【作者】 赵罕；

【导师】 郑勇奇；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2012， 博士

【摘要】 白皮松（Pinus bungeana Zucc.）是我国特有的乡土树种，也是重要的园林绿化和荒山造林树种，具有较高的经济和生态价值。由于其长期的过度利用，天然林资源已遭到严重的破坏；目前白皮松天然群体大多保存在生境条件恶劣、人迹罕至的山顶及其附近，群体间隔离和片段化严重，群体内遗传多样性保存面临严峻的挑战。本研究在对白皮松的天然分布区及生境特征调查的基础上，对不同区域内群体的更新及种群结构进行了评价，然后利用分子标记和种源试验两种手段对白皮松不同群体的遗传变异状况进行了分析；最后根据各群体的多样性分布规律、更新状况及所面临的濒危程度制定了白皮松多样性保存的策略。主要结果如下：1）通过查阅资料及实地勘察，白皮松天然分布主要集中在暖温带及北亚热带山区；白皮松的天然分布范围主要在北纬30°52’~38°25’，经度的范围主要在103°36’~115°17’。依山势走向，主要分布在太行山、吕梁山、中条山、秦岭及大巴山区；其中，大巴山区为白皮松分布的零星区域。白皮松分布区的最南界应处于湖北省的巴东县，而东南分布区的界限应该为湖北省的巴东、远安、南漳到丹江一线。白皮松垂直分布区受地形影响较大，在所调查地点中，秦岭东部的栾川、洛宁、蓝田主要分布在海拔758~1450m之间。太行山区的沁阳、陵川、榆次主要分布在海拔800~1410m之间。大巴山区的西乡、丹江口、南漳、远安主要分布在海拔443~1246m之间。吕梁山区的蒲县主要分布在1200~1360m之间。中条山区的翼城主要分布在1300~1450m之间。秦岭西部的康县、成县、天水等地主要分布在海拔1052~1517m之间。白皮松主要呈块状或片状分布，群体多以纯林形式存在或形成以白皮松为优势树种的混交林。但各分布群体隔离较为严重，仅集中分布在少数山区的山顶附近。2）通过对白皮松不同地点的天然更新状况研究，结果显示，白皮松在太行山、吕梁山、中条山及秦岭西侧群体更新效果相对较好；而在零星分布的大巴山区的南漳、丹江、远安等群体，其更新能力较差；白皮松在更新过程中大部分群体存在幼苗不足的现象。白皮松更新较好的地方为郁闭度相对较小的林窗或林缘，更新状况与周围伴生植被的生长状况关系密切。3）使用径级替代龄级的方法对不同地点的白皮松种群结构进行了分析，结果表明，不同地点白皮松在年龄结构上存在一定差异，同一地点白皮松龄级间也存在较大波动。大巴山区各群体年龄较大，群体以大于Ⅲ~Ⅳ龄级个体的分布较多，波动较大，种群处于成熟并且趋于衰退阶段。太行山、吕梁山和中条山区域群体年龄相对较小；主要分布在Ⅰ~Ⅲ龄级之间；种群暂时处于稳定的状态，但是群体缺乏后续的小龄级个体，更新存在一定的风险。秦岭两侧群体结构变化较大；群体主要分布在Ⅰ~Ⅵ龄级，除康县外其他群体均缺乏Ⅰ级更新幼苗。4）使用8个不同群体的白皮松单株，对松属其他物种已发表的309对SSR引物进行筛选，结果表明，在白皮松中能扩增出条带的引物有86条，占27.83%，其中多态性引物有7对，占2.27%，引物在白皮松中的通用性相对较小。5）对白皮松24个群体的Hardy-Weinberg平衡检验表明，只有少数的群体在7个位点中均达到平衡状态，大多数群体在一个或多个位点处于不平衡状态。白皮松大部分群体存在一定程度的近交现象，其中位点Y7只在群体P10、P11、P12中出现杂合子，其他群体均为纯合体。群体P1、P2、P3、P5、P15、P17、P18、P19、P21、P23，在其他位点也均出现杂合子完全缺失的现象。白皮松大部分群体均受到了遗传漂变、突变、迁移和选择等进化因子干扰。6）使用7对EST-SSR引物在21个白皮松天然群体的663个单株中共检测到14个多态性位点。有效等位基因数（Ne）、Shannon’s信息指数（I）、观测杂合度（Ho）、期望杂合度（He）、Nei’s多样性指数（Nei’s）平均值分别1.3097、0.3127、0.2214、0.1994、0.1963。在三个人工群体192个单株中共检测到11个多态性位点。有效等位基因数（Ne）、Shannon’s信息指数（I）、观测杂合度（Ho）、期望杂合度（He）、Nei’s多样性指数（Nei’s）平均值分别1.2263、0.2466、0.1935、0.1511、0.1487。相对于人工群体，白皮松天然群体Ne、I、Ho、He、Nei’s等多样性参数均较高。7）使用Ne、I、Ho、He、Nei’s等遗传多样性参数对不同群体白皮松多样性进行比较，结果表明，各多样性参数在群体间的排序基本相同，白皮松多样性最高的地区均为秦岭西南侧的康县和大巴山南侧的远安县，其他多样性相对较高的群体也分别位于秦岭的西侧和大巴山区；而位于秦岭东侧的河南沁阳、栾川、洛宁及太行山区、吕梁山区、中条山区的群体则均处于多样性较少的区域。白皮松多样性水平在分布区内呈规律性变化，多样性分布的中心区域主要在西部、南部，具有从西向东，从南向北依次减少的趋势。8）白皮松群体间遗传分化分析表明，白皮松群体间的遗传分化Fst平均为0.2152，基因流Nm平均为0.9119，群体间基因交流较少，群体分化较大。在遗传相似性距离为0.05处可将白皮松分为4个类群。第一个类群为P14（甘肃成县）；第二个类群为P15（甘肃徽县）群体；第三个类群包括P10（天水左家A）、P11（天水左家B）、P13(甘肃两当)、P16（甘肃康县）、P22（湖北远安）；其他群体为第四个类群。9）白皮松在北京和河南卢氏两地点的种源试验表明，白皮松适应性主要受栽培地点与采种地点距离的影响，保存率随试验地点与采种地点距离的增加而呈现出规律性减小。白皮松生长性状主要受地理分布的影响，生长速度在分布区内总体呈现出西南-东北的分布趋势。各试验点生长较快的均为天水（103.42cm、96.75cm）和两当（99.57cm、87.75cm）种源，其次是蓝田（96.61cm、85.85cm）、北京（93.17cm、81.89cm）和孝义种源（88.24cm、75.88cm）。10）通过白皮松遗传变异规律及各群体更新状况分析，制定了白皮松不同群体的保存策略。秦岭中西段群体白皮松多样性水平高，遗传分化较为剧烈，应该重点进行保存，适当增加保存群体的数量。该区域群体具有一定的天然更新能力，应主要采取原地保存的形式。大巴山群体多样性水平相对较高，但种群数量较少，更新状况不良，种群具有较大野外灭绝的风险，应采取以异地保存为主的措施。太行山、吕梁山、中条山群体遗传结构相对单一，分化较小，更新相对较好，应采取以原地保存为主的形式。秦岭东段群体遗传结构相对单一，但濒危程度较高，应以异地保存为主。更多还原

【Abstract】 Pinus bungeana is an endemic species to China, with high economic and ecologicalvalues for landscaping and afforestation. Owing to long-term over-exploitation, natural forestresources of Pinus bungeana has been severely damaged at present and natural populationsmainly found in the top of hills in poor habitats usually isolated and fragmented. Geneticdiversity in populations is facing tough challenges. On the basis of survey of the naturaldistribution and habitat, natural regeneration and population structure were evaluated; then,genetic variation of different groups were analyzed using molecular markers and provenancetest. Finally, conservation strategies were formulated according to the distribution pattern ofgenetic