白桦种群间木材纤维性状的表型变异与分子标记的遗传多态性

以东北地区的5个白桦天然种群为研究材料,在木材纤维形态性状测定的基础上,采用ISSR、RAPD分子标记研究了5个种群的遗传变异以及种群间纤维形态性状与DNA分子标记的相关性.结果表明:白桦天然林成熟材(18～31年生)的木材纤维长度在种群间差异不显著,纤维宽度和长宽比差异显著,帽儿山种群的木材纤维形态性状最好,汪清最差;利用ISSR和RAPD 2种标记检测5个白桦种群的遗传多样性,所得的研究结果基本一致,多态位点比率最高的为辽宁新宾种群,其次是帽儿山和汪清,而塔河和金山屯最低;ISSR标记的AMOVA分析表明白桦种群间的遗传变异占总遗传变异的10.82%;RAPD标记的AMOVA分析表明白桦种群间的遗传变异占总遗传变异的12.94%,研究结果均显示白桦的遗传变异主要发生在种群内部.利用2种标记技术的种群聚类结果基本一致,这一结果与基于木材纤维性状的表型聚类基本一致, 而且种群间遗传距离和地理距离之间存在一定的相关性.     

12个油茶良种的SCAR分子标记鉴别

以12个长林系列油茶Camellia oleifera良种为试材,基于特异性SCAR分子标记的开发来鉴别油茶品种。结果表明:基于4条SAPD引物、24对SRAP引物和15个ISSR引物分别对12个油茶品种进行扩增,得到了21条多态性片段,包括7条n SAPD,10条SRAP和4条ISSR片段。2条n SAPD,7条SRAP和2条ISSR多态性片段被成功转化为了11个稳定可靠的SCAR标记。长林油茶23号、长林26号、长林53号、长林56号和长林166号有1个SCAR标记,长林3号和长林55号各有2个SCAR标记,长林21号有4个SCAR标记,长林18号有6个SCAR标记,长林4号、长林27号和长林40号没有SCAR标记。这些SCAR标记可作为品种特异性的DNA指纹,辅助用于12个长林系列油茶良种的鉴别。     

山杨材性遗传与性状相关的研究

通过对山杨（Ｐｏｐｕｌｕｓｄａｖｉｄｉａｎａ）优树及４年生山杨子代林生长和材性相关性的研究结果表明：山杨木材密度与纤维性状、生长性状相互独立,纤维长度、宽度、纤维长宽比之间相关显著。纤维性状与生长性状具有一定的相关性,其相关主要是由纤维长度与材积相关决定的。     

6年生大花序桉不同种源木材纤维特性的差异分析

以广西钦廉林场6年生9个种源的大花序桉为材料,通过解析木法,测量其木材的纤维长度、纤维宽度、纤维腔径,比较9个种源6年生大花序桉的木材纤维特性。结果表明,种源间纤维长度、宽度、长宽比存在显著差异;种源的纤维形态(宽度除外)与树高、胸径生长性状相关关系不显著,据此可对各性状进行独立选择,可望培育出木材性状好又速生的种源;树高、纤维宽度与种源地的经纬度因子存在显著的相关性;9个大花序桉种源作为中大径材培育有更大的发展空间。     

桤木优良造纸材改良潜力研究

对桤木优树树高、胸径、材积、单株纤维量、纤维长、纤维长宽比、纤维含量、木材密度的变异规律和各性状间的相关关系进行了研究。结果表明:性状变异系数规律是生长性状大于材性性状;各性状间没有显著相关,各性状可独立改良;材积、纤维含量、木材密度对单株纤维量的贡献率分别为67.47%、17.02%、15.51%;各性状的改良潜力是:材积可达1倍以上,胸径48%,树高18%,木材密度可提高10%以上,纤维含量可提高4%以上,纤维长度、长宽比分别可提高7%、12%以上,生长与材性联合改良的效益在30%以上。     

“松材线虫SCAR标记与系列分子检测技术及试剂盒研制”项目通过鉴定

2006年5月，由南京林业大学森林资源与环境学院叶建仁教授主持完成的“松材线虫SCAR标记与系列分子检测技术及试剂盒研制”通过了江苏省科技厅组织的科技成果鉴定。该项研究对松材线虫与拟松材线虫特异片段进行了系统筛选，共获得了7个松材线虫DNA特异片段与5个拟松材线虫DNA特异片段，为松材线虫与拟松材线虫的分子鉴定奠定了基础。首次将2个松材线虫与2个拟松材线虫的DNA特异片段成功转化为SCAR标记，丰富了松材线虫与拟松材线虫分子标记方法。首次采用SCAR标记方法成功构建了松材线虫检测试剂盒，PCR检测过程仅需2．2小时。首次成功标记了一个可用于检测松材线虫的非放射性探针DIG-F2／R1。用该探针对线虫基因组DNA点杂交，松材线虫均表现有较强的杂交信号，而拟松材线虫、霍夫曼尼伞滑刃线虫、大核滑刃线虫、长尾属线虫等均无杂交信号。     

