罗斯的种质并没有按地理来源被清晰的分开,可能与这3个国家或地区接壤且并无明显生态条件差别有关。另外,种质繁殖后代过程中的遗传交换可能会加剧种质间的遗传相似性。同样,基于AMOVA分析产生的类群间 Φ_ST值的聚类分析也反映了相似的实验结果。虽然AMOVA分析表明地理类群间的变异仅占总变异的20.38%,但达到了极显著水平,这从另一个侧面反映了青藏高原种质类群与其他类群间存在较大的遗传差异。本结果与Chen等^{[ 43]}、陈智华等^{[ 44]}和苗佳敏等^{[ 45]}对垂穗披碱草( Elymus nutans)遗传多样性的研究结果一致,即青藏高原与其他地区的种质材料存在明显的遗传分化。特别提及的是,由于AMOVA可以计算无法估计是否处于Hardy-Weinberg遗传平衡状态或人为划分的植物类群或群体间的 Φ_ST值作为其遗传距离,因而在评价类似植物群体间和群体内的遗传关系时非常有效^{[ 46]}。如果采用POPGENE等软件强行基于H-W遗传平衡来计算人为划分群体(如地理类群)的遗传分化,可能存在较大误差。

本研究的结果表明SRAP是评价老芒麦种质资源遗传变异的有效手段。今后应增加利用SSR等标记手段以检测非编码区的遗传变异,综合评价现有老芒麦种质在全基因组水平上的遗传多样性,为种质保护、利用和品种选育提供有益的信息。

The authors have declared that no competing interests exist.

