基于松属不同树种EST序列的马尾松SSR引物开发

利用松属5个树种的EST序列,分别树种查找SSR位点,采用Primer3．0软件进行引物设计。设计的5个树种的EST-SSR引物中：所占比例最高的均为三核苷酸重复,其次是六核苷酸重复,两者之和都达到了64％以上;四核苷酸重复所占比例均为最低。从设计的引物中分别不同树种各选取20对进行引物合成和通用性检测以及马尾松群体检测,结果表明：松属5个树种的EST序列开发的马尾松SSR引物,其在马尾松中的扩增成功率为60％-80％;最高的是辐射松,最低的是海岸松,平均为72％。通用性最好的为辐射松,属内的通用性为94％;属间和科间通用性也分别达到75％和44％。不同树种序列来源的EST-SSR引物,其扩增成功率在10％-30％;最高的是北美短叶松,其次是辐射松,最低的是扭叶松和火炬松;平均为17％。     

利用RAPD和ISSR分析百日草自交系间的种质遗传多样性

首次运用RAPD与ISSR技术分析了百日草20个自交系的种质遗传多样性.结果表明：从供试材料中147条RAPD引物和44条ISSR引物筛选到具有多态性的RAPD引物12条,ISSR引物9条.RAPD引物共扩增出147条带,多态性位点数为100,多态性位点比率为68.03%,平均多样性指数为0.3559,有效等位基因数为1.4169;ISSR引物共扩增出128条带,多态性位点数为97,多态性位点比率为76.38%,平均多样性指数为0.4013和有效等位基因数为1.4728.试验表明,RAPD、ISSR及两种分子标记整合后的结果较为相似,呈极显著的正相关.聚类结果将20个自交系基本分为2类：一类群来源虹越种子集团公司;另一类群来源甘肃酒泉种子集团公司,分类与原产地相关.RAPD与ISSR标记是研究百日草种质遗传多样性有效的工具,在分析百日草的亲缘关系特别是系谱关系时,ISSR是一种多态性和重复性优于RAPD的分子标记.两种分子标记整合后既能鉴定出自交系的系谱先后关系,也能反应来源相同自交系的亲缘关系.     

用过氧化物同功酶分析鉴定松属种子的真实性

对松属６个树种种子过氧化物同功酶谱带进行分析研究，结果表明，其过氧化物同功酶谱带的数量及相对迁移率都具有特异性．对于这类种子以及幼苗形态相似而不易区别的树种，这种方法，可以作为鉴定种子真实性的指标．     

日本落叶松EST-SSR标记挖掘及特征分析

基于日本落叶松转录组测序(RNA-seq)结果,利用SSRIT工具查找SSR位点,检测到1 788个EST-SSR位点,平均长度是17.5 bp。进一步统计分析发现在落叶松中重复类型SSR位点出现次数最多的是六、三核苷酸,其余依次是五、二、四核苷酸重复类型;同时检测到656种重复基元类型,种类丰富。用Primer Premier 5.0设计500对引物,随机合成102对,以红豆杉科、柏科、松科材料为模板进行检测,结果显示在红豆杉科和柏科里的扩增率分别为95%、50%;在松科不同属的通用性为落叶松属99.01%、黄杉属67.4%、雪松属63.8%、冷杉属67.4%、云杉属70.2%和松属75.7%。利用101对SSR引物对7种不同落叶松品种亲缘关系进行了分析,获得79个多态性位点,在遗传相似系数0.69处将其区分为4个类群。     

薄壳山核桃品种亲缘关系分析与指纹图谱构建

[目的 ]基于荧光SSR标记结合高通量毛细管电泳技术,建立一种快速、高效、稳定、准确的薄壳山核桃基因分型体系,用以分析各品种间亲缘关系并构建指纹图谱,旨在为薄壳山核桃品种鉴定与新品种保护提供理论依据,也为薄壳山核桃种质资源评价、品种选育与推广提供有益参考。[方法 ]选取薄壳山核桃及其近缘种54对SSR引物进行初筛,最终确定10对标记合成荧光引物用于后续分析。基于毛细管电泳技术对25个薄壳山核桃品种进行基因分型,利用软件统计位点数据并计算各个位点的等位基因数(A)和多态信息含量(PIC)。利用SSR标记间的相互组合构建薄壳山核桃不同品种的指纹图谱。通过等位片段的转化对薄壳山核桃品种进行聚类,并分析其亲缘关系。[结果 ]10对荧光SSR标记共扩增出68条等位片段,平均为6.8个;位点Cc19最多,有12个等位基因,位点BFU-Jr19最少,有3个等位基因。多态信息含量(PIC)变化范围为0.291 0～0.843 5(平均为0.588 3)。优选的4对核心引物Cc19、PM-GA31、PM-CIN4和PM-GA41组合能完全区分25个薄壳山核桃品种。25个品种遗传相似系数在0.62～0.99之间,并聚类成2个大的类群,部分亲缘关系较近的品种聚类在一起,部分品种聚类不能与遗传背景完全对应。[结论 ]与传统的显性标记及聚丙烯酰胺凝胶电泳分型技术相比,荧光SSR标记结合毛细管电泳技术构建薄壳山核桃品种指纹图谱切实可行,通量大且速度快,结果稳定可靠,通过标记组合可有效的对不同品种进行区分。在品种亲缘关系分析中,建议增加杂交亲本数量,并在全基因组范围内选取一定数量效率更高的标记,以便最大程度地揭示薄壳山核桃品种间亲缘关系的真实水平。     

