毛细管电泳四色荧光检测法分析茶树SSR标记

将毛细管电泳四色荧光栓测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5'端加有M13尾巴序列(5'-CACGACGTTGTAAAACGAC-3')的特异正向引物、特异反向引物及带有荧光标记的通用型M13引物:为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR位点,采用蓝、绿、黑3种不同颜色的荧光染料分别对3个M13引物进行标记. 应用该方法对42个茶树品种(系)的16个SSR位点进行遗传分析的结果表明:此法具有简便、可靠、低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显著.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.     

伯乐树转录组SSR分布特征及其引物开发

基于伯乐树(Bretschneidera sinensis Hemsl.)转录组测序数据,采用生物信息学方法分析该物种转录组中SSR位点的分布特征,利用TP-M13-SSR(Simple sequence repeat with tailed primer M13)方法检测12株无亲缘关系样本SSR引物位点的多态性.结...     

磁珠富集法开发北柴胡多态性简单序列重复标记

目的:利用磁珠富集法分离北柴胡微卫星序列,以开发北柴胡微卫星引物,获得有多态性的简单序列重复(SSR)标记。方法:用生物素标记的混合探针(AC)15、(AG)15、(MAB)12和两端连接已知序列人工接头的北柴胡基因组DNA酶切片段混和后与磁珠杂交,构建微卫星序列富集的小片段插入文库;利用接头引物分别与生物素探针引物Biotin-(AC)15、Biotin-(AG)15、Biontin-(MAB)12形成3个组合,用PCR方法对文库进行初步筛选;对可能的阳性克隆子进行测序复筛,选取微卫星侧翼序列足够长的序列设计引物,用荧光标记的基因分型技术以栽培柴胡种质为材料分析其多态性。结果:开发了5对多态性SSR标记,它们在5份柴胡栽培种质中共扩增出30.70个多态性等位基因,平均每条引物可以扩增出6.14个多态性等位基因;观察等位基因数最多13个,最少3个;有效等位基因数最多11.4个,最少1.6个。同时分析了4对EST-SSR引物,比较了2种SSR标记扩增结果。结论:磁珠富集法是开发柴胡多态性SSR标记的有效方法。     

基于TP-M13-SSR指纹图谱的中国原产苹果属植物分子身份证的建立

利用TP-M13-SSR分子标记方法,构建27份中国原产苹果属植物在12个SSR位点的指纹图谱,运用条码技术生成其分子身份证。12对引物共获得251个等位基因,平均21个。引物多态性好,仅用引物CH05b06即可区分全部供试材料。27份苹果材料在12个SSR位点遗传多样性、多态性信息含量和位点杂合度的变化范围为0.6620～0.9455、0.6327～0.9211和0.6538～0.9319。基于CH05b06位点处获得的指纹谱图即可得到每份供试材料独有的分子身份证。TP-M13-SSR分子标记技术适用于苹果属植物种质资源的指纹图谱构建,利于分子基础数据库的积累。基于苹果种质资源TP-M13-SSR指纹图谱可获得每份苹果种质资源独有的分子身份证。     

基于引物扩增受阻突变技术开发水稻耐冷基因CTB4a特异性分子标记

水稻(Oryza sativa)作为热带与亚热带起源的作物对低温敏感.对水稻种质进行耐冷性鉴定,能筛选出耐冷性强的种质,发展耐冷基因分子标记,能够有效鉴别种质中耐冷基因的基因型.本研究使用芽期4℃低温处理10d对41份水稻材料进行芽期耐冷鉴定,对品种的芽期耐冷能力进行评价,获得了参试材料中除了'昆明小白谷'之外的芽期耐冷性最强的品种'南特号'.对已克隆的耐冷基因CTB4a开发分子标记,能够辅助选择水稻的耐冷育种.水稻孕穗期耐冷基因CTB4a来源于'昆明小白谷',能够影响水稻抵抗低温的能力.参照公布的CTB4a序列信息,从中挑选出序列中的作用位点SNP(单核苷酸多态性,single nucleotide polymorphism),结合引物扩增受阻突变技术(Penta-primer amplification refractory mutation system,PARMS),用Primer 6.0设计引物,建立CTB4a基因荧光分子标记GM-CTB4a,使用荧光分子标记GM-CTB4a对41份水稻品种进行鉴定,使用酶标仪在'昆明小白谷'中检测到利用标记扩增产物中包含'昆明小白谷'特异SNP、T碱基引物携带的FAM荧光信号,在另外40份品种的扩增产物中检测到包含作用位点的C碱基引物携带的HEX荧光信号.本研究利用设计的分子标记,鉴定了 41份水稻品种的耐冷性和基因型.比对分析耐冷性和基因型鉴定结果,说明我们开发的分子标记GM-CTB4a特异性较强,具有实际应用价值.研究结果为利用水稻孕穗期耐冷基因CTB4a培育强耐冷水稻品种奠定坚实基础.     

