Development and characterization of microsatellite markers for Echinopsis rhodotricha and cross-amplification in other species of Cactaceae

Located in central South America, the Chaco is a large subtropical dry forest characterized by plants with xeromorphic features, including numerous cacti. In the Brazilian Chaco at the northeasternmost part of the Chaquenian region, Echinopsis rhodotricha (Cactaceae, Trichocereeae) is one of the species threatened by intense deforestation caused by expanding pastureland. This study characterizes the microsatellite loci isolated from E. rhodotricha and cross-amplification in thirteen other cactus species. Twelve microsatellite loci were developed from an enriched genomic library. Eight of these were polymorphic and characterized in 48 individuals from three E. rhodotricha populations. The loci showed a mean of 3.2 alleles per locus and overall levels of expected and observed heterozygosities ranging from 0.00 to 0.83 and 0.00 to 0.66, respectively. Five loci showed significant departures from the Hardy–Weinberg equilibrium and also exhibited signs of null alleles. Cross-amplification in other Cactaceae species was successful, ranging from one (Ferocactus latispinus, Cacteae and Harrisia adscendens, Trichocereeae) to twelve loci (Echinopsis calochlora, Trichocereeae). The development of these microsatellite markers will contribute to investigations of population structure, genetic diversity, and gene flow in E. rhodotricha populations, as well as in other cactus species, providing information useful for the creation and delimitation of conservation areas in the Brazilian Chaco region.     

High ISSR Variation in 14 Surviving Individuals of Euryodendron excelsum (Ternstroemiaceae) Endemic to China

Euryodendron excelsum is a critically endangered Ternstroemiaceae species endemic to southern China, with only 14 individuals surviving in Ba Jia Zhen of Yangchun, Guangdong Province. Intersimple sequence repeat (ISSR) markers were used to assess genetic variation and relationships among these individuals. Population genetic parameters were estimated by a Bayesian approach as well as conventional methods. Of the 225 loci generated by 21 primers, 147 (65.33%) were polymorphic. Compared with other species of Theaceae and related families, a high level of genetic variation was identified in E. excelsum (Nei’s gene diversity, 0.2458; Shannon’s index, 0.3626). An unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed that the 14 individuals were mainly clustered into three groups, a conclusion further supported by principal coordinate analysis. Based on these results, a management and conservation strategy for E. excelsum was proposed.     

Guidelines for the restoration of the tropical timber tree <Emphasis Type="Italic">Anacardium excelsum</Emphasis>: first input from genetic data

Translocation of trees has been used as a common method to mediate genetic conservation and restoration of forests. However, very few programs include strategies developed to recover or maintain the genetic diversity of the translocated species. Anacardium excelsum is a tree native to the tropics of America that is extensively used in forestry. In Colombia, restoration of forests through the translocation of native species has regained importance, and A. excelsum has been recently included in the National Strategy for Plant Conservation. Thus, in order to define the level of genetic structure and the level of genetic diversity within certain regions where remnants of the seasonally dry tropical forests (SDTF) of Colombia have been retained, we genotyped 106 trees using nuclear inter-simple sequence repeats (ISSR) and sequenced two non-coding chloroplast loci for these specimens. Our ISSR dataset revealed the existence of a gradient in genetic diversity within A. excelsum with the most diverse remnants encountered in the south of the country, while the localities sampled in the Caribbean coast and in the Chicamocha canyon were less diverse. Chloroplast loci also pointed out the very low genetic diversity of A. excelsum from the Chicamocha canyon and we propose to prioritize this area within future conservation programs. Both chloroplast and nuclear markers supported the existence of genetic divergence between distinct regions of Colombia, uncovering genetic differences between inter-Andean, Caribbean, and Chicamocha canyon A. excelsum remnants. Hence, we advise to choose the provenance of seeds or plants carefully before translocation and to consider minimal mixing of material from different regions when initializing restoration programs for A. excelsum, in Colombia.     