日本落叶松纸浆材优良家系多性状联合选择

以12年生日本落叶松自由授粉家系子代测定林为研究对象,对生长、干形和材性性状进行相关分析、通径分析及间接选择研究,利用综合选择指数法开展了日本落叶松纸浆材优良家系选择,并为湖北等亚热带高海拔山区筛选出一批生长、干形和材性兼优的家系,最后提出了日本落叶松纸浆材选育指标及优良家系利用途径。研究表明:生长性状与纤维长度之间存在显著正相关关系,与壁腔比之间存在显著负相关关系,与木材密度、1%NaOH抽出物、综纤维素含量间相关不显著;木材密度与主枝长之间,纤维长度与主枝长、主枝粗和新枝粗之间,晚材壁腔比与主枝粗、主枝长、皮厚、冠幅、新枝数之间,1%NaOH抽出物与主枝长之间存在显著正相关关系。各控制性状对单木材积直接作用的大小依次为:胸径、树高、主枝长、壁腔比、主枝粗、皮厚、冠幅、晚材纤维长和早材纤维长,其中胸径和树高2个性状对单株材积性状的直接作用占材积总变异的63%,虽然冠幅、壁腔比等性状对单木材积的直接控制作用很小,但这些性状通过胸径对单木材积具有很大的间接遗传控制作用,由这9个性状组成的控制系统可以说明单木材积变异的98.66%。     

尾叶桉与赤桉正反交杂种F1代生长、材性及其化学组分与抗风性能的分析

以广东江门尾叶桉(Eucalyptus urophylla)与赤桉(Eucalyptus camaldulensis)正反析因交配杂种F_1代9年生测定林为材料,用Excel计算各杂交组合抗风指数、生长性状、材质形状以及木素、综纤素的均值;用R软件对这些性状进行单因素方差分析、相关性分析以及多性状指数综合评价。单因素方差分析表明正交与反交组合杂种的生长性状如树高、胸径、材积和冠幅等差异都不显著;而材质形状如基本密度和纤维宽度差异则较为显著,但纤维长度和纤维长宽比差异不显著;另外,正反交组合杂种的木素和综纤素含量的差异也较为显著。相关性分析表明,抗风指数与木材基本密度及纤维长呈正显著相关性,而与纤维长宽比呈负显著相关性;但抗风指数与树高、胸径、材积、冠幅、纤维宽、木素含量及综纤素含量之间的相关性都不显著;木材基本密度及纤维长与抗风指数的显著相关性为抗风优质的桉树选择提供参考。综合评价结果表明,当各变量权重相同时,正交组合中综合得分前3名的是杂交组合77、81和82;反交组合中综合得分前3名的是杂交组合50、51和29;同时还选出5株优良单株29-3、51-4、77-1、77-3及50-2,为今后大规模人工制种提供优质亲本材料。     

苹果柱型性状的SRAP分析

为了更深入研究苹果柱型基因,为苹果树型性状改良奠定基础,以舞姿与富士杂交的F1代群体为材料,利用SRAP分子标记技术与BSA法相结合的方法分析了苹果柱型性状。筛选出了1对SRAP引物组合M10E4,其在柱型池与普通型池间表现出分离,分离片段约为310bp。选用柱型与普通型的极端类型进行验证,柱型群体中多数单株都出现此片段,有2株出现交换,普通型群体中有3株出现交换,交换率为7.14%,用Mapmaker/Exp3.0软件对标记与目标性状进行连锁分析,测算遗传距离为7.7cM,并将此标记命名为M10E4-310。研究结果表明,利用SRAP技术初步找到了与柱型基因连锁较紧密的标记位点。     

SSR标记与欧洲黑杨材性性状的关联分析

对引进的92个1年生和65个4年生欧洲黑杨无性系进行材性测定,并结合171个SSR标记进行材性数量性状位点的关联分析.结果表明:1年生和4年生欧洲黑杨的木材基本密度分别为0.308 8 g·cm-3和0.355 2 g·cm-3,纤维长分别为601.3 μm和649.4 μm,纤维宽分别为21.81 μm和23.11 μm,微纤丝角分别为26.5°和26.1°.4年生欧洲黑杨苯醇抽提物、综纤维素、α纤维素以及木质素含量分别为2.32%、78.62%、41.48%和20.93%.通过SSR标记位点与性状间的关联分析,共获得与1年生材性性状关联的SSR标记位点10个,其中基本密度3个,微纤丝角5个,纤维长2个,纤维宽4个,贡献率6.43%～23.18%;与4年生欧洲黑杨材性性状关联的SSR标记位点8个,其中基本密度、纤维长、木质素各1个,微纤丝角、综纤维素含量和α纤维素含量各2个,贡献率7.41%～28.56%.     