利用电子PCR分析草菇基因组SSR标记多态性

【目的】利用电子PCR分析草菇基因组SSR标记多态性,并通过PCR验证分析结果的可靠性。【方法】利用MISA程序定位草菇基因组SSR位点并结合Primer3.0程序设计SSR分子标记引物,运用电子PCR进行SSR分子标记多态性分析,基于分析结果进行PCR验证。【结果】随机选取658对SSR引物在草菇同核体菌株V23-1和PD19中进行真实PCR检测,结果表明28.6%的SSR引物具有多态性。数据分析表明,如果SSR标记来源于电子PCR产物长度没有差异的类型,仅4.8%的SSR引物在真实PCR中表现出多态性;如果SSR标记来源于电子PCR产物长度差异大于或者等于3 bp的类型,其中至少48.3%的SSR引物在真实PCR中表现出多态性。【结论】PCR验证结果表明利用电子PCR可以提高SSR多态性引物的筛选效率。     

华仁杏EST-SSR标记引物设计与开发

随着生物科技的进步,ESTs（表达序列标签）已经成为开发SSR（简单重复序列）标记的重要资源。本文利用NCBI公共数据库下载蔷薇科EST序列22 458条,使用SSRHunter1.3软件进行了SSR搜索,从中获得22 527条SSR,应用Primer5.0软件设计并经由Oligo7.0软件检测,共得到61对EST-SSR引物。利用这些引物对8个华仁杏品种进行了PCR扩增及检测,得到10对能产生清晰多态性条带的EST-SSR标记,标明了10对引物的序列,为进一步开展华仁杏SSR分子标记辅助育种研究奠定了基础。     

兼性无融合生殖龙须草SSR引物开发及杂交后代的检测

以来自中国5个省份12个居群的龙须草为材料,通过锚定PCR开发复合SSR引物、数据库检索和近缘物种SSR引物的引用等3种方法开发龙须草的SSR引物.结果显示:在锚定PCR开发复合SSR位点所设计的33对引物中筛选到有效扩增引物13对,表现多态性的引物有6对;利用基因特异序列法得到多态性引物3个;所用18条同源物种引物中有83%的SSR引物在供试的12个龙须草居群中都有清晰的SSR条带,其中56%的引物表现出多态性.3种方法在龙须草12个居群中共筛选得到了19对多态性引物,多态性比率约为73%.利用筛选得到的SSR多态性引物对来自2个居群的杂交F1代进行检测,鉴别出只有母本带型的无融合生殖后代和具有双亲带型的杂交后代,在子代中还出现偏父本带型、亲本缺失带型和变异带型等,证明所开发的SSR引物可以揭示龙须草生殖方式的复杂性,适用于龙须草遗传分析及亲缘关系鉴定.     

椭圆叶花锚简化基因组的SSR信息分析及SSR引物开发

基于RAD-seq简化基因组测序技术获得椭圆叶花锚（Halenia ellipitica D.Don）简化基因组水平上的序列信息并开发SSR分子标记。利用SR search软件检测而得到双端各有至少100 bp的五种类型的SSR（二、三、四、五、六核苷酸）位点共6 201个,并成功设计其中3 865个SSR引物。在能成功设计引物的SSR位点中,三核苷酸SSR位点最多;重复序列长度包括17种（12~36 bp）;重复序列的基序共达316种,其中五核苷酸基序种类最多（91种）。从中挑选65对可对应五种SSR类型的引物,经梯度PCR检验后,利用椭圆叶花锚的4个居群32个个对可扩增的引物进行PCR和聚丙烯酰氨凝胶电泳检测,其中14对引物能在绝大多数个体中扩增,且13对具多态性。13个多态性位点的等位基因数量均值为5.462,多态性较高且不连锁（P<0.05）;其中10个位点在多数居群中偏离哈迪温伯格平衡（P<0.01）且存在较高的纯合子数量（观测杂合度H_o均值0.226）;近交系数在-0.443~1,均值为0.656;基因流N_m为0.474。     

结合通用引物快速简便鉴定阳性克隆的PCR方法

通用引物与载体多克隆住点两端序列互补,将目的片段构建入栽体pGEM-T Easy中,利用载体通用引物M13F和M13R结合片段特异引物进行菌液PCR反应.用PCR产物电泳结果来筛选阳性克隆并鉴定目的片段插入方向,同时能有效对短插入片段重组子进行筛选与鉴定.最终以DNA测序结果来验证,与测序结果一致,显示通用引物PCR方法对阳性克隆的筛选和鉴定优于传统酶切和普通PCR鉴定方法,能够弥补传统方法的不足,且简便快速,可作为筛选和鉴定阳性克隆的有效手段.     