参考文献

View Option

[1]	Moreno-González J, Cubero J I. Selection strategies and choice of breeding methods[A]. In: Hayward M D, Bosemark N O, Romagosa I. Plant Breeding, Principles and Prospects[M]. London: Chapman & Hall, 1993: 281-313. [本文引用:1]
[2]	Dewey D R. Cytogenetics of Elymus sibiricus and its hybrids with Agropyron tauri, Elymus canadensis, and Agropyron caninu[J]. Botanical Gazette, 1974, 135: 80-87. [本文引用:1]
[3]	Bowden W M, Cody W J. Recognition of Elymus sibiricus L. from Alaska and the district of Mackenzie[J]. Bulletin of the Torrey Botanical Club, 1961, 88: 153-155. [本文引用:1] [JCR: 0.625]
[4]	道来提, 麦耒. 新疆老芒麦的驯化[J]. 中国草地, 1988, (4): 48-50. [本文引用:1]
[5]	王元富, 盘朝邦, 杨智永. 川草1号老芒麦选育报告[J]. 四川草原, 1994, (4): 7-13. [本文引用:1]
[6]	张众, 王比德, 吴渠来, 等. 农牧老芒麦的选育推广及其栽培利用[J]. 中国草地, 1995, (4): 29-32. [本文引用:1]
[7]	袁庆华, 张吉宇, 张文淑, 等. 披碱草和老芒麦野生居群生物多样性研究[J]. 草业学报, 2003, 12(5): 44-49. [本文引用:2]
[8]	Melchinger A E, Graner A, Singh M, et al. Relationships among winter and spring cultivars revealed by RFLP’s[J]. Crop Science, 1994, 34: 1191-1199. [本文引用:1] [JCR: 1.513]
[9]	Li G, Quiros C F. Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica[J]. Theoretical and Applied Genetics, 2001, 103: 455-461. [本文引用:4] [JCR: 3.658]
[10]	Williams J G K, Kubelik A R, Livak K J, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers[J]. Nucleic Acids Research, 1990, 18: 6531-6535. [本文引用:1] [JCR: 8.278]
[11]	Vos P, Hogers R, Bleeker M, et al. AFLP: A new technique for DNA finger printing[J]. Nucleic Acids Research, 1995, 23: 4407-4414. [本文引用:1] [JCR: 8.278]
[12]	Ferriol M, Pico B, Nuze F. Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP markers[J]. Theoretical and Applied Genetics, 2003, 107: 271-282. [本文引用:1] [JCR: 3.658]
[13]	Budak H, Shearman R C, Parmaksiz I, et al. Molecular characterization of buffalograss germplasm using sequence-related amplified polymorphism markers[J]. Theoretical and Applied Genetics, 2004, 108: 328-334. [本文引用:3] [JCR: 3.658]
[14]	Vand emark G J, Ariss J J, Bauchan G A, et al. Estimating genetic relationships among historical sources of alfalfa germplasm and selected cultivars with sequence related amplified polymorphisms[J]. Euphytica, 2006, 152: 9-16. [本文引用:1] [JCR: 1.643]
[15]	李杰勤, 王丽华, 詹秋文, 等. 20个黑麦草品系的SRAP遗传多样性分析[J]. 草业学报, 2013, 22(2): 158-164. [本文引用:1]
[16]	Han X Y, Wang L S, Shu Q Y, et al. Molecular characterization of tree peony germplasm using sequence-related amplified polymorphism markers[J]. Biochemical Genetics, 2008, 46: 162-179. [本文引用:1] [JCR: 0.938]
[17]	Budak H, Shearman R C, Parmaksiz I, et al. Comparative analysis of seeded and vegetative biotype buffalograsses based on phylogenetic relationship using ISSRs, SSRs, RAPDs, and SRAPs[J]. Theoretical and Applied Genetics, 2004, 109: 280-288. [本文引用:1] [JCR: 3.658]
[18]	Doyle J J. DNA protocols for plants-CTAB total DNA isolation[A]. In: Hewitt G M, Johnston A. Molecular Techniques in Taxonomy[M]. Berlin, Germany: Springer-Verlag, 1991: 283-293. [本文引用:1]
[19]	Nei M, Li W H. Mathematical model for studying genetic variation in terms of restriction endonucleases[J]. Proceedings of the National Academy of Sciences, 1979, 76: 5269-5273. [本文引用:1]
[20]	Mantel N. The detection of disease clustering and a generalized regression approach[J]. Cancer Research, 1967, 27: 209-220. [本文引用:1] [JCR: 8.65]
[21]	Yap I, Nelson R J. WinBoot: a program for performing bootstrap analysis of binary data to determine the confidence limits of UPGMA-based dendrograms[C]. Manila, Philippines: International Rice Research Institute (IRRI), 1995. [本文引用:1]
[22]	Felsenstein J. Confidence limits on phylogenesis: an approach using the bootstrap[J]. Evolution, 1985, 39: 783-791. [本文引用:1]
[23]	Rolf J F. NTSYS-pc: Numerical Taxonomy and Multivariate Analysis System, Version 2. 1[M]. New York, USA: Exeter Software, Setaukel, 2000. [本文引用:1]
[24]	Powell W, Morgante M, Andre C, et al. The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) marker for germplasm analysis[J]. Molecular Breeding, 1996, 2: 225-238. [本文引用:1] [JCR: 3.251]
[25]	Schneider S, Roessli D, Excoffier L. ARLEQUIN version 3. 1: A Software for Population Genetics Data Analysis[M]. Switzerland : Genetics and Biometry Laboratory, University of Geneva, 2006. [本文引用:1]
[26]	Excoffier L, Smouse P E, Quattro J M. Analysis of molecular variance inferred from metric distances among DNA haplotypes: applications to human mitochondrial DNA restriction data[J]. Genetics, 1992, 131: 479-491. [本文引用:1] [JCR: 4.389]
[27]	Mizianty M. Variability and structure of natural populations of Elymus caninus (L. ) L. based on morphology[J]. Plant Systematics and Evolution, 2005, 251: 199-216. [本文引用:1] [JCR: 1.312]
[28]	Díaz O, Sun G L, Salomon B, et al. Levels and distribution of allozyme and RAPD variation in populations of Elymus fibrosis (Schrenk) Tzvel. (Poaceae)[J]. Genetic Resources and Crop Evolution, 2000, 47: 11-24. [本文引用:1] [JCR: 1.593]
[29]	马啸, 周永红, 于海清, 等. 野生垂穗披碱草种质的醇溶蛋白遗传多样性分析[J]. 遗传, 2006, 28(6): 699-706. [本文引用:1]
[30]	Ruiz M, Aguiriano E. Analysis of duplication in the Spanish durum wheat collection maintained in the CRF-INIA on the basis of agro-morphological traits and gliadin proteins[J]. Genetic Resources and Crop Evolution, 2004, 51: 231-235. [本文引用:1] [JCR: 1.593]
[31]	Zhang X Q, Salomom B, von Bothmer R. Application of rand om amplified polymorphic DNA markers to evaluate intraspecific genetic variation in the Elymus alaskanus complex (Poaceae)[J]. Genetic Resources and Crop Evolution, 2002, 49: 397-407. [本文引用:1] [JCR: 1.593]
[32]	Sun G L, Díaz O, Salomon B, et al. Genetic diversity in Elymus caninus as revealed by isozyme, RAPD and microsatellite markers[J]. Genome, 1999, 42: 420-431. [本文引用:1] [JCR: 1.668]
[33]	Agafonov A V, Baum B R, Bailey L G, et al. Differentiation in the Elymus dahuricus complex (Poaceae): evidence from grain proteins, DNA, and crossability[J]. Hereditas, 2002, 135: 277-289. [本文引用:1] [CJCR: 0.928]
[34]	Xu D H, Ban T. Phylogenetic and evolutionary relationships between Elymus humidus and other Elymus species based on sequencing of non-coding regions of cpDNA and AFLP of nuclear DNA[J]. Theoretical and Applied Genetics, 2004, 108: 1443-1448. [本文引用:1] [JCR: 3.658]
[35]	Zeng B, Zhang X Q, Lan Y, et al. Evaluation of genetic diversity and relationships in orchardgrass (Dactylis glomerata L. ) germplasm based on SRAP markers[J]. Canadian Journal of Plant Science, 2008, 88: 53-60. [本文引用:1] [JCR: 0.716]
[36]	鄢家俊, 白史且, 张新全, 等. 青藏高原老芒麦种质基于SRAP标记的遗传多样性研究[J]. 草业学报, 2010, 19(1): 173-183. [本文引用:1]
[37]	Hamrick J L, Godt M J W. Conservation genetics of endemic plant species[A]. In: Avise J C, Hamrick J L. Conservation Genetics, Case Histories from Nature[M]. New York: Chapman and Hall, 1996: 281-304. [本文引用:1]
[38]	孙涛, 刘左军, 刘凤梅, 等. 钝裂银莲花不同居群遗传多样性的ISSR分析[J]. 草业学报, 2013, 22(3): 259-265. [本文引用:1]
[39]	Max K H, William C S, Bruce W. The origin of isolating mechanism in flowing plants[A]. In: Max K H. Evolutionary Biology[M]. New York: Plenum Press, 1978: 185-317. [本文引用:1]
[40]	曾亮, 袁庆华, 王方, 等. 冰草属植物种质资源遗传多样性的ISSR分析[J]. 草业学报, 2013, 22(1): 260-267. [本文引用:1]
[41]	Johnson R C, Johnston W J, Golob C T, et al. Characterization of the USDA Poa pratensis collection using RAPD markers and agronomic descriptors[J]. Genetic Resources and Crop Evolution, 2002, 49: 349-361. [本文引用:1] [JCR: 1.593]
[42]	张镱锂, 李炳元, 郑度. 论青藏高原范围与面积[J]. 地理研究, 2002, 21(1): 1-8. [本文引用:1]
[43]	Chen S Y, Ma X, Zhang X Q, et al. Genetic variation and geographical divergence in Elymus nutans Griseb. (Poaceae: Triticeae) from West China[J]. Biochemical Systematics and Ecology, 2009, 37: 716-722. [本文引用:1] [JCR: 1.153]
[44]	陈智华, 苗佳敏, 钟金城, 等. 野生垂穗披碱草种质遗传多样性的SRAP研究[J]. 草业学报, 2009, 18(5): 192-200. [本文引用:1]
[45]	苗佳敏, 张新全, 陈智华, 等. 青藏高原和新疆地区垂穗披碱草种质的SRAP及RAPD分析[J]. 草地学报, 2011, 19(2): 127-134. [本文引用:1]
[46]	Fu Y B, Peterson G W, Williams D, et al. Patterns of AFLP variation in a core subset of cultivated hexaploid oat germplasm[J]. Theoretical and Applied Genetics, 2005, 530: 530-539. [本文引用:1] [JCR: 3.658]

