A new set of mono‐ and dinucleotide chloroplast microsatellites in Fagaceae

A new set of 14 chloroplast microsatellites, represented by mono‐ and dinucleotide repeats, was optimized in the three main species of the Fagaceae (Castanea sativa, Fagus sylvatica and Quercus petraea). The intraspecific variation was tested in some natural populations. The polymorphic microsatellites displayed two or three variants. Conservation of the primer pairs was checked on an additional set of six species of the Fagaceae and on Fraxinus excelsior. All the primer pairs produced a fragment of the expected size in the Fagaceae species while no amplification was obtained with 36% of the primers in F. excelsior.     

Universal primers for the amplification of chloroplast microsatellites in grasses (Poaceae)

Attempts to design truly universal primers to amplify chloroplast microsatellites have met with limited success due to nonconservation of repeat loci across widely divergent taxa. We have used the complete chloroplast genome sequences of rice, maize and wheat to design five pairs of primers that amplify homologous mononucleotide repeats across the Poaceae (grasses). Sequencing confirmed conservation of repeat motifs across subfamilies and a preliminary study in Anthoxanthum odoratum revealed polymorphism at two loci with a haplotype diversity value of 0.495. These primers provide a valuable tool to study cytoplasmic diversity in this extensively studied and economically important range of taxa.     

Chloroplast microsatellites in Prunus,Rosaceae

Ten chloroplast microsatellite markers were developed from Japanese plum (Prunus salicina) based on nucleotide sequences of c. 4300 bp from six chloroplast regions. Out of 10 microsatellites, seven markers contained mononucleotide repeats. Almost all microsatellites displayed discrete amplified fragments for 17 species in Prunus. The microsatellites generated 46 different fragment types and differentiated all used species. Polymorphism was also observed within species for all microsatellites.     

Polymorphic chloroplast simple sequence repeat primers for systematic and population studies in the genus Hordeum

In this study we report the development of primers to amplify polymorphic chloroplast simple sequence repeats in the genus Hordeum , which includes cultivated barley ( H. vulgare ssp. vulgare ) and its wild progenitor H. vulgare ssp. spontaneum . Polymorphic products were amplified in a wide range of Hordeum spp. and intraspecific variation was detected in both cultivated and wild barley. A decrease in cytoplasmic diversity was observed between sspp. spontaneum and vulgare as well as between ssp. vulgare landraces and cultivars, which is characteristic of domestication processes in many crop species. We also observed possible evidence for reticulate evolution of H. brachyantherum polyploids, with apparent multiple cytoplasmic introgressions during successive polyploidization events.     

Identification and characterization of (GA/CT) n - microsatellite loci from Quercus petraea

In this study a size selected genomic library from Quercus petraea was screened for (GA/CT)_n-microsatellite sequences. The resulting loci were analysed by PCR for their usefulness as molecular markers in Q. petraea and Q. robur. 17 out of 52 tested primer pairs resulted in the amplification of a polymorphic single-locus pattern. The number of alleles found per locus varied from 6 to 16. Combining the genetic variation observed for the characterized loci provides a unique genotype for all the individuals tested. Using intraspecific controlled crosses of Q. robur trees Mendelian inheritance could be shown for five loci.     

Characterization and primer development for amplification of chloroplast microsatellite regions of Fraxinus excelsior

This study reports the characterization of chloroplast DNA (cpDNA) variation of Fraxinus excelsior at five loci and the successful development of primer pairs for the amplification of three of these containing mononucleotide microsatellites. We detected high levels of haplotype variation among provenances of Fraxinus around Europe and within Ireland.     

Chloroplast simple sequence repeat markers for evolutionary studies in the sexually deceptive orchid genus Chiloglottis

The orchids in the genus Chiloglottis are pollinated exclusively by sexual deception. Extensive sequencing (> 19.5 kb) of noncoding chloroplast DNA revealed that simple sequence repeats (cpSSRs) were abundant, enabling a set of 41 cpSSR markers to be developed. All markers were polymorphic across the genus. Polymorphism reflected variation at both mononucleotide repeats and indels. For a subset of four taxa with 40 samples each, locus polymorphism varied from 46 to 81%, while the number of haplotypes ranged from seven to 21 per taxon. Extensive differentiation among the taxa was detected. These cpSSRs markers will enable novel insights into the evolution of this unique genus.     