松树脂溃疡病菌及其近缘种AFLP分析

利用AFLP分子标记技术对松树脂溃疡病菌及其近缘种进行DNA指纹分析,筛选出10对多态性较高的引物.上述引物在供试的9种17个菌株中共扩增出298条带,其中有多态性带283条,多态性位点百分率为94.97%.每对引物产生的多态性位点百分率在89.29%-100%之间,表明松树脂溃疡病菌及其近缘种间在DNA水平上存在广泛变异.根据AFLP标记结果计算松树脂溃疡病菌及其近缘种间的相似系数,对它们的亲缘关系进行定量描述.聚类分析结果表明,菌株间的聚类与生物学种分类的结果一致.分析引物E-AT/M-CAA绘制的镰刀菌AFLP指纹图谱遗传多样性,确定李瑟组镰刀菌各交配群的特异带、差异带,区分供试的7个交配群.     

基于ISSR的日本落叶松2代优树遗传多样性研究

为分析80株日本落叶松2代优树间的遗传多样性,本次试验利用ISSR分子标记的方法对采自辽宁省抚顺市清原县大孤家国营林场的80个样本进行了DNA的提取与检测、ISSR反应体系的优化建立及引物筛选、ISSR扩增及电泳检测。结果发现:筛选出的8个ISSR引物共扩增出281条DNA条带,全部为多态性条带,多态性百分率为100%,平均每条引物扩增的多态性条带数为35.1。ISSR扩增结果的遗传相似性在0.8479~0.9142之间,其中编号为C46和C94的遗传相似系数最低,表明二者亲缘关系最远;编号为C96和C97的遗传相似系数最高,表明二者亲缘关系最近。基于日本落叶松ISSR遗传相似性系数进行聚类,当相似系数为0.856时可将80个日本落叶松样本划分为5个类群。     

湛江高桥红海榄群落样方遗传相似性特征研究

遗传相似性特征及分布的研究对保护生物多样性、维护森林生态服务功能有积极的意义。选择广东湛江高桥红海榄Rhizophora stylosa纯林的400 m²固定样地，采用简单重复序列间区扩增ISSR和相关序列扩增多态性SRAP两种分子标记技术开展研究。用筛选出的10个ISSR引物和14个SRAP引物组合进行PCR扩增，分别共扩增出73个和111个清晰可读的位点，多态性位点数分别是60和88，多态性位点百分率分别是82.2%和79.3%。样方红海榄遗传相似系数变异范围为0.327～0.741，平均值为0.563，差值为0.414。在相似系数0.55的水平下，分为3个类群，遗传相似度为第一类群>第二类群>第三类群，遗传相似变动度第三类群>第二类群>第一种类群。在相似系数0.60的水平上，可分为8个类群。相似系数0.55的水平下红海榄聚集度越强，遗传相似度越高，遗传相似变动度越低。高桥红海榄群落样方分属于不同的类群，在遗传上有差异性，存在一定的基因交流；亲缘关系为远和较近的分布区间，遗传相似度和变动度中等，第三类群为优势种。初步证明红海榄聚集度与遗传相似度成正比，与遗传相似变动度成反比。     

利用ISSR标记对12种五针松亲缘关系的研究

采用ISSR 标记技术,对12 种五针松的亲缘关系进行分析。利用筛选出的12 个ISSR 引物共检测到117 个位点,多态条带比率(PPB)在9.40%～33.33 %之间,遗传分化最高的是偃松,最低的是柔枝松。12 种五针松的基因多样性(Ht)为26.21%,其中种内基因多样性(Hs)为7.66%,种间基因多样性(Dst)为18.55%,五针松种间变异占总变异的70.78%。遗传距离聚类,将12 种五针松分为2 个类群,第一类群包括乔松、华山松、海南五针松、华南五针松、北美乔松、山白松和云南五针松;第二类群包括美国白皮松、偃松、、柔枝松、西伯利亚红松和红松。图1 表3 参6。     

