Development and characterization of chloroplast microsatellite markers for <Emphasis Type="BoldItalic">Pinus massoniana</Emphasis> and their application in <Emphasis Type="BoldItalic">Pinus</Emphasis> (Pinaceae) species

Pinus massoniana is one of the important afforestation and pioneer tree species, which is widely distribute in southern China. Chloroplast simple sequence repeat markers (cpSSRs) have been widely used in studies of tree genetics, phylogenetic and breeding. We sequenced the whole chloroplast genome sequences of P. massoniana using PCR and Sanger sequencing. A total of 71 cpSSRs were identified, among which mononucleotide repeats were predominant (70.42%). Seventeen primer pairs were developed and amplification tests were conducted with 15 P. massoniana individuals. Also, cross-species amplification tests were conducted among 15 individuals per Pinus species, including P. elliottii, P. bungeana, P. armandii, P. caribaea, P. tabulaeformis, P. taiwanensis and P. yunnanensis which revealed polymorphic information content ranging from 0.2 to 0.8 and average of haploid diversity (h) ranging from 0.29 to 0.63. In addition, the polymorphic cpSSRs were useful in distinguishing the sampled pine species, and could be powerful tool in phylogenetic studies.     

Identification of Japanese <Emphasis Type="Italic">Lymantria</Emphasis> species (Lepidoptera: Lymantriidae) based on DNA sequences

We examined the utility of the cytochrome c oxidase subunit I (COI) and II (COII) genes of mitochondrial DNA (mtDNA) as a tool to identify nine Japanese Lymantria species, including four Asian gypsy moth species [Lymantria dispar japonica (Motschulsky), Lymantria umbrosa (Butler), Lymantria albescens Hori and Umeno, and Lymantria postalba Inoue]. In phylogenetic trees constructed for the COI and COII genes using the maximum likelihood methods, we could identify seven out of the nine Lymantria species (L. albescens and L. postalba could not be identified). These results suggest that the DNA sequences of the COI and COII genes are useful for identifying Japanese Lymantria species.     

Development of microsatellite markers for the carnivorous plant <Emphasis Type="Italic">Genlisea aurea</Emphasis> (Lentibulariaceae) using genomics data of NGS

Genlisea aurea A.St.-Hil. is a carnivorous plant endemic species to Brazil in the Lentibulariaceae family. Very few studies have addressed the genetic structure and conservation status of G. aurea and the Lentibulariaceae. Microsatellites markers are advantageous tools that can be employed to predict the vulnerability of Lentibulariaceae species. Therefore, the development of molecular markers focusing the population analyses of Genlisea for future genetic studies and conservation actions are essential. Thus, we developed simple sequence repeats (SSRs) based on in silico analyses of G. aurea draft genome assembly. We characterized 40 individuals from several populations and identified 12 loci that were polymorphic, with heterozygosity between 0.123 and 0.650. We demonstrated that the G. aurea SSR markers work cross-species in Genlisea filiformis, G. repens, G. tuberosa and G. violacea. These markers will be important for future population, phylogeographic and conservation studies in G. aurea and other Genlisea species.     

Genetic relationships of Chukchi charr <Emphasis Type="Italic">Salvelinus andriashevi</Emphasis> and Taranetz charr <Emphasis Type="Italic">Salvelinus taranetzi</Emphasis>

On the basis of sequence analysis of the mitochondrial DNA (mtDNA) control region (CR), cytochrome b (Cytb), and cytochrome oxidase-1 (CoI) genes, the relationships of endemic species Salvelinus andriashevi Berg, 1948, represented by the only population from Lake Estikhed (Chukotka), were estimated. The data on the genealogical analysis of mtDNA haplotypes supported phylogenetic closeness of S. andriashevi and S. taranetzi. It was also demonstrated that the specimens of Chukchi charr, along with Salvelinus sp. 4 (Lake Nachikinskoe), S. krogiusae (Lake Dal’nee), S. boganidae and S. elgyticus (Lake El’gygytgyn), and S. a. erythrinus from Canada’s Northwest Territories (NWT) belonged to the Arctic group of Taranetz charr. The problem of coincidence of taxonomic differentiation of charrs of the genus Salvelinus based on morphological and genetic analyses is discussed.     

