Allelic and population variation of microsatellite loci in aspen (Populus tremuloides)

To develop a robust basis for inferences about population genetics and evolution, this work assayed 192 aspens (Populus tremuloides) from 11 sites in Wisconsin, USA, for allelic and population variation at 16 microsatellite loci distributed across the Populus genome. Frequency distributions of fluorochrome-labeled alleles resolved by capillary electrophoresis were analyzed for relationships to repeat size and number. Population-level statistics were compared with those of other studies, especially in Populus. All loci were polymorphic, varying widely in the number of alleles per locus (mean = 8.25, range 2-20). Expected and observed heterozygosities were high (0.45 and 0.41, respectively), with little differentiation among populations (F(ST) = 0.006-0.045) and a moderate level of inbreeding (F(IS) = 0.09), intermediate among levels reported in studies based on isozymes. Contrary to several other reports, allele frequencies clustered tightly around the modal frequency, and the genetic diversity (measured as alleles per locus or as expected heterozygosity) was not related to either the repeat unit size or to the number of repeats.     

Trinucleotide repeat microsatellite markers for black poplar (Populus nigra L.)

Using an enrichment procedure, we have cloned microsatellite repeats from black poplar (Populus nigra L.) and developed primers for microsatellite marker analysis. Ten primer pairs, mostly for trinucleotide repeats, produced polymorphic fragments in P. nigra. Some of them also showed amplification in other poplar species. (P. deltoides, P. tricocarpa, P. tremula, P. tremuloides, P. candicans, P. lasiocarpa). The best six loci were tested on 23 P. nigra genotypes collected across Europe. The microsatellites produced up to 12 alleles per locus in this set, with observed heterozygosity between 0.32 and 0.91.     

Microsatellite marker development,mapping and applications in rice genetics and breeding

Microsatellites are simple, tandemly repeated di- to tetra-nucleotide sequence motifs flanked by unique sequences. They are valuable as genetic markers because they are co-dominant, detect high levels of allelic diversity, and are easily and economically assayed by the polymerase chain reaction (PCR). Results from screening a rice genomic library suggest that there are an estimated 5700-10 000 microsatellites in rice, with the relative frequency of different repeats decreasing with increasing size of the motif. A map consisting of 120 microsatellite markers demonstrates that they are well distributed throughout the 12 chromosomes of rice. Five multiple copy primer sequences have been identified that could be mapped to independent chromosomal locations. The current level of genome coverage provided by these simple sequence length polymorphisms (SSLPs) in rice is sufficient to be useful for genotype identification, gene and quantitative trait locus (QTL) analysis, screening of large insert libraries, and marker-assisted selection in breeding. Studies of allelic diversity have documented up to 25 alleles at a single locus in cultivated rice germplasm and provide evidence that amplification in wild relatives of Oryza sativa is generally reliable. The availability of increasing numbers of mapped SSLP markers can be expected to complement existing RFLP and AFLP maps, increasing the power and resolution of genome analysis in rice.     

Development of microsatellite markers in Dendrobium fimbriatum Hook, an endangered Chinese endemic herb

As an endangered endemic herb, Dendrobium fimbriatum, is under threat from numerous impacts. In order to analyse the genetic diversity and structure of this endangered species, we provide details of 10 microsatellite loci (out of 15 primer pairs designed) which showed polymorphic for D. fimbriatum. These loci were used to screen 25 individuals from across the species' geographical range. Ten loci were polymorphic with 2 to 19 alleles; three loci were monomorphic, while the rest produced no amplification fragments. These loci will be used to investigate population genetic structure, genetic diversity, conservation, and individual authentication in the endangered D. fimbriatum.     