1993

0.0

... 根据遗传变异的信息,可以确定具有优异农艺性状的亲本组合^[1],拓宽植物品种的遗传基础,防止其育种进程中的渐进性遗传衰退 ...

1974

0.0

... 老芒麦(Elymus sibiricus),别名西伯利亚野麦草,是披碱草属的模式种,为多年生、疏丛型、自花授粉的异源四倍体禾草,具有StStHH的染色体组构成^[2] ...

1961

0.625

0.0

... 它是欧亚大陆的广布种,在北美的阿拉斯加和加拿大北部也有少量分布^[3] ...

1988

0.0

1994

0.0

1995

0.0

2003

0.0

... 袁庆华等^[7]发现21份老芒麦种质在13个农艺和形态学性状上存在高度变异 ...

... 1 SRAP标记的多态性和变异形态-农艺性状和生化标记(同工酶和种子贮藏蛋白)是评价植物种内遗传多样性的常用手段^{[7,27,28,29,30]} ...

1994

1.513

0.0

... DNA分子标记的发展,极大地增加了研究遗传多样性的方法,它可以揭示整个基因组上可能存在的大量变异^[8] ...

2001

3.658

0.0

. 2001, 103:455-461 DOI:10.1007/s001220100570

1.Department of Vegetable Crops, University of California, Davis, CA 95616, USA e-mail: cfquiros@ucdavis.edu Tel.: 530-752-1734, Fax: 530-752-9659 US US

We developed a simple marker technique called sequence-related amplified polymorphism (SRAP) aimed for the amplification of open reading frames (ORFs). It is based on two-primer amplification. The primers are 17 or 18 nucleotides long and consist of the following elements. Core sequences, which are 13 to 14 bases long, where the first 10 or 11 bases starting at the 5′ end, are sequences of no specific constitution (”filler” sequences), followed by the sequence CCGG in the forward primer and AATT in the reverse primer. The core is followed by three selective nucleotides at the 3′ end. The filler sequences of the forward and reverse primers must be different from each other and can be 10 or 11 bases long. For the first five cycles the annealing temperature is set at 35°C. The following 35 cycles are run at 50°C. The amplified DNA fragments are separated by denaturing acrylamide gels and detected by autoradiography. We tested the marker technique in a series of recombinant inbred and doubled-haploid lines of Brassica oleracea L. After sequencing, approximately 45% of the gel-isolated bands matched known genes in the Genbank database. Twenty percent of the SRAP markers were co-dominant, which was demonstrated by sequencing. Construction of a linkage map revealed an even distribution of the SRAP markers in nine major linkage groups, not differing in this regard to AFLP markers. We successfully tagged the glucosinolate desaturation gene BoGLS-ALK with these markers. SRAPs were also easily amplified in other crops such as potato, rice, lettuce, Chinese cabbage (Brassica rapa L.), rapeseed (Brassica napus L.), garlic, apple, citrus, and celery. We also amplified cDNA isolated from different tissues of Chinese cabbage, allowing the fingerprinting of these sequences.