新疆枣资源亲缘关系的ISSR和RAPD分析

利用RAPD和ISSR标记对24份新疆主产区枣资源进行分析。9条RAPD引物共在75个位点获得了扩增,多态性位点比例为62.7%;8条ISSR引物共有48个位点获得了扩增,多态性位点比例为56.3%。基于RAPD和ISSR标记,利用UPGMA法分别构建了24份枣资源的亲缘关系图谱。结果表明,RAPD和ISSR的聚类结果相似但是不完全相同,在大类的分析上2种方法的聚类结果表现出差异,但是在细节的表现上趋向一致;综合2种方法的聚类结果,以相似系数0.80为标准,将供试的24份新疆枣种质划分为4大类群。     

Variable dormancy in seed of Pinus contorta

Seeds of lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.) collected southwest of Grande Praire, Alberta at three different times and altitudes between August 20, 1984 and August 15, 1985 showed variable responses to stratification. The responses were related to seed ripeness, as judged through evaluations of cone moisture contents, and with the length of time that the cones had remained on the trees. Unripe seeds started to lose germination in the third year after pollination when on the tree. Mature seeds which lost germination by late fall 1984 regained it by the next fall, while most unripe seeds did not. Immature seeds increased their need for stratification over time while mature seeds did not. Seeds should be left on the tree until the end of the third year to insure maximum viability because they do not reach maturity until then.     

红松不同种源种子储藏后对苗木生长的影响

红松是东北地区的重要造林树种之一,主要依靠培育实生苗造林.育苗中经常采用陈年种子,经对6个种源、7个批次的红松种子进行1年生苗木生长情况的测定,认为红松不同种源种子的储放时间长短与苗木生长的变化关系差异极显著;结果表明,有好的种源做保证,有利于苗木正常地生长.     

Viability of litter-stored Pinus taeda L. seeds after simulated prescribed winter burns

Stratified loblolly pine (Pinus taeda L.) seeds were placed at three depths in a reconstructed forest floor and subjected to simulated prescribed winter burns. Within the forest floor, pine seeds were placed at the L/upper-F interface, upper-F/lower-F interface, and lower-F/mineral-soil interface. Wind was generated by electric box-fans. Seeds that survived the burns were transferred to moist sand flats for 30-day germination tests. About 96% of seeds placed at the two upper layers in the forest floor were either destroyed by the fires or failed to germinate. Germinative capacity of seeds placed at the lower-F/mineral-soil interface averaged 79% as compared to 97% for unburned control seeds.     

Effects of Seedbed Substrate on Moisture Conditions and Germination of Pinus sylvestris Seeds in a Clearcut

The objective of the investigation was to describe moisture conditions and germination of Scots pine (Pinus sylvestris L.) seeds in different seedbed substrates (feather mosses, mineral soil, humus and ground organic material) with and without irrigation during 2 weeks, in a clearcut in northern Sweden. Without irrigation no seeds germinated, which could be explained as an effect of the low moisture conditions of the seedbed substrates. This was shown in the seeds' low moisture content, with highest values in mineral soil, 10-20%, and for the other seedbed substrates 5-15%. The moisture conditions were highest during the night and lowest in the afternoon. With irrigation, moisture conditions improved (seed moisture content 20-50%) and diurnal changes were moderated. Seeds germinated in all substrates with most seedlings in mineral soil (60% of sown germinable seeds). The moisture conditions of the seedbed substrates proved to be in poor agreement with the seeds' requirement for germination, especially during warm weather with no precipitation.     

Germination and early seedling growth of Pinus densata Mast. provenances

We studied seed germination and early seedling growth of Pinus densata to explore the range of variability within the species and to inform afforestation practices.Phenotypes were evaluated at a forest tree nursery under conditions that support Pinus yunnanensis, one of the presumed parental species of P. densata. Seeds were collected from 20 open-pollinated trees within each of eight autochthonous populations representing the natural distribution of P. densata in China to assess variation in germination traits and early seedling growth, and to examine the relationships among these traits. Results showed that seeds from all populations germinated and seedlings established successfully. There were significant differences among populations in 13 of 14 traits evaluated. Seed germination and early seedling growth were strongly related to seed size and seed weight. Bigger seeds germinated earlier and faster than small seeds, and seedling size was positively correlated with seed size. Some germination traits were strongly and significantly correlated with climatic variables associated with the provenance of the studied populations. Based on these observations, we conclude there were large, significant, and biologically important differences among P. densata populations in seed germination and seedling growth traits. The observed variability probably reflects a high degree of adaptive differentiation among populations that is likely to be relevant for future afforestation.     

