【作者】 王玉山；

【导师】 邢世岩；

【作者基本信息】 山东农业大学 ， 森林培育， 2011， 博士

【摘要】 侧柏(Platycladus orientalis (L.) Franco)起源于中国,种质资源极为丰富,是我国重要的荒山造林绿化树种。针对侧柏缺乏良种的现实,本文根据分子系统学、群体遗传学的原理和方法,利用AFLP标记结合表型性状,从分子和形态两个水平对侧柏种源遗传多样性进行评价,探讨其遗传变异规律,为侧柏优良种源选择、种源区划分、种子调拨及遗传改良等提供依据。主要研究结果如下:1.通过对17省(市、自治区)41个侧柏种源的生长性状、21个种源的球果形态进行研究,结果表明,生长性状的变异系数在5.41%~47.55%间,其中以分枝角的变异系数最大(28.74%),树高的变异系数最小(10.73%)。种子的变异范围为4.77%~47.42%,其中单粒重的变异系数最大,为30.28%,种子厚为11.63%、种子宽为10.42%,种子长的变异最小(CV=8.67%)。生长性状的变异系数比种子性状的变异系数大,说明种子的遗传稳定性高。生长性状、球果形态的变异系数和相对极差在种源间分别存在极显著差异,表现出丰富的遗传多样性。2.侧柏种源间生长性状表型分化最大的性状是树高(Vst=46.82%),分化最小的是冠幅(Vst=20.92%),生长性状的平均表型分化系数为30.12%,即生长变异的69.88%来自种源内。种子的表型分化系数范围为16.78%~27.66%,平均表型分化系数为22.92%,即种子的变异77.08%来自种源内。说明侧柏表型变异种源内远高于种源间的,种源内变异是侧柏种源的主要变异来源。3.生长和种子性状与经纬度的灰色关联分析表明,各性状与纬度的关联度大于经度,说明侧柏的表型变异是以纬向变异为主。对44个种源8个种实性状与种源点经纬度进行典型相关分析,揭示出侧柏种源种实性状的变异受纬度和经度双重控制,变异的基本模式是以纬向变异为主的类型,经纬度可解释种球变异量的38.48%。4.空间相关分析表明,不同生长性状的全局Moran’s I系数在-0.1878~0.1921间,种子的全局Moran’s I范围为-0.4842~0.7518,自相关系数多不显著,表现为近距离时呈现一定的相关性,随距离增大,表现为空间不相关或空间随机性。胸径的Moran’s I系数多大于树高的,说明胸径的空间变异性大于树高的空间变异性。各种源生长性状的G系数在-1.097~5.6426间,种子的G系数在-2.1899~1.9142间,且负值多,表明侧柏种源表型性状的空间聚集不明显,大多数种源与较小的种源相邻,即随纬度增加,表型性状呈变小的趋势。部分北方、南方种源的G系数为0,与周围种源无空间相关性,呈现随机分布状态。5.半方差函数结果说明胸径和树高的块金系数分别为58.72%、47.78%,两者均具有中等的空间相关性,分枝角的块金系数达90.6%,其空间相关性很弱。“Krige”分析反映出侧柏种源的生长性状存在一定的空间结构,呈现出渐变、双向渐变和不规则斑块的非随机分布模式。表明侧柏种源间表现为多数生长性状缺乏空间结构和部分生长性状具特定空间结构的复合模式。6.树高和胸径遗传力在两地点表现不同,平阴点树高遗传力0.7049~0.9557、胸径遗传力0.7007~0.8858,枣庄点树高遗传力0.2684~0.8102、胸径遗传力0.3262~0.9424,平阴点树高遗传力大于胸径的遗传力,枣庄点则是胸径遗传力多大于树高的遗传力。总体来说,23年生时胸径、树高的遗传力低于幼龄林遗传力。不同林龄生长量与经纬度相关分析表明:随着林龄的增大高生长与经度相关系数逐步缩小,与纬度相关系数有增大趋势;侧柏种源胸径生长量与经度相关系数逐步增大趋势,与纬度相关系数有减小趋势。7.以26个种源的254个单株为试材,利用荧光AFLP标记对侧柏种源的分子遗传多样性和遗传结构进行了研究,结果表明:8对引物扩增的平均多态位点数为100.4,多态位点百分率为46.47%。各种源扩增的多态位点数(A)差异较大,全国种源的平均多态位点数为83.6~118.5个,多态位点百分率(PPA)为38.72%~54.86%。山东种源扩增的平均多态位点数为54.5个~124.8个,PPA为25.23%~57.75%。四种源区扩增的多态位点数和多态位点百分比相比较,南部种源(A = 104.5,PPA = 48.35%)>北部种源(A = 104.2,PPA = 48.25%)>中部种源(A = 102.1,PPA = 47.28%)>山东种源(A =95.4,PPA = 44.14%)。说明南部种源的遗传多样性最为丰富,北部种源次之,中部和山东种源的遗传多样性水平较低。8.不同侧柏种源的遗传多样性有显著差异,观测等位基因(Na)的变幅为1.2622~1.6122,有效等位基因(Ne)变幅为1.0983~1.2519,Nei’s基因多样度(H)变幅为0.0635~0.1531,Shannon信息指数(I)变幅为0.1025~0.2371。26个侧柏种源的平均Na=1.5311、Ne=1.2022、H =0.1305、I =0.2102。侧柏种源种级水平观测的等位基因数为Na = 2.000,有效等位基因数为Ne = 1.2406,Nei’s基因多样度为H = 0.1605,Shannon信息指数为I = 0.2695,四个参数在种级水平上显著高于种源水平。在区域水平上,南部种源的Nei’s基因多样度、Shannon信息指数(H=0.1434、I = 0.2366)高于北部种源(H = 0.1425、I = 0.2305)和中部种源(H = 0.1377、I = 0.2283),但无显著性差异,显著高于山东种源(H = 0.1285、I = 0.2227),说明南、北部种源的杂合体多,遗传多样性丰富,而山东种源的遗传多样性低。9.遗传多样性分析结果显示:种级水平总的基因多样度Ht为0.1518,种源内基因多样度HS为0.1106,种源间的遗传分化系数Gst为0.2703,也就是说总变异的27.03%来源于种源间遗传变异,72.97%属于种源内的遗传变异。分子方差(AMOVA)分析结果显示,种源间的遗传变异占总变异的14.02%,种源内变异占74.86%,区域间变异占11.12%,表明侧柏种源出现了较高程度的遗传分化,遗传多样性主要存在于种源内。10.遗传分化系数计算得到种级水平的基因流Nm = 1.3804,说明侧柏4个种源区间存在适度的基因交流,但各区域的基因流显著高于全分布区的基因流(1.3804),其中北部种源的基因流最高,达3.8442,中部种源为3.3679,南部种源为2.5390,山东种源为2.1078,说明尺度小的区域内,侧柏种源的基因交流频繁。11. 26个侧柏种源间的遗传一致度介于0.8422~0.9919之间,平均为0.9380;遗传距离为0.0086~0.1717,平均值0.0647,说明种源间具有较高的基因相似度,遗传距离小。基于侧柏样本间的Nei遗传距离进行UPGMA聚类,26种源可分为5大类,大致按地域聚在一起:第一类为南部种源,包括云南文山、福建南平、贵州贵阳3个种源;第二类为北部种源,包括北京密云、内蒙乌拉山、辽宁朝阳和新疆伊犁4个种源;第三类为西南部种源,包括四川西昌、湖北荆门和河南确山3个种源;第四类为中部的8个种源,为甘肃徽县、江苏铜山、陕西黄陵、山西石楼、宁夏银川、河北易县以及山东的枣庄、莒南8个种源;第五类为山东种源,包括费县、曲阜、泰安、博山、安丘、平度、平阴和聊城8个种源。聚类结果显示纬度(地理距离)相近的种源被聚到了一起,反映了侧柏的遗传分化和生境有着一定的相关性,经Mantel检验,种源的地理距离与遗传距离之间显示正相关,但未达到显著性水平(r=0.288 6,P = 0.0930)。更多还原