Development of 10 microsatellite loci from the Korean Ratsnake (Elaphe schrenckii) and its application across Elaphe species from South Korea, Russia, and China

We have developed 10 microsatellite loci from the Korean ratsnake Elaphe schrenckii. Polymorphism of each locus was assessed in 25 unrelated individuals for E. schrenckii from South Korea, 10 E. anomala and 10 E. schrenckii from China, and 10 E. schrenckii from Russia. E. anomala, a closely related species to E. schrenckii in China. The number of alleles per locus varied from 1–9 (mean 4.4) for Korean populations, 2–7 (mean 3.9) for Chinese E. anomala, 1–7 (mean 4.0) for Chinese E. schrenckii, and 1–4 (mean 2.5) for Russian E. schrenckii. The average expected heterozygosity was 0.500, 0.534, 0.487, and 0.338 for E. schrenckii from South Korea, E. anomala from China, E. schrenckii from China, and E. schrenckii from Russia, respectively. The microsatellite markers used in this study may be applied to other Elaphe species for population analysis, eliminating the time-consuming task of finding new loci. These selected markers are useful for assessing population structure, intraspecific variation, and conservation and management of E. schrenckii.     

Discovery of four natural clones in a crayfish species Procambarus clarkii

Self-cloning is quite rare in shrimp, lobsters, crayfish and crabs. Here we report the discovery of four natural clones of red swamp crayfish (Procambarus clarkii), each containing 2-6 genetically identical individuals, during the genotyping of 120 individuals with five microsatellites. The four clones were heterozygote at most of the five microsatellite loci. Phylogenetic analysis using microsatellite genotypes suggests recent origin of the four clones. Sequencing a part of the mitochondrial gene Cox I confirmed that the four clones were from the species Procambarus clarkii.     

Isolation and characterization of 19 polymorphic microsatellite loci from Neosalanx taihuensis,a rapidly invasive and adaptative species

Neosalanx taihuensis can very rapidly adapt to new environmental conditions and quickly becomes the dominant species when introduced to lakes and reservoirs. To better understand the genetic mechanism responsible for this remarkable ability to invade and adapt to diverse ecosystems, 19 polymorphic microsatellite loci were isolated from the genomic DNA of N. taihuensis. These microsatellite loci were identified from 24 individuals from an invasive population sampled in Fuxian Lake, Yunnan Plateau, Southwest China. The number of alleles per locus ranged from 2 to 14. Observed and expected heterozygosities ranged from 0.0417 to 0.8636 and from 0.3112 to 0.9202, respectively. The polymorphism information content (PIC) varied from 0.2583 to 0.8928. These microsatellite loci will be essential in performing molecular analyses of genetic diversity and genetic structure in the future. This will ultimately facilitate better understanding of the genetic mechanisms that allow N. taihuensis to successfully invade and adapt to new ecosystems.     

Isolation and characterization of thirteen microsatellite loci in four diploid species of Ranunculus cantonensis polyploid complex

Ranunculus is a cosmopolitan genus, and the evolution of the genus has been influenced by both polyploidy and hybridization. The interactions between some species of Ranunculus are regarded as a natural model system for studying hybridization. Four diploid species of the R. cantoniensis polyploid complex have relatively little reproductive isolation from each other, and they form a complicated hybrid pool in a multispecies hybrid zone. To clarify the genetic structure and spatiotemporal dynamics of gene flow in this multispecies hybrid zone, 13 polymorphic microsatellite loci were isolated from R. silerifolius var. dolicanthus. The loci's characteristics were observed in 30 individuals. The number of alleles per locus ranged from 6 to 20, and the PIC values ranged from 0.350 to 0.914. Eleven loci were significantly biased from the Hardy–Weinberg equilibrium. The 13 microsatellite loci were shown to be successfully amplified in three other related species. These polymorphic microsatellites will be powerful molecular tools for further studies of the Ranunculus genetic structure and gene flow.     