diversity, regeneration status and the ecological risks. The main conclusions are asfollows:1, Through literature review and field survey, natural distribution of Pinus bungeana weremanly in the Mountains of warm temperate and northern subtropical zone; the range of naturallatitudinal was between30°52’~38°25’ and longitudinal was between103°36’~115°17’. Thedistribution was mainly in the Taihang Mountain, Lvliang Mountain, Qinling Mountain andDaba Mountain and distribution in Daba Mountain was scattered. Southern boundary of Pinusbungeana was found in Badong county, Hubei Province. Southeast boundary of the distributionwas found from Badong county, Yuan’an county, Nanzhang county to Danjiangkou city inHubei Province. Vertical distribution of Pinus bungeana was largely fluctuated due to theterrain. In counties of Luanchuan, Luoning, Lantian in eastern Qinling Mountain, Pinusbungeana were mainly distributed in elevations between758~1450meters. In counties ofQinyang, Lingchuan, Yuci in Taihang Mountain, Pinus bungeana were mainly distributed inelevations between800~1410meters. In counties of Xixiang, Nanzhang, Yuan’an and city ofDanjiangkou in Daba Mountain, Pinus bungeana were mainly distributed in elevationsbetween443~1246meters. In Pu county in Lvliang Mountain were mainly distributed inelevations between1200~1360meters. In Yicheng county in Zhongtiao Mountain were mainlydistributed in elevations between1300~1450meters. In counties of Kang, Cheng and Tianshuicity in Qinling Mountain, Pinus bungeana were mainly distributed in elevations between 1052~1517meters. Fragment was the main shape in distribution of Pinus bungeana andpopulations exist mainly in mountaintop and nearby place in forms of pure forest or dominantspecies in mix forest.2, Study of natural regeneration in different populations showed that: natural regenerationwere better in Taihang Mountain, Zhongtiao Mountain Lvliang Mountain and the west side ofQinling Mountain but poor in areas of Nanzhang county, Yuan’an county, Xixiang county andDanjiangkou city in Daba Mountain; shortage of seedlings exist in most of the populations andthe better regeneration place within population were mainly in forest gap and forest edge.Regeneration was closely associated with growth status of other vegetation.3, Population structure was analysed using method of diameter classes instead of ageclasses, the result showed that: Age classe structure was different in different locations and alsofluctuated in the same location. In Daba Mountain, individuals were older and distributedmainly in Ⅲ~Ⅳ age classes. In this region, populations were in a stage of mature and tend todecline. In Taihang Mountain, Lvliang Mountain and Zhongtiao Mountain, individuals wasyoung and mainly distributed between Ⅰ~Ⅲ age classes. Populations in this region werestable temporarily, but have some risks for lack of regeneration seedlings. In Qinling Mountain,the structure of populations changes larger, individuals mainly distributed betweenⅠ~VI ageclasses and lack of first class seedlings.4,309pars of SSR primes of other species in Pinus were screened using8individuals indifferent populations. The result showed that:86pars of primes can be successfully amplifiedout of309pairs of SSR primers, accounting for27.83%, and7were polymorphic primersaccounting for2.27%; universal primers of other Pinus were relatively low in Pinus bungeana.5, The Hardy-Weinberg equilibrium test indicated that most of the populations were in anunbalanced state at one or more loci and inbreeding existed in most of the populations. At