Development of simple sequence repeat (SSR) markers and their use in identification of Dendrobium varieties

The aim of this study was to develop simple sequence repeat (SSR) markers for Dendrobium varieties/species, many of which have medicinal and horticultural values. Two genomic DNA libraries of Dendrobium Sonia enriched with GA repeats and CA repeats were constructed. Fourteen polymorphic SSR markers were identified when screened against 42 popular commercial Dendrobium hybrids. The average allele number was 12.0 ± 1.9 and the observed heterozyosity was averaged at 0.70. All 42 hybrids tested, except for two tissue culture mutants, were uniquely identified with the markers used. Sibling hybrids were closely clustered. Hybrids were also closer to parents. These SSR markers can be used for molecular ecology research, genetic mapping and marker‐assisted breeding. They can also help protection for new Dendrobium varieties.     

Microsatellite markers developed using a next‐generation sequencing technique for Neotrogla spp. (Psocodea: Prionoglarididae), cave dwelling insects with sex‐reversed genitalia

The genus Neotrogla (Psocodea: Prinoglarididae) comprises four named species from Brazil. Females of this cave‐dwelling insect are characterized by a conspicuous penis‐like intromittent organ, termed a gynosome, which is inserted into the vagina‐like male genitalia during copulation. Another evolutionarily novel structure, the spermathecal plate, enables a female to simultaneously store two freshly deposited spermatophores (consisting of sperm and possibly nutritious substances) in her sperm storage organ (spermatheca). It is unknown whether the two spermatophores are derived from two different males. To investigate the mating ecology and population genetic structures of these insects with sex‐reversed genitalia, 16 novel highly polymorphic microsatellite loci were isolated and characterized based on ~2,275 Mbp genomic sequences from an undescribed Neotrogla species. Our first screening detected 99,888 candidate loci. Similar to other hemipteroid insects studied thus far, AAT motif microsatellites were conspicuously dominant. We further screened 99 sequences, for which 50 pairs of polymerase chain reaction primers were successfully designed. Sixteen of these primers successfully amplified products of the expected size in the 11 Neotrogla sp. individuals collected from two caves. The number of alleles per loci varied from two to nine, with no significant deviation from the Hardy–Weinberg equilibrium in either population. Although the caves sampled were only approximately 1 km apart, significant genetic differentiation was detected between the two populations. In total, 13, 12, 13 and 11 loci were cross‐amplified in N. aurora, N. brasiliensis, N. curvata and N. truncata, respectively, indicating the applicability of these microsatellite loci for metapopulation genetic studies in multiple Neotrogla species.     

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。     

山杨杂种无性系的SSR分子标记遗传多样性

采用5对SSR引物对52个山杨杂种无性系进行了遗传多样性检测,结果表明在研究的5个位点上SSR标记多态位点百分率为100%,平均等位基因数为4.4个,有效等位基因数最多的位点是PTR7,最少的位点为PTR12;欧美山杨杂种的遗传多样性最丰富,相比之下,中美山杨杂种遗传变异最低;聚类分析表明,在一定的遗传距离基础上,欧美山杨杂种和欧洲山杨首先聚为一类,然后又与中美山杨杂种聚类,最后是中国山杨。研究表明来自芬欧美山杨杂种具有较高的遗传多样性,这对我国山杨遗传资源的扩大,以及未来山杨杂交育种,杂种优势的利用都是重要的。     

Polymorphic tetranucleotide microsatellite DNA loci from the southern dusky salamander (Desmognathus auriculatus)

We describe polymerase chain reaction (PCR) primers and amplification conditions for seven tetranucleotide microsatellite DNA loci isolated from the southern dusky salamander (Desmognathus auriculatus). Primers were tested on 16 individuals from one population in Aiken County, South Carolina. We detected an average of 6.57 alleles per locus, an observed heterozygosity range of 0.44–0.94, and high polymorphic information contents (mean of 0.68).     

