【作者】 鄢家俊；

【导师】 白史且； 张新全；

【作者基本信息】 四川农业大学 ， 草业科学， 2009， 博士

【摘要】 老芒麦(Elymus sibiricus L.)别名西伯利亚披碱草,是禾本科(Gramineae)小麦族(Triticeae)披碱草属(Elymus)的多年生优良牧草,是披碱草属的模式种。老芒麦在北半球温带地区分布较广,在我国特别是青藏高原地区有丰富的野生资源分布。由于其高产优质和对寒冷干旱气候的良好适应性,近年来,老芒麦已经成为青藏高原地区栽培利用最为广泛的当家草种之一。试验在调查我国青藏高原地区野生老芒麦种群生态分布的基础上,以该地区应用广泛的老芒麦国审品种“川草1号”(Elymus sibiricus L.cv.chuancao No.1)和“川草2号”(Elymus sibiricus L.cv.chuancao No.2)为对照,对收集到的54份野生资源从种群生态与形态学、生产性能、种子醇溶蛋白和DNA分子标记等方面进行系统的遗传多样性研究和优异种质筛选;同时利用穗部性状、SRAP和SSR分子标记对该地区东南缘的13个老芒麦自然居群进行了遗传变异和群体遗传结构分析。主要结果如下:1、对青藏高原老芒麦野生种群生态分布、生境类型、群落组成和形态学变异研究表明:(1)老芒麦在青藏高原地区分布广泛,群落生境初步划分为:高山亚高山草甸型、河谷草地型和森林灌丛型;群落组成以:高山红柳+老芒麦+发草,沙棘+老芒麦+蒿类,老芒麦+锦鸡儿+鹅观草,老芒麦+披碱草+多节雀麦4种类型最多。(2)野生老芒麦种质形态学性状具广泛变异,其中与牧草产量和种子产量相关的形态性状变异较大,而与分类相关的指标则变异程度较小。(3)聚类分析将不同形态的老芒麦聚为3大类群,聚类结果除与海拔有一定关系外,与其地理分布一致性不明显。(4)主成分分析表明内外颖长、内外颖芒长、旗叶宽、倒二叶片长、株高、内外稃长、外稃芒长、内外稃宽、穗中部节上每小穗的小花数、穗长、叶色、茎粗、灰度和穗中部节上的小穗数是引起老芒麦形态分化的主要指标。2、物候期观测将供试材料分为早熟型和晚熟型两大类,生育期最短的是SAG205119和SAG205151,仅为115天,最长的是老芒麦品种“川草2号”,为129天。在试验所在地,老芒麦于6月初到7月中旬出现生长高峰期,株高呈直线上升趋势。不同材料的单株产量存在一定的差异,鲜草产量为35.66g/株～87.59g/株,单株平均鲜重为58.16g,干草产量为11.09g/株～29.18g/株,单株平均干重为17.96g。所有老芒麦材料的茎叶比为1.84～2.71,平均值为2.17,粗蛋白含量为8.27%～14.79%,平均值为10.96%。大部分材料的茎叶比低于对照而粗蛋白含量高于对照,说明青藏高原野生老芒麦具有较高的牧草品质。聚类分析将所有材料聚为高产优质和表现一般两大类型,其中的6份材料SAG205167、SAG205179、SAG204089、SAG205230、SAG205124和SAG204451的单株鲜草产量和牧草品质都高于对照品种,开发利用价值较大,而对照品种在本次试验中表现出优良性状退化的现象。3、基于酸性聚丙烯酰胺凝胶电泳(A-PAGE)的醇溶蛋白标记对54份野生老芒麦种质进行遗传多样性分析。供试材料共分离出42条带纹,多态率达92.86%。4个电泳分区(α、β、γ和ω)的平均Shannon指数为0.4627,Nei-Li遗传相似系数(GS)变异范围为0.2424～0.9767,平均值为0.5822。说明供试野生老芒麦材料具有较为丰富的醇溶蛋白遗传多样性。对所有材料的聚类分析发现,在GS为0.562的水平上供试材料可聚成4个大类,绝大部分来自于相同或相似生态地理环境的材料聚成一类,主成分分析显示了相似的结果。基于Shannon多样性指数估算了老芒麦5个地理类群内和类群间的遗传分化,发现地理类群内和地理类群间的遗传变异分别占总变异的68.17%和31.83%。对各地理类群基于Nei’s无偏估计的遗传一致度的聚类分析表明,各地理类群间的遗传分化与其所处的地理生态环境具有较高的相关性。4、采用SRAP分子标记技术,对52份野生老芒麦材料进行遗传多样性分析,筛选出的16对随机引物组合共扩增出318条清晰可辨的条带,其中多态性条带275条,占86.48%;每对引物扩增出14～27条带纹,平均为19.88条,多态性信息(PIC)含量为0.122～0.326之间,平均为0.24,SRAP标记效率(MI)为4.26;材料间的遗传相似系数(GS)范围在0.5064到0.9586之间,平均值为0.7921;52份种质的Nei’s遗传多样性(He)为0.2270,Shannon’s指数(Ho)为0.3472;这些结果说明供试野生老芒麦在分子水平具有较为丰富的遗传多样性。对所有材料的聚类分析和主成分分析发现,在GS=0.80的水平上,供试材料可聚为5类,大部分来自相同或相似生态地理环境的材料聚为一类。对5个老芒麦地理类群基于Shannon’s指数的遗传分化估算发现,类群内遗传变异占总变异的65.29%,而类群间遗传变异占总变异的34.71%。对各生态地理类群基于Nei氏无偏估计的遗传一致度聚类分析表明,各生态地理类群间的遗传分化与其所处的生态地理环境具有一定的相关性。5、利用SSR标记技术对52份老芒麦材料的遗传变异及亲缘关系进行了研究。18对SSR引物共扩增出236条清晰的条带,其中多态性条带204条,多态性位点率(PPB)为86.44%;每对引物扩增出7～20条带纹,平均为13.1条,多态性信息(PIC)含量为0.267～0.471之间,平均为0.35,SSR标记效率(MI)为3.98;材料间的遗传相似系数(GS)为0.622到0.895之间,平均GS值为0.766;52份种质的Nei’s遗传多样性指数(He)为0.3286,Shannon’s指数(Ho)为0.4851,表明供试材料之间差异明显,具有较为丰富的遗传多样性。根据研究结果进行聚类分析和主成分分析,可将52份老芒麦材料分成5大类,具有相同地理来源或相似生境的材料趋向于聚为一类。6、对采集自青藏高原东南缘的13个野生老芒麦居群在原生境下的15项穗部性状变异进行了研究。Shannon指数分析表明,13个居群在穗部性状上具有丰富的遗传多样性(He=1.7937)且居群内遗传变异(69.28%)大于居群间(30.72%);聚类分析将这13个居群分为三个组;主成分分析表明单穗长和宽、单穗重、小穗长、内外稃长和每穗轴节小穗数等是造成13个居群老芒麦穗部特征差异的主要因素;相关分析的结果表明海拔、纬度、经度和降水量对青藏高原野生老芒麦居群穗部性状变异贡献较大,而年均温对此影响不大。根据研究结果提出了老芒麦资源的收集和保护策略。7、基于SRAP和SSR分子标记分析了青藏高原东南缘8个老芒麦自然居群遗传变异及群体遗传结构。16对SRAP引物在90个单株中共扩增出384条可统计条带,其中多态性条带334条,占86.98%。16个SSR位点共检测出等位变异221个,平均每个位点13.8个,其中具有多态性的位点数192个,占86.88%。两种分子标记检测到老芒麦居群水平的基因多样性(He)分别为0.1092和0.1296,而物种水平的基因多样性达0.2434和0.3732。基于两种标记的的Nei’s遗传分化指数Gst(0.5525和0.5158)表明老芒麦居群出现了较大程度的遗传分化,居群间的基因流非常有限,分别为0.4050和0.4694。Shannon指数的群体分化系数(56.43%和53.19%)和分子方差变异(AMOVA)分析(58.64%和52.41%)结果与Nei’s遗传分化指数基本一致,均显示老芒麦的遗传变异主要分布在居群间,居群内变异相对较小。基于聚类分析结果表明各居群间存在较为明显的地理分化,8个居群分化为采集地范围内的南、北和中部3个分支。通过对老芒麦遗传多样性和遗传结构的分析提出了对该物种遗传多样性的保护策略。更多还原