【Abstract】 Orientalis (Platycladus orientalis (L.) Franco) originates in China.It’s germplasm resource is very abundant. Orientalis is an important tree species of afforestation on the rocky barren.According to the reality of the lack of good provenances. The diversity of four orientalis intraspecific populations was analyzed using morphological traits, the genetic diversity and population genetic structure for four geo-ecological groups using principles of molecular systematic and fluorescent-AFLP marker techniques were studied by morphology traits and molecular markers, respectively, in order to provide science evidence for conservation and utilization, to select good provenance and to divid provenance region and to allocate seed.. The main results are as follows:1. The diversity of growth traits of 41 provenances and cones of 21 provenances in 17 provinance of P.orientalis were investigated. The results indicated: There were significant differences among and within provenances. The growth traits have great diversity with the variation coefficient (CV), the range varied from 5.41% to 47.55%. The variation coefficient of branching angle was highest (28.74%), and the one of tree height was lowest; The variation coefficient of seeds was from 4.77% to 47.42%, and the variation coefficient of the seed thickness was the highest, the one of the seed length was the lowset. The variation coefficient of the seed traits is lower than the growth traits, this showed that the genetic stability of seeds is higher.There are siginificantly diversity of the variation coefficient and the relative range in the cone shape and the growth traits, the analysis indicated that there are abundant genetic diversity in the P.orientalis.2. The largest morphological differentiation coefficient of the growth traits among the orientalis was the tree height(Vst= 46.82%), the smallest was the crown(Vst = 20.92%). The average morphological differentiation coefficient of the growth traits was 30.12%, in means that the Vst within the provenance was 69.88%;The range of the morphological differentiation coefficient was from 16.78% to 27.66%. The average of the Vst is 22.92%, the genetic variation among provenance accounted for 77.08% of total variations. This suggested that the variation of morphological traits among provenance of orientalis is much higher than within provenance.3. The result of grey correlative analysis between the growth traits, the seed traits and the latitude, longitude indecates: The correlation of between all traits and the latitude was greater than the longitude, this showed that the morphological diversity of the orientalis mainly came from the latitudinal variation. The typical correlation analysis between the 44 provenances with 8 different traits of the cone showed: the geography variation of oreitalis was a variation that latitude mainly brought on. It’s variation mode was two-way variation from latitude and longitude.4.Spatial correlation analysis showed that global moran’s I of growth characters is from -0.1878 to 0.1921,the global moran’s I of seed is from -0.4842 to 0.7518, most autocorrelation coefficient is not significant,the result showed that presents certain correlation over short distance, while presented spatial uncorrelation or spatial random with distance increases. Global moran’s I of DBH is highter than tree height,indicated that variation of DBH is highter than tree height.Local G coefficient of growth characters is from-1.097 to 5.6426,while the seed’s is from -2.1899 to 1.9142,and most of them is negative, showed a lack of spatial structure,around provenance is smaller. Local G coefficient of few of northern and southern provenance is 0,they are randomly distributed.5. The semivariance analysis showed that the nugget of DBH growth and height growth were respectively 58.72% and 47.78%, both of which had moderate spatial correlation. While the nugget of branch angle was as high as 90.6%,and the spatial correlation was very weak. When analysis in krige method were performed, the growth Characters of the provenances of platycladus orientalis had certain kinds of spatial structures which presenting