蜜蜂EST中的微卫星分析

为加速分子标记在蜜蜂遗传、进化与行为等方面的利用,分析了简单重复序列(Simple Sequence Repeats,SSRs)在蜜蜂EST中的分布频率与密度。所分析的蜜蜂EST数据集包含15869条序列,总长为7．9Mb。结果显示,蜜蜂ESTs中SSRs的频率为1／0．52kb,其中6碱基重复基序占总SSRs的45．0％,是最丰富的重复单元,而2、1、3、4与5碱基重复基序分别占总SSRs的17．9％、14．1％、11．6％、9．2％和2．2％。同时,在各种SSRs重复单元中,富含A碱基的重复单元占据优势地位,如：A、AT、AG、AC、AAT、AAG、AAC、AAAT、AAAG、AAAAG、AAAAT、AATAT、AAAAAG和AAAAAT重复基序,而富含G碱基的重复单元在基因编码区中含量较低。进一步分析显示：蜜蜂SSRs在冗余与非冗余EST数据集中的分布频率与密度相似,仅存在极小的偏差,表明可从现有的部分ESTs数据中方便地获取有效的微卫星标记。     

巴氏蘑菇微卫星序列的分离及其对Co60辐射诱变菌株的鉴别

根据链霉素磁珠和生物素特异结合的特性,用生物素标记的二聚核苷酸重复序列探针从巴氏蘑菇的基因组中分离微卫星序列。将结合于链霉素磁珠上的标记探针同两端连接已知序列人工接头的巴氏蘑菇DNA酶切片段杂交。洗脱未杂交DNA片段后,用磁珠富集的片段建立微卫星文库。挑取522个菌落用对应重复序列为引物进行PCR筛选,得到48个阳性克隆,经测序有32个菌落含微卫星序列。微卫星富集效率为阳性克隆数的67%,总克隆数的6%。除去重复或无效的微卫星序列,在设计出的12对用于鉴别85个巴氏蘑菇的Co60辐射变异株微卫星引物中,有4对引物总共扩增出明显的变异菌株17个。证明有些微卫星位点可用于巴氏蘑菇辐射变异品种的指纹筛选与鉴别。     

Development of multiplex sets of simple sequence repeat DNA markers covering the soybean genome

Multiplexing involves the analysis of several markers in a single gel lane that is based on the allele size range of marker loci. Multiplex SSR marker analysis is conducted with primers that are labeled with one of three dyes. The development of an SSR multiplex system requires estimates of the allele size range of markers to strategize primer labeling and for grouping markers into multiplex sets. A method is presented that describes the development of multiplex sets of SSR markers in soybean (Glycine max (L.) Merr.) by the selective placement of primer sites and by the analysis of diverse germplasm. Primer sites were placed at specific distances from the SSR to adjust the allele size range of marker loci. The analysis of pooled DNA samples comprising diverse soybean genotypes provided robust estimates of the allele size range of marker loci that enabled the development of multiplex sets. Eleven multiplex sets comprising 74 SSR markers distributed across the 20 linkage groups of soybean were developed. Multiplex sets constructed from the analysis of diverse soybean germplasm should have a wide range of genotyping applications. The procedures used in this study were systematic and rapid and should be applicable for multiplex development in any species with SSR marker technology.     

Isolation and characterization of microsatellite markers in Dendrobium officinale,an endangered herb endemic to China

As an invaluable herb, Dendrobium officinale has been in severe danger since 1950 because of human exploitation and habitat deterioration. In order to investigate the genetic diversity and structure of this species, we isolated and characterized 12 microsatellite loci derived from two microsatellite‐enriched libraries. Twenty‐two individuals from Leye population were analysed. These loci were polymorphic and displayed three to 12 alleles per locus. The observed and expected heterozygosities ranged from 0.150 to 0.624 and from 0.162 to 0.605, respectively. These are the first microsatellite loci characterized from D. officinale that will contribute to research on the conservation, genetic diversity, population structure and individual authentication.     