... 序列相关扩增多态性(sequence related amplified polymorphisms,SRAPs)是一种简单的分子标记系统,已经在植物分子作图和基因定位上被证明其有效性^[9] ...

... 正向引物的CCGG 序列可以优先与具有丰富GC碱基的外显子序列退火结合,而反向引物的AATT 序列可优先与具有丰富AT碱基的内含子和启动子序列退火结合^[9] ...

... SRAP标记已经应用于芸苔属(Brassica)^[9]、南瓜属(Cucurbita)^[12]、野牛草(Buchloe dactyloides)^[13]、紫花苜蓿(Medicago sativa)^[14]、黑麦草(Lolium)^[15]和芍药属(Paeonia)^[16] ...

... SRAP标记综合RAPD标记的简便性和AFLP标记的高多态性和稳定性于一身,其上下游通用引物可以搭配使用,极大地提高了引物的利用效率,是一种进行遗传多样性评价、品种鉴定和系统进化研究的有效工具^[9,13] ...

1990

8.278

0.0

... 从技术的可操作性来看,每个引物5'端的10个随机碱基序列可以促进任意序列的扩增,即类似于RAPD(random amplified polymorphic DNA,随机扩增多态性DNA标记)标记的结果^[10] ...

1995

8.278

0.0

. 1995, 23:4407-4414

AFLP: A new technique for DNA finger printing

<Affiliation>Keygene N.V., Wageningen, The Netherlands.

A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.

PMID: 7501463

... 引物3'端的3个选择性碱基可以对模板DNA进行选择,与在AFLP(amplified fragment length polymorphism,扩增片段长度多态性)选择性扩增中使用的引物的3'端很相似^[11] ...

2003

3.658

0.0

. 2003, 107:271-282 DOI:10.1007/s00122-003-1242-z

Genetic diversity of a germplasm collection of Cucurbita pepo using SRAP and AFLP markers

1.Center for Conservation and Breeding of the Agricultural Diversity (COMAV), Polytechnic University of Valencia, Camino de Vera 14, Valencia 46022, Spain

2004

3.658

0.0

. 2004, 108:328-334 DOI:10.1007/s00122-003-1428-4

Molecular characterization of buffalograss germplasm using sequence-related amplified polymorphism markers

1.Department of Agronomy and Horticulture University of Nebraska Lincoln NE 68583 USA

Buffalograss [Buchloe dactyloides (Nutt.) Englem] germplasm has a broad resource of genetic diversity that can be used for turfgrass, forage and conservation. Buffalograss is the only native grass that is presently used as a turfgrass in the Great Plains region of North America. Its low growth habit, drought tolerance and reduced requirement for fertilizer and pesticides contribute to interest in its use. The objectives of this study were to use sequence-related amplified polymorphism (SRAP) markers in the evaluation of genetic diversity and phenetic relationships in a diverse collection of 53 buffalograss germplasms, and to identify buffalograss ploidy levels using flow cytometry. Based on their DNA contents, buffalograss genotypes were grouped into four sets, corresponding to their ploidy levels. Thirty-four SRAP primer combinations were used. This is the first report of the detection of differentiating diploid, tetraploid, pentaploid and hexaploid buffalograss genotypes, representing diverse locations of origin, using SRAP markers. Cluster analysis by the unweighted pair-group method with arithmetic averages based on genetic similarity matrices indicated that there were eight clusters. The coefficients of genetic distance among the genotypes ranged from 0.33 up to 0.99 and averaged D=0.66. The genetic diversity estimate, He, averaged 0.35. These results demonstrated that genotypes with potential traits for turfgrass improvement could readily be distinguished, based on SRAP. The use of PCR-based technologies such as SRAP is an effective tool for estimating genetic diversity, identifying unique genotypes as new sources of alleles for enhancing turf characteristics, and for analyzing the evolutionary and historical development of cultivars at the genomic level in a buffalograss breeding program.

... 24%,低于已报道的禾本科植物野牛草(95%)^[13]和鸭茅(Dactylis glomerata)(84 ...

2006

1.643

0.0

. 2006, 152:9-16 DOI:10.1007/s10681-006-9167-7

Estimating genetic relationships among historical sources of alfalfa germplasm and selected cultivars with sequence related amplified polymorphisms

1.USDA-ARS, Vegetable and Forage Crops Research Unit, Prosser, WA 99350, USA 2.Department of Plant Pathology, University of Wisconsin, Madison, USA 3.USDA-ARS, Soybean Genomics and Improvement Lab., Beltsville, MD 20704, USA

Fifteen alfalfa populations consisting of six public cultivars and nine historically recognized sources of alfalfa germplasm in North American cultivars were examined using sequence related amplified polymorphisms (SRAPs). Three bulk DNA samples from each population were evaluated with fourteen different SRAP primer pairs. This resulted in 249 different amplicons, of which over 90% were polymorphic. A dendrogram from the analysis suggests that the public cultivars are quite diverse from all the historical sources of germplasm. The highest mean genetic similarity among the nine original sources of Medicago germplasm was 0.85 between PI 536535 (Peruvian) and 536536 (Indian), while the lowest (0.47) was between PI 560333 (M. falcata) and 536539 (African). The highest mean genetic similarity among the nine original sources of Medicago germplasm and the public alfalfa cultivars was 0.78 between PI 536532 (Ladak) and Vernal, while the lowest (0.59) was between PI 536539 (African) and Oneida. Relationships based on SRAP analysis appear to generally concur with expected relationships based on fall dormancy. This report demonstrates that SRAPs are a promising marker system for detecting polymorphisms in alfalfa.