TP-M13-SSR技术及其在苹果种质资源遗传多样性研究中的应用

利用在SSR扩增产物检测过程中的一种基于荧光测序技术的高通量低成本分析技术体系TP-M13-SSR,对苹果种质资源的25份地方品种进行了遗传多样性研究,得到其遗传多样性、多态性信息含量和位点杂合度的变化范围分别为0.5032～0.8448、0.3952～0.8268和0.4400～0.9600。UPGMA法聚类分析将25个苹果品种分为2大类,与地理起源和亲缘关系相关。这种方法具有经济、灵敏、高效等优点,在苹果遗传多样性研究中得到成功应用。本文讨论了TP-M13-SSR技术的优缺点,以及在果树种质资源遗传多样性研究中的应用潜力。     

Analysis of the genetic diversity of garlic (Allium sativum L.) by simple sequence repeat and inter simple sequence repeat analysis and agro-morphological traits

The genetic diversity of 39 garlic accessions was investigated using eight simple sequence repeat (SSR) primer combinations and 17 inter-simple sequence repeat (ISSR) primer combinations. A total of 109 polymorphic loci were detected among these accessions, with an average of 4.63 polymorphic loci per SSR primer combination and 4.29 polymorphic loci per ISSR primer combination. The mean effective number of alleles, the mean Nei's genetic diversity, and the mean Shannon's information index for SSR were 1.4799, 0.2870, and 0.4378, respectively; and those for ISSR were 1.4847, 0.2898 and 0.4415, respectively. Cluster analysis, using the unweighted pair-group method with arithmetic averages (UPGMA) based on the allele frequency data, classified the accessions into three groups. The results of principal component analysis (PCA) were consistent with those of the cluster analysis. PCA showed that each of these three groups exhibited significant variation in agro-morphological traits. These findings suggest that the eight SSR and 17 ISSR primers identified could define valuable markers for genetic diversity for use by plant breeders.     

Comparison of SSR and cytochrome P-450 markers for estimating genetic diversity in Picrorhiza kurrooa L.

Picrorhiza kurrooa L., a high altitude medicinal plant, is known for its drug content called Kutkin. In the present study, DNA-based molecular marker techniques, viz. simple sequence repeats (SSR) and cytochrome P-450 markers were used to estimate genetic diversity in Picrorhiza kurrooa. Twenty five accessions of Picrorhiza kurrooa, collected from ten different eco-geographical locations were subjected to 22 SSR and eight cytochrome P-450 primer pairs, out of which 13 SSR markers detected mean 5.037 alleles with a mean polymorphic information content (PIC) of 0.7718, whereas eight cytochrome P-450 markers detected mean 5.0 alleles with a mean PIC of 0.7596. Genetic relationship among the accessions was estimated by constructing the dendrograms using SSR and cytochrome P-450 data. There was a clear consistency between SSR and cytochrome P-450 trees in terms of positioning of most Picrorhiza accessions. SSR markers could cluster various Picrorhiza kurrooa accessions based on their geographical locations whereas cytochrome P-450 markers could cluster few accessions as per their geographical locations. The Mantel test between SSR and cytochrome P-450 markers revealed a good fit correlation (r = 0.6405). The dendrogram constructed using the combined data of SSR and cytochrome P-450s depicted two clusters of accessions based on its eco-geographical locations whereas two clusters contained the accessions from mixed eco-geographical locations. Overall, the results of the present study point towards quiet high degree of genetic variation among the accessions of each eco-geographic region.     

Development of linkage map in F2 population of selected parents with respect to Macrophomina phaseolina resistance trait using screened polymorphic RAPD and developed SCAR markers of jute

Two molecular markers (RAPD and simple sequence repeat (SSR)) were applied on 12 Corchorus capsularis jute samples. Two of them were Macrophomina phaseolina-resistant and the remaining eight were M. phaseolina-susceptible accessions. Eleven SSR primer combinations out of 18 gave the polymorphic results between M. phaseolina-resistant and -susceptible accessions. Five pairs of sequence characterised amplified region (SCAR) primers designated as SCP-4, SCS-3, SCS-13, SCG-10 and SCU-10 were designed based on the polymorphic loci obtained between JRC-412 and CIM-036. Only SCU-10 and SCS-13 produced polymorphic markers corresponding to OPU-10 and OPS-13 amplified from ‘CIM-036’ and JRC-412, respectively. RAPD and SCAR markers were employed for construction of a linkage map using a set of 67 F2 population of a cross between JRC-412 and CIM-036 as a mapping population. Nine markers were assigned to two linkage groups (LGs) covering a total length of 628.4 cM with an average distance of 28 cM between markers.     