Aggressiveness of Fusarium oxysporum and F. verticillioides isolates on stone and scots pine under greenhouse conditions

Scots pine (Pinus sylvestris) and Stone pine (Pinus pinea) are two important species used in re-forestation that are subject to damage by damping-off fungi in forest nurseries. Twenty-two isolates of Fusarium oxysporum and F. verticillioides from diseased seedlings of eight different hosts were tested for aggressiveness on seeds and seedlings of both pine species, including the effects on seedling emergence and mortality. Scots pine was more susceptible to damping-off than Stone pine, as indicated both by reduced seedling emergence and elevated seedling mortality. The impact of F. oxysporum and F. verticillioides on seeds and seedlings did not differ significantly for either pine species. Our findings support previous studies that found that these are damping-off pathogens on the studied pines. Whereas most isolates proved to be pathogenic, some isolates of both Fusarium species showed to be non-pathogenic.     

4种榆科珍稀树种的种苗形态研究及其栽培效果

对琅琊榆、糙叶榆、长序榆、栗叶榆等4种榆科珍稀树种从种子(翅果)到幼苗的形态做了探索性研究,对其形态通过观察、记载而绘制成图。此4种榆科树种的苗木定植3年后经评价,表明在福建来舟林场已引种成功。     

Correlation of seedling size and branch number with disease resistance of Pinus thunbergii seedlings to Bursaphelenchus xylophilus

Japanese black pine (Pinus thunbergii) seedlings resistant to pine wood nematode (PWN; Bursaphelenchus xylophilus) are routinely selected in Japanese field inoculation trials. Correlations between morphological factors (such as height, stem diameter at ground level and number of branches on seedlings) and disease resistance were examined to improve the production efficiency of 1‐year‐old black pine seedlings for inoculation. Family relatedness and environmental conditions strongly affected seedling resistance; accordingly, logistic regression analysis was used to separate effects of these two variables. Height and stem diameter at ground level significantly correlated with disease resistance in seedlings inoculated with PWN. Because (a) interactions between stem diameter at ground level and environmental condition were significant and (b) height did not interact with any other factor, it was concluded that height of 1‐year‐old Japanese black pine seedlings independently correlated with PWN resistance. Thus, field inoculation tests should use tall seedlings to achieve enhanced survival rates.     

Heat-shock response of Pinus and Picea seedlings

The effect of a short period of elevated temperature, or heat shock, on protein synthesis was investigated in 2-day-old seedlings of jack pine (Pinus banksiana Lamb.), loblolly pine (P. taeda L.), lodgepole pine (P. contorta Dougl.), black spruce (Picea mariana (Mill.) BSP), and white spruce (P. glauca (Moench) Voss.). In all species, heat shock led to increased [(35)S]methionine incorporation into heat-shock proteins (hsp's) with molecular masses of 83 and 72 kDA. Heat shock also induced synthesis of several low molecular weight proteins that were absent from control seedlings. The low molecular weight hsp's produced by pine seedlings had molecular masses of 27, 24.6, 20.5 and 17.5 kDa, whereas those produced by spruce seedlings had molecular masses of 27.2, 19.8, 18.4, 17.2 and 16.0 kDa. All of the low molecular weight hsp's showed isoelectric variants. Heat shock led to increased [(35)S]methionine incorporation into a group of low molecular weight hsp's that were also present in control seedlings.     

A mycobacterium isolated from tissue cultures of mature Pinus sylvestris interferes with growth of Scots pine seedlings

We isolated a rapidly growing, pigment-producing mycobacterium from senescent tissue cultures derived from mature Scots pine (Pinus sylvestris L.). The bacterium was found in three senescent suspension cultures and in a senescent protoplast culture. Growth of Scots pine cells had ceased in all of these cultures. Exogenous contamination was eliminated by rigorous surface sterilization of the buds with hypochlorite before aseptic removal of the bud scales. Based on biochemical and physiological properties and DNA sequence comparisons, the isolated mycobacterium did not belong to any known species. Its sequence most closely resembled those of Mycobacterium obuense (97%) and M. aichiense (96%). Tissue browning was frequently observed in callus or suspension culture of Scots pine. Because the effect of the mycobacterium on growth of undifferentiated tissues that were browning was difficult to evaluate, we applied the bacterium to Scots pine seeds in aseptic conditions. Seedlings grown in the presence of the mycobacterium had shorter hypocotyls than control seedlings and seedlings cocultivated with a Pseudomonas strain known to be harmless to plants. However, hypocotyl growth of seedlings cocultivated with another mycobacterium, M. chlorophenolicum, was similar to that observed in the presence of the isolated mycobacterium. Phenylalanine ammonia-lyase activity of seedlings cocultivated with the mycobacterium isolate was significantly higher than that of control seedlings or seedlings cocultivated with M. chlorophenolicum or Pseudomotnas fluorescens. We believe that this is the first report of the isolation of mycobacteria from tissue cultures of a tree. Our finding that the mycobacterium may interfere with the growth of Scots pine in vitro warrants further study.