【Abstract】 As the type species of the genus Elymus, E. sibiricus L. (Siberian wildrye) is a perennial, high quality forage indigenous to eurasia,. and in China especially in the Qinghai-Tibetan Plateau, there are abundant natural resources of E. sibiricus. E. sibiricus is an important component of native grasslands and has been recently developed as a major cultivated forage in Qinghai-Tibetan Plateau, owing to its good palatability and excellent capability of adaptation to cold and drought condition.Based on the field investigation on ecological distribution and growth habits of E sibiricus populations in the Qinghai-Tibetan Plateau of China, fifty-four wild accessions were collected and tested both in assessment of genetic diversity and germplasm appraisal, according to population ecology, morphological, production performance, gliadin markers and DNA markers, by a comparison with two representative varieties E sibiricus L. cv. Chuancao No. 1 and E sibiricus L. cv. Chuancao No. 2. In addition, employing ear characters diversity, SRAP and SSR markers, we studied the population structure and genetic variation among E. sibiricus populations from the eastern alpine region of Qinghai-Tibetan Plateau. The main results showed as follows:1. According to studying on distribution characteristics, habitat types, community composition and morphological variation of wild E. sibiricus, below results were obtained: (1) E sibiricus were widely distributied in Qinghai-Tibetan Plateau, its habitat types could be classified into three types: alpine and subalpine meadow type, valley grassland type and forest-shrub type; the main community composition including four types: Salix cheilophila var.microstachyoides +E. sibiricus + Deschampsia caespitosa, Hippophae rhamnoides + E. sibiricus + Artemisia; E. sibiricus+ Caragana+ Roegneria, E. sibiricus + E. nutans+ Bromus plurinodes. (2) Natural E. sibiricus produces rich morphological diversity. Among the 30 observed properties, morphological variances most significantly occured in the traits related with forage and seed yield, as for classification index, there are less variation. (3) Based on the phenotypic characteristics, 37 accessions were clustered into three morphological types, and the cluster result don’t agree well with geographical distribution but related to altitude. (4) Principlal component analysis results indicated that 17 of the characters measured: Outer and inner glume length, awn length of outer and inner glume, boot leaf width, length of the second leaf from the inflorescence, plant height, lemma length and width, palea length and width, awn length of lemma, floret number in each spikelet, ear length, leaf color, internode diameter, gray grade and spikelet number were the main sources of morphological differentiation of E. sibiricus accessions. 2. Diversities were also detected exist in production performance of different E. sibiricus accessions. Twenty-three accessions could be divided into early maturing type and late maturing type on the basis of phenological observation. In all of the accessions, SAG205119 and SAG205151 showed the shortest growth period (115d), and "E. sibiricus cv Chuancao No. 2" showed the longest growth period (129d). E. sibiricus present the growth peak from beginning of June to the middle of July at the test place. The yield and quality characterizes of the twenty-three accessions also significantly different, the fresh yield was 35.66-87.59g/plant, with an average of 58.16g/plant, the hay yield was 11.09-29.18g/plant, and with an average of 17.96g/plant. The stem-leaf ratio was 1.84-2.71, with an average of 2.17, the crude protein content was 8.27%-14.79%, with an average of 10.96%, most of the accessions have the lower stem-leaf ratio and higher crude protein content than the CK, its means that the E. sibiricus accessions from Qinghai-Tibetan Plateau posses the high quality. Based