2013

0.0

. 2013, 22(2):158-164

<Affiliation>Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire, USA. (Current address for Dr. de Bruijn) MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, United Kingdom.

The transcription factor Runx1 marks all functional hematopoietic stem cells (HSCs) in the embryo and is required for their generation. Mutations in Runx1 are found in approximately 25% of acute leukemias and in familial platelet disorder, suggesting a role for Runx1 in adult hematopoiesis as well. A comprehensive analysis of Runx1 expression in adult hematopoiesis is lacking. Here we show that Runx1 is expressed in functional HSCs in the adult mouse, as well as in cells with spleen colony-forming unit (CFU) and culture CFU capacities. Additionally, we document Runx1 expression in all hematopoietic lineages at the single cell level. Runx1 is expressed in the majority of myeloid cells and in a smaller proportion of lymphoid cells. Runx1 expression substantially decreases during erythroid differentiation. We also document effects of reduced Runx1 levels on adult hematopoiesis.

PMID: 14990855

2008

0.938

0.0

. 2008, 46:162-179 DOI:10.1007/s10528-007-9140-8

Molecular characterization of tree peony germplasm using sequence-related amplified polymorphism markers

1.Chinese Academy of Sciences Beijing Botanical Garden, Institute of Botany 20 Nanxin Cun, Xiangshan Haidian District, Beijing 100093 P.R. China 2.Graduate School of the Chinese Academy of Sciences Beijing 100049 P.R. China 3.Institute of Subtropical Botany Key Laboratory of Molecular Physiology and Biochemistry Xiamen Fujian 361006 P.R. China 4.University of Miyazaki Faculty of Agriculture Miyazaki 889-2192 Japan

This study examined 63 tree peony specimens, consisting of 3 wild species and 63 cultivars, using sequence-related amplified polymorphism (SRAP) markers for the purpose of detecting genomic polymorphisms. Bulk DNA samples from each specimen were evaluated with 23 SRAP primer pairs. Among the 296 different amplicons, 262 were polymorphic. The maximum parsimony, neighbor-joining, and unweighted pair-group method using arithmetic average trees were largely in congruence. In the three trees, the wild species Paeonia ludlowii and P. delavayi formed separate clusters with strong bootstrap support, and P. ostii was closely related to all cultivars. The cultivars were divided into groups with various corresponding bootstrap values. The genetic similarity among the genotypes ranged from 0.02 to 0.73. These results demonstrate that SRAP markers are effective in detecting genomic polymorphisms in the tree peony and should be useful for linkage map construction and molecular marker assisted selection breeding.

2004

3.658

0.0

. 2004, 109:280-288 DOI:10.1007/s00122-004-1630-z

Comparative analysis of seeded and vegetative biotype buffalograsses based on phylogenetic relationship using ISSRs, SSRs, RAPDs, and SRAPs

1.Department of Agronomy and Horticulture University of Nebraska Lincoln NE 68583 USA

Buffalograss [Buchloe dactyloides (Nutt.) Englem.] is the only native grass that is being used extensively as a turfgrass in the Great Plains region. Its low-growth habit, drought resistance, and low-maintenance requirement make it attractive as a turfgrass species. Our objective was to obtain an overview on the genetic relatedness among and within seeded and vegetative biotype buffalograsses using inter-simple sequence repeats (ISSRs), random amplified polymorphic DNA (RAPDs), sequence-related amplified polymorphisms (SRAPs), and simple sequence repeats (SSRs) markers that were derived from related species (maize, pearl millet, sorghum, and sugarcane). Twenty individuals per cultivar were genotyped using 30 markers from each marker system. All buffalograss cultivars were uniquely fingerprinted by all four marker systems. Mean genetic similarities were estimated at 0.52, 0.51, 0.62, and 0.57 using SSRs, ISSRs, SRAPs, and RAPDs, respectively. Two main clusters separating the seeded-biotype from the vegetative-biotype cultivars were produced using UPGMA analysis. Further subgroupings were unequivocal. The Mantel test resulted in a very good fit (SRAP=0.92, ISSR=0.90) to good fit (RAPD=0.86, SSR=0.88) of cophenetic values. Comparing the four marker systems to each other, RAPD and SRAP similarity indices were highly correlated (r=0.73), while Spearman rsquo

s rank correlation coefficient between RAPDs and SSRs was r=0.24 and between ISSRs and SSRs was r=0.66. A genotype-assignment analytical approach might be useful for cultivar identification and property rights protection. Polymorphic SRAPs were abundant and demonstrated genetic diversity among closely related cultivars.

... Budak等^[17]发现在揭示遗传关系相近的野牛草品种间的遗传多样性时SRAP标记比SSR(simple sequence repeats,简单重复序列)、ISSR(inter-simple sequence repeat,简单序列重复区间扩增多态性)和 RAPD标记更加有效 ...