Characterization of new microsatellite loci from the razor clam (Sinonovacula constricta) and transferability to related species

Simple sequence repeats (SSRs) have been widely used in marine animals, but were seldom used directly to test intra- and inter-specific transferability in the populations of related species. 19 new pairs of microsatellite primers were obtained from Sinonovacula constricta. The allele number of these markers ranged from 3 to 15. The observed and expected heterozygosities changed from 0.033 to 1.000 and from 0.033 to 0.912, respectively. Three loci (SC3-8, SC3-19, and SC4-17) significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction, and none of the loci showed linkage disequilibrium. In addition, the transferability of all polymorphic SSRs was assessed in three closely related species. The results showed that four loci were informative in Solen strictus (SC2-11, SC2-12, SC4-19 and SC2-2) and Cultellus attenuates (SC2-2, SC4-9, SC4-19 and SC3-11), respectively, and two loci (SC2-2 and SC2-11) were informative in Solen grandis. These loci will be useful for the evaluation of genetic diversity, connectivity among related species and stock management.     

Characterization of 13 polymorphic microsatellite loci in the Japanese land leech

We developed 13 polymorphic microsatellite loci of the Japanese land leech (Haemadipsa japonica; Haemadipsidea) using an Illumina MiSeq sequencing approach. A total of 42,064 nuclear DNA contigs were filtered for microsatellite motifs, among which 30,873 simple sequence repeat loci were identified. From these sequences, we selected 30 primer sets, and 13 of these loci were successfully amplified. Polymorphism of the 13 loci was tested using 16 individuals sampled from sixteen populations across Japan. The number of alleles and polymorphism information content varied from 5 to 17 and 0.335 to 0.883, respectively, and observed and expected heterozygosity values ranged from 0.143 to 0.875 and 0.349 to 0.893, respectively, indicating that these loci are polymorphic. Furthermore, we established useful multiplex PCR using these loci. The 13 microsatellite loci described in this paper are the first nuclear microsatellite markers for a land leech species.     

Potentials and limitations of the cross-species transfer of nuclear microsatellite marker in six species belonging to three sections of the genus Populus L.

The genus Populus is classified into six different sections, and depending on the declaration of hybrids, the number of species varies between 22 and 85. Species within one section, and sometimes between sections, are crossable to each other, resulting in many naturally but also artificially produced hybrids. Morphological attributes for a clone characterisation are often difficult to evaluate when different poplar species or even hybrids are crossed; thus, molecular markers are needed to characterise the different species. Taking advantage of the large microsatellite resource developed for Populus trichocarpa, however, amplification of these microsatellite markers in other Populus species either often fails, or in the case of amplification, unrelated genomic regions are amplified. To meet this obvious problem of the species transferability of microsatellite markers, in total, 305 microsatellite loci, mainly from P. trichocarpa but also few from Populus tremuloides and Populus nigra, were tested for their transferability to diverse genotypes of six species belonging to three sections of the genus Populus. Ultimately, 209 microsatellite loci could be amplified with varying sizes in the different species. The PCR products of selected loci were separated in a polyacrylamide gel and sequenced to assure that the expected loci were derived from the database genome of P. trichocarpa. The present results constitute a large study for microsatellite transferability for Populus species. The documented microsatellite loci can be applied to species-, hybrid- and clone-specific diagnostic approaches or as universal markers for comprehensive ecological studies.     

Amplistroma erinaceum,a new species and its anamorph from Panama

The new species Amplistroma erinaceum, collected on wood of Anacardium excelsum, is characterized by light cream-coloured, rostrate to spinose stromata, and immersed perithecia and long ostiolar necks. Its relationship to other Amplistroma species within the Amplistromataceae (incertae sedis, Ascomycota) are studied based on morphological, cultural, and molecular sequence data of the large subunit ribosomal DNA (28S rDNA).     