on the production performance, all of the accessions were clustered into two types, one is high yield and quality, the other is generally showing. Among the former type, six accessions SAG205167, SAG205179, SAG204089, SAG205230, SAG205124 and SAG204451 showed a higher fresh yield and quality than CK, which hold a greater potentiality for further application. As for the "E. sibiricus cv Chuancao No.1" and "E. sibiricus cv Chuancao No.2", the excellent characteristics of them seems were degenerating in this study.3. Acid polyacrylamide gel electrophoresis (A-PAGE) was employed to detect the gliadin genetic diversity among 54 wild accessions of E. sibiricus collected from Qinghai-Tibetan plateau. A total of 42 bands were detected in all accessions, of which 92.86% were polymorphic. The average number of Shannon index to four electrophoretic zones (α,β,γ,ω) was 0.4627. The Nei-Li genetic similarity coefficient of the tested accessions ranged from 0.2424 to 0.9767, and the average was 0.5822. These results suggested that there was a rich genetic polymorphism among the tested wild resources of E. sibirucus. 54 wild accessions can be clustered into 4 groups at GS = 0.562 level on dendrogram. The principal coordinates (PCA) reflected almost the same relationships among the studied materials as showed in cluster analysis. Moreover, the accessions from the same origin frequently clustered into one group. Genetic differentiation of between and within five eco-geographical groups of E. sibiricus is estimated by Shannon’s diversity index, which shown that 68.17% genetic variance existed within group, and 31.83% genetic variance was among groups. The unweighted pairwise groups method using arithmetic average (UPGMA) cluster analysis based on Nei’s unbiased measures of genetic identity was assayed for five geographical groups of E. sibiricus, which indicated that there was a significantly positive correlation between genetic differentiation and geographical habits among the five groups.4. Sequence-related amplified polymorphism (SRAP) molecular markers were used to detect the genetic diversity of 52 wild accessions of Elymus sibiricus L. collected from Qinghai-Tibetan plateau. A total of 318 fragments were identified with 16 SRAP primers sets, of which 86.48% were polymorphic. For each primer set, there were 14-27 fragments were detected, with an average of 19.88, the polymorphism information content (PIC) was 0.122-0.326, with an average of 0.24, and the marker index (MI) of SRAP was 4.26. The genetic similarity (GS) coefficient of the tested accessions ranged from 0.5064 to 0.9586, with an average of 0.7921. The Nei’s index of diversity (He) at the species level was 0.2270, and the Shannon’s index (Ho) was 0.3472. These results suggested that there was rich genetic diversity among the tested wild resources of E. sibiricus. The results demonstrated a strong geographic effect on molecular variation of the local E. sibiricus as indicated by unweighted pairwise groups method using arithmetic average (UPGMA), and 52 wild accessions were clustered into five group at GS=0.80 level on dendrogram. Genetic differentiation between and within five eco-geographical groups of E. sibiricus was estimated by Shannon’s diversity index, which showed that 65.29% genetic variance existed within group, and 34.71% genetic variance was among groups. Based on Nei’s unbiased measures of genetic identity, UPGMA cluster analysis measures of five eco-geographical groups of E. sibiricus, indicated that there was a correlation between genetic differentiation and eco-geographical habits among the groups.5. The genetic