1991

0.0

... 基因组DNA提取参照Doyle^[18]描述的CTAB(hexadecyl trimethyl ammonium bromide,十六烷基三甲基溴化铵)法进行 ...

1979

0.0

. 1979, 76:5269-5273

Mathematical model for studying genetic variation in terms of restriction endonucleases

A mathematical model for the evolutionary change of restriction sites in mitochondrial DNA is developed. Formulas based on this model are presented for estimating the number of nucleotide substitutions between two populations or species. To express the degree of polymorphism in a population at the nucleotide level, a measure called "nucleotide diversity" is proposed.

PMID: 291943

... 11x软件分析二元矩阵,计算各种质间的Nei和Li^[19]遗传相似系数,利用SAHN模块,基于UPGMA法进行聚类分析,构建聚类树形图 ...

1967

8.65

0.0

... 11x软件基于聚类树矩阵计算其协表征矩阵(cophenetic matrix),再利用Mantel检验,计算遗传相似性(GS)矩阵与协表征矩阵间的相关系数,从而反映聚类结果与GS矩阵的符合度^[20] ...

0.0

... 利用WINBOOT 软件^[21]基于bootstrap重抽样法,对聚类树形图节点分支的可靠性进行了检测^[22],设定重复抽样次数为1000 ...

1985

0.0

... 利用WINBOOT 软件^[21]基于bootstrap重抽样法,对聚类树形图节点分支的可靠性进行了检测^[22],设定重复抽样次数为1000 ...

2000

0.0

... 的条带)的频率^[23] ...

1996

3.251

0.0

2006

0.0

... 1软件进行了分子变异方差分析(AMOVA)^[25],计算类群间的遗传分化系数#cod#x003a6 ...

1992

4.389

0.0

... 统计量)^[26] ...

2005

1.312

0.0

. 2005, 251:199-216 DOI:10.1007/s00606-004-0262-8

Variability and structure of natural populations of Elymus caninus (L.) L. based on morphology

1.W. Szafer Institute of Botany Polish Academy of Sciences Lubricz 46 31–512 Kraków Poland

Great variability involving 36 quantitative and 31 qualitative characters, as well as the habitats occupied has been shown in 18 populations of Elymus caninus from Poland, based on the detailed population and statistical analyses of morphological characters. The variability of these characters proves that the intraspecific units which have been distinguished on the basis of these characters do not have systematic significance. The two morphotypes of E. caninus ( ldquo

pauciflorum rdquo

and

caninus

) described in this study will provide the basis for further genetic studies aimed at establishing their significance for considerations in the systematics of the species.

2000

1.593

0.0

. 2000, 47:11-24 DOI:10.1023/A:1008719119756

Levels and distribution of allozyme and RAPD variation in populations of Elymus fibrosis (Schrenk) Tzvel. (Poaceae

1.The Swedish University of Agricultural Sciences 2.The Swedish University of Agricultural Sciences

To study the magnitude and nature of genetic variation in E. fibrosus, the levels and distribution of allozyme and RAPD variations were investigated in populations collected from Finland and Russia. The results obtained from the allozyme and RAPD studies were compared to each other in 10 of the populations. The allozyme analysis showed that 6 of 12 presumed loci (50%) were polymorphic within the species, while the mean number of polymorphic loci within populations was 4.8%. The mean number of allele per locus for the species was 1.5 and 1.05 across the populations. Genetic diversity at the species level was low (H_es = 0.025), and the mean population genetic diversity was even lower (H_ep = 0.007). Both these values were much lower than the average for other Elymus and self-fertilising species. The largest proportion of the total allozyme diversity was found among, rather than within the populations (G_ST = 0.70). The allozyme genetic distances between the populations did not reflect geographic distances. Cluster and principal coordinates analyses revealed the same allozyme relationship patterns among the populations. A comparison of allozyme and RAPD variation in 10 of the populations showed differences in the amount of genetic variation. The RAPD analysis revealed higher levels of variation (A_p = 1.19, P_p = 20.3 and H_ep = 0.09) than the allozyme one) A_p = 1.06, P_p = 5.8 and H_ep = 0.008). For both markers, the largest proportion of the total gene diversity was found among the populations studied (G_st = 0.63 for RAPDs and G_st = 0.65 for allozyme). In contrast to the allozyme analysis, the RAPD based genetic distances did reflect geographic distances. The cluster and principal coordinates analyses showed different grouping of populations for each data set. There was a positive, but not significant, correlation (r = 0.41) between the genetic distance matrices resulting from these markers. Regional comparison revealed that the Finnish populations had a higher diversity than the Russian ones. Generally, this study indicates that E. fibrosus contains low genetic variation in its populations. The results are discussed in the context of conservation of the species.

2006

0.0

2004

1.593

0.0

. 2004, 51:231-235 DOI:10.1023/B:GRES.0000024024.59141.e3

Analysis of duplication in the Spanish durum wheat collection maintained in the CRF-INIA on the basis of agro-morphological traits and gliadin proteins

1.Instituto Nacional de Investigacion y Tenologia Agraria y Alimentaria, Centro de Recursos Fitogeneticos

A total of 106 potential duplicate cases involved 277 accessions were detected on the basis of passport data in the durum wheat collection maintained in the CRF-INIA. Similarity between accessions was measured by agro-morphological traits. The 90% of the agro-morphological duplication were verified with gliadin proteins, allowing identification of similar material with greater refinement than agro-morphological data. However, the results indicated not to decide for rationalisation only on the basis of molecular data.