Phylogeny and character evolution of the Neotropical fern genus <Emphasis Type="Italic">Stigmatopteris</Emphasis> (Dryopteridaceae)

We did a phylogenetic analysis on 21 of the 23 species of Stigmatopteris, a neotropical montane genus of wet forest floors. The analysis was based on four plastid markers: rbcL, rps4-trnS, trnG-trnR, and trnL-trnF. Two of the most closely related families to the Dryopteridaceae (Didymochlaenaceae and Hypodematiaceae) and 12 dryopteroid genera were used as outgroups. Eight morphological characters were mapped on the resulting tree. Stigmatopteris was recovered as monophyletic and sister to a clade formed by polystichoid ferns such as Arachniodes, Dryopteris, and Polystichum. Synapomorphies that distinguish Stigmatopteris are 1-pinnate-pinnatifid laminae, visible punctate glands in the mesophyll, the presence of hydathodes, uniseriate cilia on the scale margins, and loss of indusia. Within the genus are two main clades. The first consists of four species characterized by pinnae long-decurrent in at least the distal third of the lamina. The second consists of the remaining 17 species in the analysis and has no known morphological synapomorphies. Nested within this second clade is a subclade of six species endemic to southeastern Brazil. Anastomosing veins, a character often used in keys to distinguish the species, evolved three times within the genus.     

Tandem repeats analysis for the high resolution phylogenetic analysis of <Emphasis Type="Italic">Yersinia pestis</Emphasis>

Background

Yersinia pestis, the agent of plague, is a young and highly monomorphic species. Three biovars, each one thought to be associated with the last three Y. pestis pandemics, have been defined based on biochemical assays. More recently, DNA based assays, including DNA sequencing, IS typing, DNA arrays, have significantly improved current knowledge on the origin and phylogenetic evolution of Y. pestis. However, these methods suffer either from a lack of resolution or from the difficulty to compare data. Variable number of tandem repeats (VNTRs) provides valuable polymorphic markers for genotyping and performing phylogenetic analyses in a growing number of pathogens and have given promising results for Y. pestis as well.

Results

In this study we have genotyped 180 Y. pestis isolates by multiple locus VNTR analysis (MLVA) using 25 markers. Sixty-one different genotypes were observed. The three biovars were distributed into three main branches, with some exceptions. In particular, the Medievalis phenotype is clearly heterogeneous, resulting from different mutation events in the napA gene. Antiqua strains from Asia appear to hold a central position compared to Antiqua strains from Africa. A subset of 7 markers is proposed for the quick comparison of a new strain with the collection typed here. This can be easily achieved using a Web-based facility, specifically set-up for running such identifications.

Conclusion

Tandem-repeat typing may prove to be a powerful complement to the existing phylogenetic tools for Y. pestis. Typing can be achieved quickly at a low cost in terms of consumables, technical expertise and equipment. The resulting data can be easily compared between different laboratories. The number and selection of markers will eventually depend upon the type and aim of investigations.

     

Assessment of genetic diversity in <Emphasis Type="Italic">Mucuna</Emphasis> species of India using randomly amplified polymorphic DNA and inter simple sequence repeat markers

Genus Mucuna which is native to China and Eastern India comprises of perennial climbing legume with long slender branches, trifoliate leaves and bear green or brown pod covered with soft or rigid hairs that cause intense irritation. The plants of this genus are agronomically and economically important and commercially cultivated in India, China and other regions of the world. The high degrees of taxonomical confusions exist in Mucuna species that make authentic identification and classification difficult. In the present study, the genetic diversity among the 59 accessions of six species and three varieties of M. pruriens has been assessed using DNA fingerprinting based molecular markers techniques namely randomly amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and combined dataset of RAPD and ISSR. Also, genetic relationship among two endemic species of Mucuna namely M. imbricata and M. macrocarpa and two varieties namely IIHR hybrid (MHR) and Dhanwantari (MD) with other species under study was investigated by using cluster analysis and principal coordinate analysis. The cluster analysis of RAPD, ISSR and combined dataset of RAPD and ISSR clearly demonstrated the existence of high interspecific variation than intra-specific variation in genus Mucuna. The utility and efficacy of RAPD and ISSR for the study of intra species and interspecies genetic diversity was evident from AMOVA and PCoA analysis. This study demonstrates the genetic diversity in Mucuna species and indicates that these markers could be successfully used to assess genetic variation among the accessions of Mucuna species.     