Evaluation of Genetic Diversity in Lemons and Some of Their Relatives Based on SRAP and SSR Markers

Genetic diversity was evaluated by sequence-related amplified polymorphism (SRAP) and simple sequence repeat (SSR) markers among 45 lemons (Citrus limon (L.) Burm. f.), five citrons (Citrus medica L.), four rough lemons (Citrus jambhiri Lush), and two Citrus volkameriana accessions. Twenty-one SRAP primer combinations produced a total of 141 (77%) polymorphic fragments with an average of 6.7 fragments per primer combinations whereas 13 SSR primers produced a total of 26 (76%) polymorphic fragments with an average of 2.0 per primer. The unweighted pair-group method arithmetic average analysis as assessed with combined SRAP and SSR data demonstrated that the accessions had a similarity range from 0.65 to 1.00. Rough lemons and C. volkameriana accessions were relatively closely related. In lemon group, accessions from hybrid origin were distant from the others. We also applied principal components analysis (PCA) for a better presentation of relation among the accessions studies. Using PCA, 88.7% of the total variation in the original dimensions could be represented by just the two dimensions defined by the first two PCs. Although nearly all accessions could be distinguished, there was a low level of genetic diversity detected among lemon cultivars.     

Development and characterization of microsatellite markers from tropical forage Stylosanthes species and analysis of genetic variability and cross-species transferability

A limited number of functional molecular markers has slowed the desired genetic improvement of Stylosanthes species. Hence, in an attempt to develop simple sequence repeat (SSR) markers, genomic libraries from Stylosanthes seabrana B.L. Maass & 't Mannetje (2n=2x=20) using 5' anchored degenerate microsatellite primers were constructed. Of the 76 new microsatellites, 21 functional primer pairs were designed. Because of the small number of primer pairs designed, 428 expressed sequence tag (EST) sequences from seven Stylosanthes species were also examined for SSR detection. Approximately 10% of sequences delivered functional primer pairs, and after redundancy elimination, 57 microsatellite repeats were selected. Tetranucleotides followed by trinucleotides were the major repeated sequences in Stylosanthes ESTs. In total, a robust set of 21 genomic-SSR (gSSR) and 20 EST-SSR (eSSR) markers were developed. These markers were analyzed for intraspecific diversity within 20 S. seabrana accessions and for their cross-species transferability. Mean expected (He) and observed (Ho) heterozygosity values with gSSR markers were 0.64 and 0.372, respectively, whereas with eSSR markers these were 0.297 and 0.214, respectively. Dendrograms having moderate bootstrap value (23%-94%) were able to distinguish all accessions of S. seabrana with gSSR markers, whereas eSSR markers showed 100% similarities between few accessions. The set of 21 gSSRs, from S. seabrana, and 20 eSSRs, from selected Stylosanthes species, with their high cross-species transferability (45% with gSSRs, 86% with eSSRs) will facilitate genetic improvement of Stylosanthes species globally.     

Isolation of EST-derived microsatellite markers for genotyping the A and B genomes of wheat

Genetic variation present in 64 durum wheat accessions was investigated by using three sources of microsatellite (SSR) markers: EST-derived SSRs (EST-SSRs) and two sources of SSRs isolated from total genomic DNA. Out of 245 SSR primer pairs screened, 22 EST-SSRs and 20 genomic-derived SSRs were polymorphic and used for genotyping. The EST-SSR primers produced high quality markers, but had the lowest level of polymorphism (25%) compared to the other two sources of genomic SSR markers (53%). The 42 SSR markers detected 189 polymorphic alleles with an average number of 4.5 alleles per locus. The coefficient of similarity ranged from 0.28 to 0.70 and the estimates of similarity varied when different sources of SSR markers were used to genotype the accessions. This study showed that EST-derived SSR markers developed in bread wheat are polymorphic in durum wheat when assaying loci of the A and B genomes. A minumum of ten EST-SSRs generated a very low probability of identity (0.36×10⁻¹²) indicating that these SSRs have a very high discriminatory power. EST-SSR markers directly sample variation in transcribed regions of the genome, which may enhance their value in marker-assisted selection, comparative genetic analysis and for exploiting wheat genetic resources by providing a more-direct estimate of functional diversity. Received: 19 December 2000 / Accepted: 17 April 2001