2002

1.593

0.0

1999

1.668

0.0

2002

0.0

0.928

. 2002, 135:277-289

Differentiation in the Elymus dahuricus complex (Poaceae): evidence from grain proteins, DNA, and crossability

<Affiliation>Central Siberian Botanical Garden (CSBG SB RAS), Zolotodolinskaya st., 101, Novosibirsk, 630090, Russia. agalex@csbg.nsc.ru

The status of the taxa Elymus dahuricus Turcz. ex Griseb., E. excelsus Turcz. ex Griseb., E. woroschilowii Probat. and E. tangutorum (Nevski) Hand.-Mazz., all of which are sometimes treated as members of E. dahuricus s.l. or as constituting the E. dahuricus complex, has been investigated using morphological, biochemical (SDS-PAGE of endosperm proteins) and molecular (DNA-AFLP) variation characteristics. Populations include accessions from Siberia, Far East (Russia), Tien-Shan (Kirghizstan), and Tibet and Sichuan Provinces (China). Variation in the morphological characteristics and endosperm proteins patterns have been found in all populations of the complex. DNA variation was very low within populations but was different among populations; this was attributed to genetic drift. Seed fertility of artificial hybrids and F2-F3 progenies in 38 combinations was analyzed. All biotypes studied form a common recombination genepool (RGP). The reproductive compatibility of hybrid combinations decreases with increased geographical separation of the parents. Differences in protein patterns and in genetic variation drawn from DNA-AFLPs and phenotypical segregation in F2 on series of diagnostic characters does not support existing taxonomic treatments of the E. dahuricus complex in Southern Russia, and Central-Asian area. The E. dahuricus complex seems to be a polymorphic species having a wide geographical range and wide genetic variation. The different species need probably be relegated to infraspecific rank. We have refrained from making the necessary combinations pending examination of additional specimens, including specimens of taxa not included in this study and the type specimens of all taxa involved.

PMID: 12152347

2004

3.658

0.0

. 2004, 108:1443-1448 DOI:10.1007/s00122-004-1588-x

Phylogenetic and evolutionary relationships between Elymus humidus and other Elymus species based on sequencing of non-coding regions of cpDNA and AFLP of nuclear DNA

1.Biological Resources Division Japan International Research Center for Agriculture Sciences (JIRCAS) 1-1 Ohwashi, Tsukuba 305-8686 Ibaraki Japan

Species of the genus Elymus are closely related to some important cereal crops and may thus serve as potential alien genetic resources for the improvement of these crops. E. humidus is indigenous to Japan and is well adapted to a humid climate. However, the phylogenetic and evolutionary relationships between E. humidus and other Elymus species are unclear. To elucidate these relationships, we examined the sequences of three non-coding regions of chloroplast DNA (cpDNA) and the amplified fragment length polymorphism (AFLP) variation of nuclear DNA in E. humidus and other related species. A total of 15 sequence mutations from the three non-coding regions, trnL-trnF, trnF-ndhJ(C), and atpB-rbcL, covering approximately 1,800 bp, were detected in the Elymus species. A phylogenic tree resulting from the cpDNA sequence data revealed that all the species containing the St nuclear genome (St, StH, StY, and StHY) formed a well-supported clade that is remote from the Hordeum species (H). This result strongly supports the finding that Pseudoroegneria is the maternal genome donor to the genus Elymus. In addition, E. humidus showed the closest relationship with the cpDNA genome of the Pseudoroegneria species. The AFLP analysis detected 281 polymorphic bands with 11 AFLP primer combinations. The AFLP result showed that E. humidus is relatively closer to E. tsukushiensis. However, the cpDNA sequencing results indicated that E. humidus and E. tsukushiensis have different cytoplasmic origins. Our results suggest that the evolutionary process between E. humidus and E. tsukushiensis is not monophyletic, although the two species have similar morphological characters and adaptability.

2008

0.716

0.0

... 4%)^[35],而且也低于对青藏高原地区老芒麦SRAP研究的结果(86 ...

2010

0.0

... 48%)^[36] ...

1996

0.0

... 一般来说,相对于异花授粉和常异花授粉植物而言,自花授粉植物具有较高的群体间遗传变异和较低的群体内遗传变异^[37] ...

2013

0.0

. 2013, 22(3):259-265

<Affiliation>The Walther Oncology Center, James Whitcomb Riley Hospital for Children, Indiana University Medical Center, Indianapolis, Indiana, USA.