松茸SCAR标记的建立

松茸群全球共报道15个种1变种,我国记载5个种1变种(臧穆,1990).长白山区有2种,即松茸(Tricholoma matsutake)和假松茸(T.bakamatsutake),同属于口蘑属.松茸是与松属(Pinus)树木共生的一种外生菌根菌,不仅菌肉肥厚、营养丰富、风味绝佳,而且具有抗癌作用(傅伟杰等,2005).     

与地被菊株型匍匐性连锁RAPD标记的SCAR转化

地被菊(Dendranthema grandiflorum)是20世纪80年代由陈俊愉院士首先提出的菊花新品种群(王彭伟等,1990;Chen et al.,1995),因株型低矮或匍匐、多分枝、抗性强、适应性广、成型快、覆盖能力强、着花繁密,同时具备绿化、美化、彩化和香化功能,园林应用前景广阔(于忠香,2004).     

Rapid detection of self-biting disease of mink by specific sequence-characterized amplified regions

Self-biting disease occurred in most farmed fur animals in the world. The mechanism and rapid detection method of this disease has not been reported. We applied bulked sergeant analysis (BSA) in combination with RAPD method to analyze a molecular genetic marker linked with self-biting trait in mink group. The molecular marker was converted into sequence-characterized amplified regions (SCAR) marker for rapid detection of this disease. A single RAPD marker A8 amplified a specific band of 263bp in self-biting minks, which was designated as SRA8-250, and non-specific band of 315bp in both self-biting and healthy minks. The sequences of the bands exhibited 75% and 88% similarity to Canis familiarizes major histocompatibility complex (MHC) class II region and Macaca mulatta MHC class I region, respectively. A SCAR marker SCAR-A8 was designed for the specific fragment SRA8-250 and validated in 30 self-biting minks and 30 healthy minks. Positive amplification of SCAR-A8 was detected in 24 self-biting minks and 12 healthy minks. χ2 test showed significant difference (p<0.01) in the detection rate between the two groups. This indicated that SRA8-250 can be used as a positive marker to detect self-biting disease in minks. Furthermore, the finding that self-biting disease links with MHC genes has significant implications for the mechanism of the disease.     

DNA fingerprinting of Populus trichocarpa clones using RAPD markers

Nine trees from a single, natural population of black cottonwood (Populus trichocarpa Torr. et Gray) in Alaska were screened for randomly amplified polymorphic DNA (RAPD) markers with ten different 10-base random oligonucleotide primers in order to evaluate the use of RAPD analysis for distinguishing black cottonwood clones. Nine primers amplified the genomic DNA targets; two primers were able to differentiate all clones. Eight clones were distinguished among the nine tree samples assayed. Two trees showed identical banding patterns with all primers used; therefore it is suggested that these trees are from the same clone. The RAPD fingerprinting method is simple and powerful-one primer can distinguish different clones, while the use of multiple primers reduces fingerprint similarity and resolves discrepancies.     

核桃早实基因的SCAR标记

根据与核桃早实性状连锁的RAPD标记OPB-08958序列,设计SEA-F/SEA-R和SEB-F/SEB-R2对SCAR引物。在亲本、杂交后代和栽培品种(系)中的检测结果表明:SEA-F/SEA-R引物对在早实亲本‘绿园’中能稳定扩增出SCAR-SEA958特异标记;在‘绿园’ב绿丰’杂交F1后代群体中,SCAR-SEA958标记与核桃早实性状共分离,与核桃早实性状间的遗传距离为1.99cM;用晚实优系‘T-12’和栽培品种‘元丰’及‘T-12’ב元丰’杂交F1后代,检测SCAR-SEA958标记的分离情况,SCAR-SEA958与核桃早实性状共分离;在9个核桃栽培品种(系)中,SCAR-SEA958在8个供试品种的7个早实品种上出现,在1个早实品种‘鲁光’和1个晚实优系‘T-12’上没有扩增出条带。SCAR-SEA958标记在不同遗传背景下有较高的稳定性。     

Development of AFLP and RAPD markers linked to a locus associated with twisted growth in corkscrew willow (Salix matsudana 'Tortuosa')