Genome size and the accumulation of simple sequence repeats: implications of new data from genome sequencing projects

The relationship between the level of repetitiveness in genomic sequences and genome size has been re-investigated making use of the rapidly growing database of complete eubacterial and archaeal genome sequences combined with the fragmentary but now large amount of data from eukaryotic genomes. Relative simplicity factors (RSFs), which measure the repetitiveness of sequences, were calculated and significantly simple motifs (SSMs), which identify the kinds of sequences that are repeated, were identified. A previously reported correlation between genome size and repetitiveness was confirmed, but it was shown that the higher RSFs seen in eukaryotic genomes also reflect a generally higher level of repetitiveness independent of genome size differences. Differences in genome size are responsible for about 10% of the variance in RSF seen between species. The spectrum of SSMs seen within a genome differed markedly within the eubacteria but less so in eukaryotes and, particularly, in archaea. Species with SSM spectra that differ from the norm tend also to have high RSFs for their genome size and to be pathogens that make use of repetitive sequences to avoid host defence responses. Some of the variance in repetitiveness seen in other species may therefore also reflect the action of selection, although other forces such as variation in the effectiveness of mechanisms for regulating slippage errors of replication, may also be important.     

Development of microsatellite markers for the red‐listed wood‐decay fungus Phlebia centrifuga

Seven polymorphic microsatellite markers were developed for the wood‐decay basidiomycete Phlebia centrifuga. The primers were identified using two techniques, based on intersimple sequence repeats (ISSR) and amplified fragment length polymorphism (AFLP), respectively. The markers were screened on 27 isolates from Europe and North America. Two markers varied only on a worldwide scale, but not within Europe. The other five showed variation on both scales. These markers will now be used to characterize populations of P. centrifuga, which is red‐listed as near‐threatened in its natural habitat due to human disturbance.     

Development of genomic resources for Wenchengia alternifolia (Lamiaceae) based on genome skimming data

Wenchengia alternifolia (Lamiaceae), the sole species of the genus Wenchengia is extremely rare and is currently listed as Critically Endangered (CR) on the IUCN Red List. The species had long been considered endemic to Hainan Island, China and was once believed to be extinct until a small remnant population was rediscovered at the type locality in 2010. Four more populations were later found on Hainan and in Vietnam. In order to develop genomic resources for further studies on population genetics and conservation biology of this rare species, we identified infraspecific molecular markers in the present study, using genome skimming data of five individuals collected from two populations on Hainan Island and three populations in Vietnam respectively. The length of plastome of the five individuals varied from 152,961 bp to 150,204 bp, and exhibited a typical angiosperm quadripartite structure. Six plastid hotspot regions with the Pi > 0.01 (trnH-psbA, psbA-trnK, rpl22, ndhE, ndhG-ndhI and rps15-ycf1), 1621 polymorphic gSSRs, as well as 1657 candidate SNPs in 237 variant nuclear genes were identified, thereby providing important information for further genetic studies.     

Characterization of new microsatellite loci from Vitis vinifera and their conservation in some Vitis species and hybrids

We present here characterization data for seven new microsatellite markers designed from new microsatellite loci isolated from a microsatellite‐enriched DNA library from Vitis vinifera. The observed heterozygosity varied from 0.73 up to 0.93 and the number of alleles per locus ranged from 12 to 26. This high polymorphism makes these new markers interesting for use in genotyping studies and completing the set of microsatellite markers already available for V. vinifera. Additionally these seven new markers appear to be conserved in four other Vitis species and 15 Vitis hybrids used as rootstocks for V. vinifera cultivation.     

Identification and characterization of 43 novel polymorphic EST-SSR markers for arum lily,Zantedeschia aethiopica (Araceae)

• Premise of the study: A new set of microsatellite or simple sequence repeat (SSR) markers from expressed sequence tags (ESTs) was developed for arum lily (Zantedeschia aethiopica), which is one of the most iconic and widely recognized ornamental plants in the world. • Methods and Results: Using 2175 unigenes derived from 4283 random ESTs in arum lily, 166 primer pairs were designed and tested for amplification in 24 accessions from Asia, Europe, and Africa. A total of 43 loci were polymorphic, with the number of alleles per locus ranging from two to 10. The observed heterozygosity, expected heterozygosity, and polymorphism information content ranged from 0.2313 to 0.8480, 0.3034 to 0.8648, and 0.1015 to 0.7364, respectively. • Conclusions: These novel polymorphic EST-SSR markers will facilitate future studies of genetic variation and molecular-assisted breeding systems in arum lily.