Oct-4 is a transcriptional regulator required to maintain the totipotentiality of embryonic stem (ES) cells. Downregulation of its activity is required for proper differentiation of the blastocyst during uterine implantation. Uterine implantation and subsequent vascularization increase oxygen exposure of the developing embryo, thereby altering the intracellular reduction-oxidation status. We tested whether Oct-4 could be regulated by these changes in reduction-oxidation status. We found that Oct-4 DNA binding was exquisitely sensitive to abrogation by oxidation but that the DNA binding of another ES cell transcription factor, FoxD3, was much less sensitive to oxidation. The reducing enzyme Thioredoxin (but not Ape-1) could restore DNA-binding activity of Oct-4. Thioredoxin was less effective at restoring the DNA-binding ability of FoxD3. It was also found that Thioredoxin (but not Ape-1) could physically associate with cysteines in the POU domain of Oct-4. Finally, overexpressing normal Thioredoxin increased the transcriptional activity of Oct-4, while overexpressing a mutant Thioredoxin decreased the transcriptional activity of Oct-4. These data imply that ES cell transcription factors are differentially sensitive to oxidation and that Thioredoxin may differentially regulate ES cell transcription factors.

PMID: 15153603

... 加上种质间在繁殖后代过程中存在一定的基因流(花粉流)^[38],这两种因素可能会在一定程度上降低种质间的遗传差异,导致种质间较高平均GS值的出现 ...

1978

0.0

... 地理隔离、生态隔离和繁殖隔离会极大地影响植物的种内遗传多样性^[39] ...

2013

0.0

... 2 供试种质间及其地理类群间的遗传关系根据表现所有供试种质间遗传关系的聚类树形图和主向量空间分布图来看,各种质的聚类分布与地理来源有较强的关系^[40] ...

2002

1.593

0.0

... 种质的地理来源代表了不同的宏观生态环境,而这些不同的生态环境条件是造成种质间的遗传变异的主要因素^[41] ...

2002

0.0

... 由于青藏高原的高海拔及它被喀喇昆仑山、阿尔金山和祁连山等著名高山与其他种质的来源地区隔离开来^[42],造成其植物种质可能具有特定的生态地理适应性 ...

2009

1.153

0.0

. 2009, 37:716-722 DOI:10.1016/j.bse.2009.12.005

Genetic variation and geographical divergence in Elymus nutans Griseb. (Poaceae: Triticeae) from West China

Abstract

Elymus nutans Griseb. as an important forage grass and gene pools for improving cereal crops, widely distributes in west China. However, little is known about its genetic and geographical patterns. Inter-simple sequence repeat (ISSR) markers were applied to assess genetic diversity and geographical divergence among 63 E. nutans accessions from west China. The cluster analysis separated the accessions into several groups with geographical origins. The molecular variance analysis (AMOVA) showed that the proportion of variance explained by within- and among-geographical group diversity was 43.2% and 56.8%, respectively. Based on pairwise genetic distances (Φ_ST) between geographical groups, the relationships were congruent with the cluster of accessions. The distinct geographical divergence of E. nutans was revealed between Qinghai-Tibet Plateau and Xinjiang. The ecogeographical conditions such as climate and mountain ranges and elevation behaved as the crucial factors for genetic divergence. Furthermore, the study also indicated that Qinghai-Tibet Plateau might be the diversity differentiation center of E. nutans. The result will facilitate the breeding program and germplasm collection and conservation.

... 本结果与Chen等^[43]、陈智华等^[44]和苗佳敏等^[45]对垂穗披碱草(Elymus nutans)遗传多样性的研究结果一致,即青藏高原与其他地区的种质材料存在明显的遗传分化 ...

2009

0.0

2011

0.0

2005

3.658

0.0

. 2005, 530:530-539 DOI:10.1007/s00122-005-2044-2

Patterns of AFLP variation in a core subset of cultivated hexaploid oat germplasm

1.Plant Gene Resources of Canada, Saskatoon Research Centre Agriculture and Agri-Food Canada 107 Science Place Saskatoon S7N 0X2 SK Canada 2.Cereal Research Centre Agriculture and Agri-Food Canada 195 Dafoe Road Winnipeg R3T 2M9 MB Canada

Many core collections have been developed from large collections of crop germplasm, but most of these have not been characterized, particularly using molecular techniques, for germplasm management and utilization. We have attempted to characterize a structured sample representing a world collection of 11,622 cultivated hexaploid oat accessions in the hope of understanding the genetic structure of the world collection. The amplified fragment length polymorphism (AFLP) technique was applied to screen 670 accessions representing 79 countries and one group of uncertain origin. For each accession, 170 AFLP polymorphic bands detected by five AFLP primer pairs were scored. Analyses of the AFLP data showed the effectiveness of the stratified sampling applied in capturing country-wise AFLP variation. The frequencies of polymorphic bands ranged from 0.11 to 0.99, with an average of 0.72. The majority (89.9%) of the AFLP variation resided within accessions of each country, and only 6.2% of the AFLP differences existed among accessions of major geographic regions. Accessions from the Mediterranean region were the most distinct, while those from Russia and the USA were the most diverse. The two distinct groups that were observed were separated largely on the basis of common oat and red oat. Red oat was genetically more diverse than its common and hull-less counterparts, and hull-less oat was more related to common oat than red oat. Landrace and non-landrace accessions displayed similar AFLP variation patterns. These patterns are significant for understanding the domestication of cultivated oat and are useful in classifying the intraspecific diversity of oat germplasm, developing specific core subsets of the oat collection, and exploring new sources of genes for oat improvement.

... _ST值作为其遗传距离,因而在评价类似植物群体间和群体内的遗传关系时非常有效^[46] ...