Secondary contact between two divergent lineages of charrs of the genus <Emphasis Type="Italic">Salvelinus</Emphasis> in the Northwest Pacific

The present-day contact zone between the Beringian and Arctic phylogenetic lineages of charrs of the genus Salvelinus in the Northwest Pacific is documented. A comparative analysis of the genetic differentiation and divergence indices for allopatric and sympatric populations of charrs and phylogenetic and genealogical analyses of the mtDNA haplotypes indicate that Lake Achchen and the Lake Pekulineiskoe are the zones of secondary contact between S. m. malma and S. taranetzi; Lake Nachikinskoe, between S. m. malma and Salvelinus sp. 4; and Lake Dal’nee, between S. m. malma and S. krogiusae. The level of divergence between phylogenetic groups of haplotypes considerably exceeds the range of intraspecific variability of S. m. malma and could not have been achieved after colonization of the lakes in conditions of sympathy. The obtained data suggest that the territory of Kamchatka was colonized by the common ancestor of the Arctic phylogroup of Taranetz charr.     

Genetic characterization of Amur sturgeon <Emphasis Type="Italic">Acipenser schrenckii</Emphasis> and its hybrid caught around Hokkaido

Genetic characterization was performed in five individuals of wild Amur sturgeon Acipenser schrenckii, and/or its presumed hybrid caught around Hokkaido, using a mitochondrial DNA (mtDNA) marker and two markers of nuclear DNA (nDNA). Genetic analyses indicated that two of the five fish had the mtDNA haplotype of Kaluga, Huso dauricus, whereas the nDNA markers indicated signs for both A. schrenckii and H. dauricus genotypes, referring to a hybrid origin. The other three fish were plausibly pure A. schrenckii. The results indicated the importance of combined usage of mtDNA and nDNA markers for correct species identification in sturgeon.     

Phylogenetic relationships among cultivated <Emphasis Type="Italic">Zanthoxylum</Emphasis> species in China based on cpDNA markers

We here present a molecular phylogenetic analysis of cultivated Zanthoxylum species which have a long history of cultivation both for economic and for chemical values in China. Three cpDNA markers, including matK, rbcL, and trnL-F, were sequenced, with the goals of untangling phylogenetic relationships and inferring biogeographic origin and patterns of distribution among Zanthoxylum species. Based on three cpDNA markers, 19 haplotypes with 64 polymorphic sites in Zanthoxylum provenances were identified in our study. A low genetic differentiation (G _ST ?=?0.271, N _ST ?=?0.373) was observed within Zanthoxylum provenances. Based on phylogenetic tree and haplotype network, all 19 haplotypes were grouped into six clusters. Our results also supported the hypothesis that the so-called “Green Huajiao” belongs to the species Zanthoxylum armatum rather than Zanthoxylum schinifolium. The results also revealed that haplotypes of two cultivated species, Zanthoxylum bungeanum and Z. armatum, most probably diverged during the Late Miocene. Ancestral area reconstruction indicated that cultivated Zanthoxylum species experienced multiple long-distance dispersal events and several vicariance events and the ancestors of Zanthoxylum first colonized Yunnan and Guizhou provinces (D). Accordingly, the current disjunct distribution of Z. bungeanum and Z. armatum may represent long-distance dispersal of ancestors popularly named “Dahongpao” and “Qinghuajiao,” respectively. It is concluded that cpDNA markers may provide a new conceptual and practical opportunity to evaluate genetic diversity and to identify local cultivars of Zanthoxylum, making it a valuable source to include into potential breeding programs.     