A set of chloroplast microsatellite primers for Eucalyptus (Myrtaceae)

We present a set of 35 chloroplast microsatellite primers for Eucalyptus. Ten of the microsatellites displayed intraspecific polymorphism, and identified nine haplotypes among 16 Eucalyptus globulus individuals. Primer conservation was high, with a polymerase chain reaction (PCR) success rate of 98% when tested on four other Eucalyptus species and seven additional myrtaceous genera. Chloroplast microsatellites have applications in phylogeographic studies, fingerprinting and progeny analysis.     

A new set of universal de novo sequencing primers for extensive coverage of noncoding chloroplast DNA: new opportunities for phylogenetic studies and cpSSR discovery

We present a new set of universal de novo sequencing primers targeting noncoding chloroplast DNA. The set of 107 polymerase chain reaction (PCR) primers span approximately 86% of the noncoding nucleotides in the large single copy region of Nicotiana tabacum, Oryza sativa and the orchid Phalaenopsis aphrodite. PCR tests confirmed the primers are effective in a wide range of monocots and dicots. More than 19.5 kb of cpDNA sequence was obtained across representative orchid genera with up to 82 chloroplast simple sequence repeats (cpSSRs) detected per genus. This primer set will facilitate both phylogenetic studies and rapid discovery of cpSSRs for plants, such as orchids, where there are limited genomic resources.     

Chloroplast simple sequence repeats (cpSSRs): technical resources and recommendations for expanding cpSSR discovery and applications to a wide array of plant species

Chloroplast microsatellites, or simple sequence repeats (cpSSRs), are typically mononucleotide tandem repeats. When located in the noncoding regions of the chloroplast genome (cpDNA), they commonly show intraspecific variation in repeat number. Despite the growing number of studies applying cpSSRs, studies of economically important plants and their relatives remain over‐represented. Thus, the potential of cpSSRs to offer unique insights into ecological and evolutionary processes in wild plant species has yet to be fully realized. This review provides an overview of the technical resources available to aid cpSSR discovery including a list of cpSSR primer sets available and cpDNA sequencing resources. Our updated analysis of 99 whole chloroplast genomes downloaded from GenBank confirms that potentially variable cpSSRs are abundant in the noncoding cpDNA of plants. Overall variation in the frequency of cpSSRs was extreme, ranging from one to 700 per genome (median = 93), while in 81 vascular plants, between 35 and 160 cpSSRs were detected per genome (median = 86). We offer five recommendations to aid wider development and application of cpSSRs: (i) When genus‐specific cpSSR primers are available, cross‐species amplification can often be fruitful. (ii) While potentially useful, universal cpSSR primers at best provide access to only a small number of variable markers. (iii) De novo sequencing of noncoding cpDNA is the most effective and efficient way to develop cpSSR markers in wild species. (iv) DNA sequencing of cpSSR alleles is essential, given the complex nature of the genetic variation associated with hypervariable cpDNA regions. (v) The reliability of cpSSR length based genetic assays need to be validated in all studies.     

Universal primers for amplification of three non-coding regions of chloroplast DNA

Six primers for the amplification of three non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA from various plant species. The primers worked for most species tested including algae, bryophytes, pteridophytes, gymnosperms and angiosperms. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology (in supplying markers) and evolution (inter- and probably intraspecific phylogenies) of plants.     

红边龙血树叶绿体基因组特征及其系统发育分析

红边龙血树(Dracaena marginata)是一种在全球广泛种植的龙血树属园艺植物,具有较高的观赏价值和药用价值。本研究首次利用高通量测序技术对红边龙血树叶片进行全基因组测序,组装得到完整的叶绿体基因组序列,并进行注释、序列特征比较和系统发育分析。结果表明,红边龙血树叶绿体基因组包含一个典型的四分体结构,长度为154926 bp,是目前已报道的龙血树属中叶绿体基因组最小的物种;共拥有132个基因,包含86个编码蛋白基因、38个转运RNA基因和8个核糖体RNA基因;密码子偏好性分析发现存在偏好使用A/U碱基结尾的现象,整体上密码子偏好性较低;共鉴定出46个简单重复序列位点和54个长重复序列,分别在大单拷贝区与反向重复区有最大检出率;种间边界分析发现边界区域基因存在相对位置差异,扩张收缩情况总体较为相似;与近缘种进行系统发育分析,红边龙血树与细枝龙血树聚为一类,关系最近,符合形态学分类特征。对红边龙血树叶绿体基因组的解析为龙血树属植物的物种鉴定、遗传多样性和叶绿体转基因工程等提供了重要数据基础。     