跨种扩增筛选大鳄龟微卫星标记及遗传多样性分析

跨种扩增是一种能够快速、有效地获得物种微卫星标记的方法。本研究利用在近缘种中已发表的微卫星DNA引物,对大鳄龟（Macroclemys temminckii）进行跨种PCR扩增,在合成的69对引物中获得8对具有多态性的微卫星位点。对PCR扩增产物进行统计,得出观测杂合度（H_o）的范围是0.041 7~0.954 5,平均为0.384 8;期望杂合度（H_E）的范围为0.041 7~0.811 8,平均为0.479 1;多态信息含量范围为0.040 0~0.759 2,平均为0.423 2;经过卡方检验后,部分微卫星位点符合哈迪-温伯格平衡。总体来说,这些位点是研究大鳄龟遗传结构的良好分子标记。     

Development of microsatellite markers for muskellunge (Esox masquinongy) and cross‐species amplification in two other esocids

We report the development of seven polymorphic microsatellite loci in muskellunge (Esox masquinongy) using an unenriched subgenomic library. Polymorphic loci exhibited 2–11 alleles with observed heterozygosities ranging from 0.190 to 0.917 (n = 24). All seven loci amplified by their respective primer pairs resulted in monomorphic products in northern pike (E. lucius) whereas three loci amplified monomorphic products in grass pickerel (E. americanus americanus); these results imply conservation of flanking sequence but loss of polymorphism between these closely related species. Only one of six microsatellite primers developed in a previous study in northern pike amplified polymorphic products in muskellunge.     

Intrageneric amplification of horse microsatellite markers with emphasis on the Przewalski's horse (E. pmewalskii)

Primer sequences flanking 13 microsatellite loci isolated from the domestic horse (E. caballus) were successfully used to amplify homologous loci in the Przewalski's horse (E. przewalskii). The results demonstrate that the level of polymorphism at all 13 loci in the Przewalski's horse was comparable to that in the domestic horse and the overall exclusion probability in the Przewalski's horse was calculated to be 0.9994. The results suggest that it should be possible to use E. caballus-derived microsatellite markers to provide parentage verification and additional valuable information to the captive management of E. przewalskii. The ability to amplify corresponding loci in the remaining five species of the genus was also confirmed, illustrating the general application of markers isolated from the domestic horse to the evaluation of polymorphism in the other six species of the genus.     

Genetic Differentiation and Conservation of 14 Surviving Individuals of Euryodendron excelsum Endemic to China

Random amplification polymorphic DNA (RAPD) markers were used to assess the genetic variations and the evolutionary relationships among all 14 individuals of a critically endangered Euryodendron excelsum (Theaceae) population distributed in Ba Jia Zhen, Yangchun, Guangdong, China. Twenty-three random primers detected 156 sites, out of which 95 (60.26%) were polymorphic loci. The number of the observed alleles was 1.6090, and the number of the effective alleles was 1.3471. Nei’s gene diversity was 0.1993, and Shannon index was 0.1534. A relatively high level of genetic variation was identified in E. excelsum. An unweighted pair group method with arithmetic mean (UPGMA) tree established from Jaccard similarity coefficients suggested that 14 individuals were clustered into two subgroups and that the No. 2 plant was genetically distant from the rest of the individuals. The UPGMA clustering was also supported by a principle components analysis of RAPD phenotypic data. The management and conservation strategy of E. excelsum was proposed based on our results. Translated from Acta Scientiarum Naturalium Universitatis Sunyatseni, 2005, 44(1) (in Chinese)     

Development and characterization of 24 chloroplast microsatellite markers for two species of <Emphasis Type="Italic">Eranthis</Emphasis> (Ranunculaceae)

Chloroplast microsatellites for two Korean endemic species, Eranthis byunsanensis and E. pungdoensis (Ranunculaceae), were isolated to address the questions of their distributional patterns and evolutionary relationships, using next-generation sequencing. Twenty-four polymorphic chloroplast microsatellite markers for these two species were developed, and then characterized in 65 individuals (55 individuals of E. byunsanensis and 10 individuals of E. pungdoensis). The number of alleles per locus ranged from 2 to 9; the average number of alleles across all the loci scored 4.792. The unbiased diversity per locus ranged from 0.089 to 0.880; the unbiased diversity averaged over all the loci was 0.646. The developed markers were successfully amplified for three congeneric species, E. stellata, E. pinnatifida, and E. longistipitata. The markers developed in this study can provide a valuable and important tool for understanding genetic variations, population structures, evolutionary histories and phylogeography of E. byunsanensis, E. pungdoensis, and related species.     