Phylogenetic relationships of Sakhalin taimen <Emphasis Type="Italic">Parahucho perryi</Emphasis> inferred from PCR-RFLP analysis of mitochondrial DNA

RFLP analysis of three amplified mtDNA fragments (Cytb/D-loop, ND1/ND2, and ND3/ND4L/ND4) was performed in the following taxa: Parahucho perryi, Hucho taimen, Brachymystax lenok, B. tumensis, Salmo salar, Salvelinus leucomaenis, and S. levanidovi. For mtDNA of P. perryi, a substantial decrease in the haplotype and nucleotide diversity was observed as a result of random genetic drift, caused by a reduction in the effective population size. Nucleotide divergence estimates between the mtDNA haplotypes were determined. Sakhalin taimen P. perryi was found to be approximately equally diverged from S. salar and from the charrs of the genus Salvelinus, by 11.0 and 10.0%, respectively. The divergence between P. perryi and H. taimen constituted 14.6%, between P. perryi and lenoks of the genus Brachymystax, 14.2%, and between H. taimen and Brachymystax, 7.7%. The analysis of possible phylogenetic relationships of the mtDNA from P. perryi among the group of taxa examined confirmed validity of the genus Parahucho. Phylogenetic reconstructions performed showed that robustness of the trees constructed for the complex of phylogenetically informative characters over three mtDNA fragments was considerably higher than that of the trees constructed for individual genes.     

New efficient extragenic microsatellite markers for hemophilia a carrier state diagnostics

In search of new efficient markers for genetic diagnostics of hemophilia A, two tri-nucleotide microsatellite repeats (STR) at chromosome X loci, which flank coagulation factor VIII gene (F8), namely STR HA472—CTT-repeat, which is localized adjacent to the GAB3 gene 163 kb apart from the 3′ end of the F8 gene and STR HA544—repeat (CTT) _x (ATT) _y located at a distance of 375 kb from the 5′ end of the F8 gene were discovered. Detailed analysis using PCR and sequencing has shown that STR HA472 contains two long variable CTT-blocks separated by small spacer CCTCCC. The location of recognition site of restriction endonuclease MnlI (CCTC) in the spacer permits to test differentially the polymorphic blocks and thus to increase the analysis informativity. STR HA544 is also represented by two polymorphic blocks (CTT and ATT), for separate amplification of which highly informative PCR amplification assays were elaborated. The study has been done using DNA samples of 212 individuals (125 women) from 48 families with hemophilia A carriers. Our results point to Mendelian inheritance of the markers studied, a high number of allelic variants and high heterozygosity, which was 90 and 100% for HA544 and HA472, respectively. This permitted us to use these data for practical gene diagnostics of the carriers and prenatal diagnostics of hemophilia A. In addition to high informativity STR HA472 and HA544 are highly important for diagnostics as they are located at a shorter distance than other known extragenic polymorphisms of the F8 gene. In contrast to dinucleotide repeats, trinucleotide repeats are readily tested, not requiring high-resolution electrophoretic systems. In addition, they are located on the opposite sites of the F8 gene. This permits to control homologous recombination events in the locus and thus to prevent diagnostic mistakes.     

Identification and functional analysis of anthocyanin biosynthesis genes in <Emphasis Type="Italic">Phalaenopsis</Emphasis> hybrids

Phalaenopsis species are among the most popular potted flowers for their fascinating flowers. When their whole-genome sequencing was completed, they have become useful for studying the molecular mechanism of anthocyanin biosynthesis. Here, we identified 49 candidate anthocyanin synthetic genes in the Phalaenopsis genome. Our results showed that duplication events might contribute to the expansion of some gene families, such as the genes encoding chalcone synthase (PeCHS), flavonoid 3′-hydroxylase (PeF3′H), and myeloblastosis (PeMYB). To elucidate their functions in anthocyanin biosynthesis, we conducted a global expression analysis. We found that anthocyanin synthesis occurred during the very early flower development stage and that the flavanone 3-hydroxylase (F3H), F3′H, and dihydroflavonol 4-reductase (DFR) genes played key roles in this process. Over-expression of Phalaenopsis flavonoid 3′,5′-hydroxylase (F3′5′H) in petunia showed that it had no function in anthocyanin production. Furthermore, global analysis of sequences and expression patterns show that the regulatory genes are relatively conserved and might be important in regulating anthocyanin synthesis through different combined expression patterns. To determine the functions of MYB2, 11, and 12, we over-expressed them in petunia and performed yeast two-hybrid analysis with anthocyanin (AN)1 and AN11. The MYB2 protein had strong activity in regulating anthocyanin biosynthesis and induced significant pigment accumulation in transgenic plant petals, whereas MYB11 and MYB12 had lower activities. Our work provided important improvement in the understanding of anthocyanin biosynthesis and established a foundation for floral colour breeding in Phalaenopsis through genetic engineering.     