a non-random distribution such as progressive change,bi-directional progressive change and irregular patch.Analysis among the provenances P. orientalis also showed a complex pattern that most growth characters seldom have any spatial structure,while A small proportion of the growth characters have spatial structure any way.6. The heritability in, behaved differently between the Pingyin and Zaozhuang sites. At Pingyin site, the heritability in height and DBH was respectively 0.7049~0.9557 and 0.7007~0.8858. At this site, the tree height had higher heritabilities. At Zaozhuang site, the heritability in height and DBH was respectively 0.2684~0.8102 and 0.3262~0.9424. At this site, the DBH had higher heritabilities than the tree height had. Totally, trees in 23 years old had lower heritabilities of both in DBH and tree heigh than young trees had.The correlation analysis between geographical coordinate and annual increment of trees in different forest ages in Platycladus orientalis had the following results: The correlation coefficient between tree height and longitude tended to become weaker with forest got older, while the correlation coefficient between tree height and latitude tended to become stronger with forest got older. Correspondingly, the correlation coefficient between tree DBH and longitude tended to become stronger with forest got older, while the correlation coefficient between DBH and latitude tended to become weaker with forest got older.7.Population genetic structure was studied using fluorescent-AFLP markers on 256 orientalis (P. orientalis (L.) Fr.) accessions collected from the Southern group, the Northern group, the Central of Chian group and the Shandong province group of fluorescent-AFLP markers. The purpose of this study was to determine the genetic structure and genotypic diversity amongst the different provenance of orientalis. The results showed that the average number of polymorphic loci (A) was 100.4, the percentage of polymorphic loci (P) was 46.47% by 8 pairs of EcoR I /MseI (Mse I - a FAM fluorescent marked primer) primers in orientalis of four groups. Analysis for the average number of polymorphic loci (A) and the percentage of polymorphic loci (P) in four orientalis groups indicated that the Sourthern group (A = 102.1,P = 47.28%)> the Northern group (A = 104.2,P = 48.25%)> the Cemtral Chian group (A = 102.1,P = 47.28%)> the Shandong Province group (A =95.4,P = 44.14%); The analysis of diversity and genetic structure from four geo-ecological groups showed that genetic diversity of orientalis provenance in the Southern Group is most abundant, then it is the Northern group; the Northern group and the Shandong Provence group were lowest.8. There was significantly different of genetic diversity among orientalis provenance, the range of is from 1.2622 to 1.6122, is from 1.0983 to 1.2519, the Shannon Index is from 0.1025 to 0.2371. The observed number of alleles (Na), the effective number of alleles (Ne), the Nei’s gene diversity (H) and Shannon information index (I) at species level were respectively 1.5311, 1.2022, 0.1305 and 0.2102. The observed number of alleles (Na), the effective number of alleles (Ne), the Nei’s gene diversity (H) and Shannon information index (I) at provenances level were respectively 2.000, 1.2406, 0.1605 and 0.2695. Four parameters at species level was high significant higher than at provenances level.At regional level, Nei’s gene diversity and Shannon information index (H=0.1434、I = 0.2366)in the Southern group was higher than those in the Northern group (H = 0.1425、I = 0.2305)and in the Central China group(H = 0.1377、I = 0.2283), there were no significant differences; but they were significant higer than those in the Shandong