Salix matsudana Koidz. cultivar 'Tortuosa' (corkscrew willow) is characterized by extensive stem bending and curling of leaves. To investigate the genetic basis of this trait, controlled crosses were made between a corkscrew female (S. matsudana 'Tortuosa') and a straight-stemmed, wild-type male (Salix alba L. Clone 99010). Seventy-seven seedlings from this family (ID 99270) were grown in the field for phenotypic observation. Among the progeny, 39 had straight stems and leaves and 38 had bent stems and curled leaves, suggesting that a dominant allele at a single locus controls this phenotype. As a first step in characterizing the locus, we searched for amplified fragment length polymorphism (AFLP) and randomly amplified polymorphic DNA (RAPD) markers linked to the tortuosa allele using bulked segregant analysis. Samples of DNA from 10 corkscrew individuals were combined to produce a corkscrew pool, and DNA from 10 straight progeny was combined to make a wild-type pool. Sixty-four AFLP primer combinations and 640 RAPD primers were screened to identify marker bands amplified from the corkscrew parent and progeny pool, but not from the wild-type parent or progeny pool. An AFLP marker and a RAPD marker linked to and flanking the tortuosa locus were placed on a preliminary linkage map constructed based on segregation among the 77 progeny. Sectioning and analysis of shoot tips revealed that the corkscrew phenotype is associated with vascular cell collapse, smaller cell size in regions near the cambium and less developed phloem fibers than in wild-type progeny. Identification of a gene associated with this trait could lead to greater understanding of the control of normal stem development in woody plants.     

SCAR markers: A potential tool for authentication of herbal drugs

Randomly Amplified Polymorphic DNA (RAPD) is easy to develop and simple molecular marker, but lack of reproducibility makes it less reliable for authentication of herbal drugs. Besides RAPD, other popular PCR and non-PCR based markers like AFLP, ISSR, SSR and RFLP are also used for authentication. However, these also have disadvantages like use of radioactive isotopes, high cost and absolute requirement of sequence information. Therefore, it is a better option to improve the reproducibility of RAPD by converting RAPD amplicons into Sequence Characterized Amplified Region (SCAR) markers. SCAR markers are easy, reliable and reproducible thus, have an advantage over RAPD markers for authentication of medicinal herbs used in the preparation of traditional medicines. These markers however, have been developed for only a few medicinal herbs. This review is an attempt to evaluate critically the role of SCAR markers in authentication of medicinal herbs used in traditional formulations.     

A male-specific SCAR DNA marker and sex ratio of seedlings in salak(Salacca zalacca var.zalacca)

A male-specific SCAR DNA marker was developed using a RAPD DNA marker specific for male plants of Salacca zalacca var. zalacca(salak palm). The marker is 1579 bp long and has a GC content of 38.5 %. Its sequence contains 1 or 2 open reading frames, indicating the marker is probably a coding region. No highly similar sequences were found in a search of the Gen Bank database.Sexes were identified using the SCAR DNA marker for three kinds of seedlings grouped by the number of seeds per fruit(1, 2 or 3). The sex ratio of female to male did not differ significantly from 1:1 for the three kinds of seedlings, implying that the number of seeds per fruit is not a reliable index to identify the sex of a seedling.     

家蚕来源肠球菌RAPD反应体系的优化

通过调整 Mg2 、DNA模板、引物、Taq DNA聚合酶和dNTPs的浓度及PCR反应循环次数,建立并优化了家蚕来源肠球菌的RAPD-PCR反应体系及条件.结果表明,在25 μl反应体系下,当Mg2 的浓度为3.5 mmol/L、DNA模板加入量为50 ng、引物浓度为15 pmol/L、Taq DNA聚合酶加入量为3U、 PCR反应循环次数为45时,扩增结果最好.在本实验室,为家蚕来源肠球菌的遗传多样性研究建立了一种最佳RAPD分析模型.     

松材线虫SCAR标记与检测技术

将松材线虫RAPD特异片段OPM05-X2100进行分离、回收,与载体pGEM-TVector连接,转化大肠杆菌并培养,对目标克隆测序。根据测序结果,用Oligo5.0软件设计引物,正向引物为M05F2(5’-CGGGT CATGG CTGGA GGTAT CGT-3’),反向引物为M05R1(5’-TGGCT CAATG GCAAA TCCTT CGTA-3’),成功地将松材线虫特异片段OPM05-X2100通过引物对M05F2/R1转化为SCAR-M05-X600。运用SCAR标记引物M05F2/R对枯死松树体内分离的92份线虫样本的DNA进行标记,并对单条线虫经简易方法提取的DNA进行检测。结果表明:引物组合对所有81份松材线虫株系均扩增出一条600bp的清晰、明亮的条带,而对8份拟松材线虫、1份霍夫曼尼伞滑刃线虫、1份大核滑刃线虫、1份长尾属线虫均无扩增产物。该对引物可以检测单条松材线虫。利用这对特异引物组合可构建松材线虫检测试剂盒,实现对松材线虫的快速检测的目标。