Phylogeography of mitochondrial DNA in South Asian Dolly Varden char <Emphasis Type="Italic">Salvelinus curilus</Emphasis> Pallas, 1814 (Salmoniformes,Salmonidae): Mediated gene introgression?

In 41 individuals of South Asian Dolly Varden char Salvelinus curilus, nucleotide sequences of tRNA-Pro gene fragment (27 bp) and mtDNA control region (483–484 bp) were analyzed. The fish were collected in 20 localities covering virtually the whole range of the species: Kuril Islands, Sakhalin Island, and Primorye. In addition, six individuals of three other char species (S. albus, S. malma, and S. leucomaenis), which are closely related to S. curilus and inhabit the Russian Far East, were examined. In all, we detected 12 different variants of mtDNA haplotypes that formed three distinct groups differing in 14–20 nucleotide positions. The first group consisted of six haplotypes found in S. curilus in Kuril Islands, Sakhalin, and Primorye (mtDNA phylogroup OKHOTSKIA). The second group comprised four haplotypes representing the mtDNA phylogroup BERING, which had been described earlier (Brunner et al, 2001); they were found in S. curilus in Kuril Islands and Sakhalin, as well as in S. albus and S. malma in Kamchatka and northern Kurils. The third group included two haplotypes detected in S. leucomaenis. The existence of two mtDNA lineages (OKHOTSKIA and BERING) in S. curilus from Kurils and Sakhalin was explained by hybridization and mtDNA transfer from S. malma to S. curilus. The absence of the BERING haplotypes in S. curilus from Primorye water reservoirs is related to the physical isolation of the Sea of Okhotsk and Sea of Japan basins in past epochs. On the basis of comparing phylogenetic trees, constructed from the data on allozyme and mtDNA variation, we suggest that in this case, a mediated transfer of mtDNA in Alpinoid chars → S. malma → S. curilus chain could occur.     

Hypervariability of the D-loop region in mitochondrial DNA of Russian sturgeon <Emphasis Type="Italic">Acipenser gueldenstaedtii</Emphasis> (Acipenseriformes,Acipenseridae)

The structure of the D-loop region in mitochondrial DNA (mtDNA) of Russian sturgeon Acipenser gueldenstaedtii from the Azov Sea population was studied with the method of direct sequencing. Interindividual heteroplasmy of the length of mtDNA in the region of D-loop realized by the presence of a different number of tandem repeats (82 pairs of bases) was found. Analysis of tandem repeats in the D-loop region in mtDNA in the studied sample (28 individuals) revealed eight mitotypes differed in the pattern of nucleotide substitution and in the number of tandem repeats (2, 3, and 4 repeats). Revealed mitotypes can be considered as potential genetic markers for different biological groups, schools, or seasonal races of A. gueldenstaedtii.     

Differentiation of <Emphasis Type="Italic">Vaccinium</Emphasis> Cultivars and Wild Clones Using RAPD Markers

The potential use of the random amplified polymorphic DNA (RAPD) technique for characterization and assessment ofgenetic relatedness was investigated in 13 wild cranberry (Vaccinium macrocarpon Ait) clones collected from Newfoundlandand Labrador, Canada. RAPD markers were also used to distinguish representatives of three different Vaccinium species.Twenty-two decamer arbitrary primers yielded informative, reproducible and polymorphic banding patterns in 13 cranberryclones and produced a total of 134 bands, of which 114 were polymorphic. The UPGMA cluster analysis separated the clonesinto two main groups: Cluster I of seven and Cluster II of six with 0.62 to 0.91 and 0.50 to 0.77 Nei and Li’s similarity range,respectively. In another experiment, a subset of eight primers was used and the RAPD markers discriminated the threeVaccinium species: cranberry, lowbush blueberry (V. angustifolium Ait) and lingonberry (V. vitis-idaea L). The RAPDpolymorphisms will be useful for Vaccinium genotype differentiation and the technology should be valuable for themaintenance of germplasm banks and the efficient choice of parents in the current Vaccinium germplasm improvementprogram.     

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.