diversity of 52 E. sibiricus accessions were evaluated by SSR markers A total of 318 fragments were identified with 18 SSR primers sets, of which 204 (86.48%) were polymorphic, and each primer generated 13.1 fragments, the polymorphism information content (PIC) per primer was 0.267-0.471, with an average of 0.35, and the marker index (MI) of SSR was 3.98. The genetic similarity (GS) coefficient of the tested accessions ranged from 0.622 to 0.895, with an average of 0.766. The Nei’s index of diversity (He) of the 52 accessions was 0.3286, and the Shannon’s index (Ho) was 0.4851. These results suggested that there was rich genetic diversity among the tested wild resources of E. sibiricus. 52 accessions could be divided into five main groups by cluster and principal component analysis, the accessions from the same region or with the same habitat type tends to were classified into the same group, indicating the geographical distribution of genetic diversity of E. sibiricus.6. The variation in 15 ear characters of 13 populations of Elymus sibiricus L. were researched in the present study. Results from the Shannon-weaver index analysis showed that there was an abundant genetic diversity (H’=1.7937) among these populations in ear characters, the genetic variation within populations (69.31%) was greater than that among populations (30.69%). Cluster analysis showed that 13 populations could be categorized into 3 groups. Principlal component analysis results indicated that 7 of the characters measured: Ear length, Ear width, Weight of single ear, Spikelet length, Palea length, Lemma length and Spikelet per rachis were the main sources of ear characters variation of 13 E.sibiricus populations. Results from the correlation analysis showed that altitude, latitude, longtitude and precipitation have remarkable influence on the ear characters variation of E. sibiricus, but mean annual temperature has little influence on it. Based on the genetic information available for E. sibiricus , some conservation strategies were proposed.7. In present study, the genetic diversity and population structure in eight natural populations of E. sibiricus from Qinghai-Tibetan Plateau of China was analyzed by means of sequence-related amplified polymorphism (SRAP) and Microcatellite markers (SSR). A total of 384 fragments were identified with 16 SRAP primers sets, of which 86.98% were polymorphic. Meanwhile, a total of 221 alleles were detected at 16 loci, with 192 (86.88%) being polymorphic, indicating considerable genetic variation at the species level. The mean gene diversity (He) was estimated to be 0.1092 and 0.1296 within populations detected by SRAP and SSR markers respectively, and 0.2434 and 0.3732 at the species level. A high level of genetic differentiation among populations was detected based on Nei’s genetic diversity analysis both in SRAP (Gst=0.5525) and SSR (Gst=0.5158) markers, and an indirect estimate of the number of migrants per generation (0.4050 by SRAP markers, 0.4694 by SSR markers) showed that gene flow was low among populations. Shannon’s index analysis and AMOVA analysis displayed the same result that mainly genetic variation of Elymus sibiricus existed among the populations. In addition, a geographical pattern of population differentiation, where the populations from south, north and middle area of sampling sites were clearly separated from each other, was revealed by cluster analysis. Based on the genetic information available for the native E. sibiricus, we proposed that it should be advisable to collect and preserve the native Elymus sibiricus germplasm pool in a larger extent.更多还原