locusY7, heterozygous were exist only in P10, P11, P12, and there were also heterozygous completeabsence in other loci. Most of the populations were subjected to effects of genetic drift,mutation, migration and the choice of evolutionary.6,14polymorphic loci were detected in663individuals among21natural populationsusing7pairs of EST-SSR primes. Mean of Ne、I、Ho、He、Nei’s were1.3097、0.3127、0.2214、0.1994、0.1963respectively.11alleles and14genotypes were detected in192artificial populations. The average of Ne、I、Ho、He、Nei’s were1.2263、0.2466、0.1935、0.1511、0.1487respectively. Compared with artificial populations, the natural populations have ahigher genetic diversity.7, Genetic diversity of different populations were compared using parameters of Ne、I、Ho、He、Nei’s. The result showed that: The ranking of populations was roughly the same usingdifferent genetic diversity parameters. P16in southwestern part of Qinling Mountain and P22in southern part of Daba Mountain have the highest genetic diversity, other populations withhigh genetic diversity were also in the west of Qinling Mountain and Daba Mountain. Thepopulations with low genetic diversity were P17、P18、P19in the east of Qinling Mountain andpopulations in Taihang Mountain、Lvliang Mountain and Zhongtiao Mountain. Centers ofgenetic diversity were mainly in the west and the south of the distribution and the geneticdiversity in distribution tended to reduce from west to east and from south to north.8, Analysis of genetic differentiation showed that: The average of Fst and Nm were0.2152and0.9119respectively, gene flow was less and genetic differentiation was high amongpopulations. Populations can be divided into four groups at the genetic distance of0.05. Thefour groups were P14; P15; P10, P11, P13, P16, P22and the remaining populations.9, Provenance trials in Beijing and Lushi of Henan Province showed that: Adaptability ofPinus bungeana was affected mainly by the distance between the growing site and the origin ofseed source and decreased in a regular pattern with increase of the distance. Growth trait ofPinus bungeana was affected mainly by the geographical distribution and changed in trend ofnorth east to south west from large to small. Growth rates from high to low were Tianshuicounty（103.42cm、96.75cm）, Liangdang county（99.57cm、87.75cm）, Lantian county96.61cm、85.85cm）, Beijing（93.17cm、81.89cm）and Xiaoyi county（88.24cm、75.88cm）.10, Conservation strategy was drafted according to the genetic variation pattern andregeneration of Pinus bungeana. In west-middle section of Qinling Mountain, the populationsof Pinus bungeana have high diversity and evident genetic differentiation, should be protectedwith priority and increase the number of populations appropriately for conservation.Populations in this region have the ability of natural regeneration, so in situ should be the mainconservation form. The Daba Mountain populations have the high level of genetic diversity;but the population sizes were small and the condition of natural regeneration was poor; so the populations in this region has the risk of extinction and ex situ should be the main conservationform. The populations in Taihang Mountain, LvLiang Mountain, and ZhongTiao Mountainhave simple genetic structure and low level of genetic differentiation, but regeneration wasrelatively good; so, in situ should be the main conservation form. Populations in the east of theQinling Mountain have simple genetic structure and regeneration was poor, facing high risk ofextinction, so ex situ should be the main conservation form.更多还原