Characterizations of Enterocytozoon bieneusi at new genetic loci reveal a lack of strict host specificity among common genotypes and the existence of a canine-adapted Enterocytozoon species

Molecular characterizations of the microsporidian pathogen Enterocytozoon bieneusi at the ribosomal internal transcribed spacer (ITS) locus have identified nearly 500 genotypes in 11 phylogenetic groups with different host ranges. Among those, one unique group of genotypes, Group 11, is commonly found in dogs. Genetic characterizations of those and many divergent E. bieneusi genotypes at other genetic loci are thus far impossible. In this study, we sequenced 151 E. bieneusi isolates from several ITS genotype groups at the 16S rRNA locus and two new semi-conservative genetic markers (casein kinase 1 (ck1) and spore wall protein 1 (swp1)). Comparison of the near full (~1,200 bp) 16S rRNA sequences showed mostly two to three nucleotide substitutions between Group 1 and Group 2 genotypes, while Group 11 isolates differed from those by 26 (2.2%) nucleotides. Sequence analyses of the ck1 and swp1 loci confirmed the genetic uniqueness of Group 11 genotypes, which produced sequences very divergent from other groups. In contrast, genotypes in Groups 1 and 2 produced similar nucleotide sequences at these genetic loci, and there was discordant placement of ITS genotypes among loci in phylogenetic analyses of sequences. These results suggest that the canine-adapted Group 11 genotypes are genetically divergent from other genotype groups of E. bieneusi, possibly representing a different Enterocytozoon sp. They also indicate that there is no clear genetic differentiation of ITS Groups 1 and 2 at other genetic loci, supporting the conclusion on the lack of strict host specificity in both groups. Data and genetic markers from the study should facilitate population genetic characterizations of E. bieneusi isolates and improve our understanding of the zoonotic potential of E. bieneusi in domestic animals.     

Novel microsatellite markers obtained from Gansu zokor (Eospalax cansus) and cross-species amplification in Plateau zokor (Eospalax baileyi)

Zokor (Mysopalacinae) is a group of subterranean rodents. Although they are regarded as important components in the ecosystems they belong to, due to their underground lifestyle, very little is known about their ecology and behavior. Development of microsatellite markers can potentially assist in addressing behavioral and ecological questions such as dispersal, mating and social systems. Here we report the development of 11 polymorphic microsatellite loci from the genome of Eospalax cansus using 454 shot-gun sequencing. Genetic diversity was assessed using DNA samples of 47 individuals of E. cansus from a single location. Cross-amplification in Eospalax baileyi was also tested, with five polymorphic markers being amplified successfully. These markers are the first published microsatellites loci from E. cansus and will be invaluable for studies addressing ecological and behavioral questions involving E. cansus and E. baileyi, and potentially other species in Mysopalacinae.     

Development and characterization of microsatellite markers for Fusarium virguliforme and their utility within clade 2 of the Fusarium solani species complex

Clade 2 of the Fusarium solani species complex contains plant pathogens including Fusarium virguliforme and closely related species Fusarium brasiliense, Fusarium crassistipitatum, Fusarium tucumaniae, which are the primary causal agents of soybean sudden death syndrome (SDS), a significant threat to soybean production. In this study, we developed microsatellite markers from a F. virguliforme genome sequence and applied them to a F. virguliforme population collection of 38 isolates from Michigan and four reference strains from other locations. Of the 225 detected microsatellite loci, 108 loci were suitable for primer design, and 12 of the microsatellite markers were determined to be highly polymorphic, amplifying on average 5.7 alleles per locus. Using these markers, F. virguliforme isolates were partitioned into three distinct clusters, but isolates were not grouped based on relatedness of sampling sites. In addition, 11 out of 12 markers were demonstrated to be highly transferrable to other closely related species.