Province group(H = 0.1285、I = 0.2227). The analysis of four population diversity and genetic structure from four provenances showed that genetic diversity of orientalis provenances in the Southern Group was most abundant, there were many heterozygous in the Southern group and the Northern group, the genetic diversity of orientalis provenances in the Shandong Province Group was lower.9. The parameters--genetic differentiation coefficient and gene flow of population genetic structure in orietnalis were analyzed in this study. The genetic diversity at the species level (Ht) was 0.1106, the genetic differentiation coefficient (Gst) among the provenances was 0.2703, This showed that orientalis genetic variation was mainly within the provenances and accounted for 72.97% of total variations, the genetic variation between provenances accounted for 27.03% of total variations. The results from AMOVA analysis showed that the orientalis genetic variation was mainly within the provenances and accounted for 74.86%. The genetic variation of within regional accounted for 11.12%. There was a high level of genetic differentiation at provenances level, and the genetic diversity was mainly at provenances level.10. The gene flow (Nm) was 1.3804 according to the genetic differentiation coefficient between groups (Nm = 1. 3804). This indicated that there are partly gene exchanges among four regional of orientalis. The gene flow (Nm) at regional level was high significant higher than at species level. Analysis for gene flow (Nm) in four orgional groups indicated that the Northern group (Nm = 3.8442) > the Central China group (Nm = 3.3679)> the Southern group (Nm = 2.5390)> the Shandong province group(Nm = 2.1078). This showed that the gene exchange was frequently within the small scale region.11. Nei’s genetic identities in 26 orientalis provenances were between 0.8422-0.9919, the average was 0.9380; Genetic distances were between 0.0086~0.1717, the average was 0.0647. It was suggested that there were higher similarity between various geo-ecological groups in orientalis and lower genetic distance.12. The results from UPGMA cluster analysis for 26 provenances showed that all the orientalis could be divided into 5 groups. Almost all provenances from the same population could be clustered into the same group. Three provenances in groupⅠwhich was call the Southern population came from Ynnan Wenshan, Fujian Nanping, Guizhou Guiyang ; There were 4 provenances found from Northern population in groupⅡ, which came from Beijing Miyun, Neimenggu Wulashan, Liaoning Chaoyang, and Xinjiang Yili; Totally 3 provenances were clustered in groupⅢ, Among them, 1 originatel from Sichuan Xichang, 1 from Hubei Jingmen, and the last one is from Henan Qunshan; The groupⅣcame from 7 provinces of the Central Chian, including Gansu Weixian, Jiangsu Tongshan, Shanxi Huangling, Shanxi Shilou, Ningxia Yinchuan, Hebei Yixian, Zaozhuang and Junan of Shandong Province; The groupⅤcame from Shandong Province, including 8 provenances, they are Feixian provenance, Qufu provenance, Tai’an provenance, Boshan provenance, Anqiu provenance, Pingdu provenance, Pingyin provenance and Liaochen provenance.The Mantel test showed: There was a positive correlation between geographical distance and genetic distance (r=0.288 6,P = 0.093 0). The results from UPGMA cluster analysis for four region groups showed that the Southern group and the Central China group was clustered together firstly and the Northern group were clustered together, at last the Shandong Province group were clustered together. This indicated further that the similarity between the Southern group and the Central China group was highest and genetic relationship was closest.更多还原