苎麻叶绿体SSR引物筛选及分析

叶绿体微卫星标记为单亲遗传(除部分裸子植物外),有独立的进化路线,它在植物遗传多样性、群体遗传结构、系统发育分析及杂种鉴定等研究上用途广泛,是研究谱系地理的有效手段。苎麻的主产地和主要分布区均在我国,但其谱系地理研究目前尚未见有报道。该研究选择来自全国不同地区的52个苎麻样本,利用聚丙烯酰胺凝胶电泳法,在23对已知通用叶绿体SSR引物中筛选出适用于苎麻谱系地理研究的SSR引物,并利用筛选到的多态引物对52个苎麻样本进行聚类分析和单倍型网络图分析。结果表明:从23对通用引物中共筛选出16对适用于苎麻的多态引物,其平均多态信息含量为0.1053,虽然以上引物多态性较低,但能够用于野生苎麻的遗传分析研究;这52个苎麻样本聚为10支,分为8个单倍型,初步分析表明叶绿体SSR遗传变异速率较慢,不适用于苎麻种内的系统发育研究,但以上引物能够检测苎麻种内单倍型变异,可用于苎麻的谱系地理研究。     

Isolation and characterization of microsatellites in the columnar cactus: Polaskia chichipe and cross‐species amplification within the Tribe Pachycereeae (Cactaceae)

Microsatellite markers were developed for the columnar cactus Polaskia chichipe from central Mexico. After an enrichment procedure and three screening steps 87% of colonies contained microsatellites. A pair of primers for 10 loci (seven polymorphic) were developed, tested and used to estimate variation in samples of 18–45 individuals from the Tehuacan Valley, Mexico. Alleles per locus ranged from two to eight (mean 5.28; SD 2.5). Range of expected heterozygosity values was 0.188–0.797 (mean 0.502; SD 0.25). These loci are particularly useful for more precise evolutionary studies, such as gene flow and breeding systems, for this cactus species.     

Parentage testing and linkage analysis in the horse using a set of highly polymorphic microsatellites

Ten (TG)_n positive clones, isolated from an equine genomic library and sequenced, contained 12–19 uninterrupted TG repeats. Primers for polymerase chain reaction (PCR) were synthesized and nine of these (TG)_n loci (HTG7-15) were successfully amplified and utilized in this study together with five previously reported equine microsatellite loci (HTG2-6). The PCR products were analysed by polyacrylamide gel electrophoresis followed by automated laser fluorescence detection or autoradiography. All microsatellites showed polymorphism and stable Mendelian inheritance. Differences in microsatellite variability between horse breeds were detected. A linkage analysis comprising HTG2-15, one coat colour gene and 16 genetic blood markers enabled addition of HTG2 to linkage group U2 and a new linkage group (U6) was established comprising the loci HTG7 and HTG12. Close linkage was excluded within a set of eight microsatellites. The estimated probability of exclusion in four breeds for a parentage test based on these eight loci varied between 0.96 and 0.99.     

Isolation and characterization of 11 microsatellite primers for a temperate reef fish, the California sheephead (Semicossyphus pulcher)

Eleven microsatellites were characterized for Semicossyphus pulcher (California sheephead) using an enrichment protocol. The number of alleles varied from three to 14 for a sample of 40 individuals from two populations. Expected heterozygosities ranged from 0.311 to 0.891. All loci but one were in Hardy-Weinberg equilibrium. No evidence for linkage disequilibrium was observed. These polymorphic microsatellites will be useful for genetic diversity and connectivity analyses of S. pulcher.     

The D4 set: primers that target highly variable intron loops in plant chloroplast genomes

Chloroplast group II introns offer high-quality, rapidly evolving single-copy loci for comparative sequence analysis. These introns feature diagnostic secondary structures with loops that are among the least evolutionarily constrained sequence in plastomes. We exploited these structures to develop universal primers that amplify and sequence the large Domain IV (D4) loop in several angiosperm introns. With a single sequence read, we recover 300-600 nucleotides of highly variable sequence across angiosperms, with rates of change that are equal to or higher than many of the best known intergenic spacers in plant chloroplast genomes.     

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。