Genetic differentiation of <Emphasis Type="Italic">Puccinia triticina</Emphasis> Erikss. in Russia

A collection of Puccinia triticina isolates was characterized for polymorphism of microsatellite loci and estimated for their differentiation by geographic origin. The collection included 20 isolates from the Ural region, 31 from West Siberia, 53 from Central Europe, 32 from the Northwest region, 32 from the Volga region, and 40 from the North Caucasus (24 from Dagestan and 16 from Krasnodar and Stavropol). The studied isolates were represented by 65 virulence phenotypes. In the polymorphism analysis of 18 microsatellite loci, 69 genotypes were determined. The index values of genetic distances (F _st, R _st, KB _c) between populations for microsatellite loci indicated differentiation of P. triticina isolates according to geographical origin, and they were clustered into three groups: (1) Asian, (2) European, and (3) North Caucasian. The North Caucasian isolates from Krasnodar and Stavropol regions were closer in similarity to European isolates than the Dagestan ones. Current analysis confirmed the assumption made earlier on the basis of the virulence analysis about the existence of several geographic fungi populations in Russia.     

Microsatellite loci polymorphism of chloroplast DNA of scots pine (Pinus sylvestris L.) in Asia and eastern Europe

The variability of four microsatellite loci of chloroplast DNA was studied in 38 populations of Pinus sylvestris in the European part of Russia, in Siberia, Kazakhstan, Transbaikalia and Mongolia. High variability was observed in all regions. In total, 152 haplotypes were identified. The level of population differentiation R _ST was 2.1%. The differentiation of three geographical groups of populations (European, Siberian-Kazakhstan and Transbaikalian-Mongolian) was insignificant (R _CT = 0.004). At the same time, some rare haplotypes were found to be specific for a certain geographical region. Distribution of the rare haplotypes, which differentiated European populations from Asian populations and Mongolian and Transbaikalian populations from the Siberian, showed the independence of the history of these regions. This corresponds more to the hypothesis that the modern area of Pinus sylvestris originated via settlement from many origins than to the hypothesized single center of the post-glacial recolonization. The distribution of the pairwise differences between the individual specimens corresponded to the model of sudden population growth. The assessments of the age of this event for Pinus sylvestris (4.5–4.7 million years), which were obtained on the basis of this model, significantly exceeded the age of the Quaternary. Therefore, the revealed population growth is hardly due to the changes in flora related with the glaciation, but rather mirrors the moment of the species formation.     

Genetic variability of brown bear (<Emphasis Type="Italic">Ursus arctos</Emphasis> L., 1758)

We perform an analysis of the diversity of the control region fragment (570 bp) and ten nuclear microsatellite loci for 36 specimens of the brown bear (Ursus arctos) from several regions of the Caucasus. Four mitochondrial haplogroups that significantly differ from each other are revealed. An assumption of reiterative brown bear colonization of the Caucasus is made. Microsatellite data analysis shows a medium level of heterozygosity and clear differentiation between Caucasian and European bears combined with a slight distinction between the Caucasian regions.     

Genome-Wide Microsatellite Identification in the Fungus Anisogramma anomala Using Illumina Sequencing and Genome Assembly

High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers (also known as Simple Sequence Repeats). Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N₅₀ of 10,384 bp and scaffold N₅₀ of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery.     

Dramatic expansion of the range of the invasive ash pest,buprestid beetle Agrilus planipennis Fairmaire, 1888 (Coleoptera,Buprestidae) in European Russia

The emerald ash borer (Agrilus planipennis Fairmaire, 1888) is a dangerous invasive pest of ashes. It was previously believed that in European Russia it occurs only in Moscow, Moscow Province, and in the eastern regions of Smolensk Province. An examination of ash trees in 12 cities of European Russia has revealed a much wider area of the emerald ash borer invasion. The pest was found in Konakovo (Tver Province), Tula, Kaluga, Orel, and Voronezh. We have found that A. planipennis damages not only Fraxinus pennsylvanica (an American species which is commonly planted in cities), but also the aborigine European ash Fraxinus excelsior. Ashes in the European forests and in the protective forest belts are badly endangered.     

Genetic Diversity of Some Pear Cultivars and Genotypes Using Simple Sequence Repeat (SSR) Markers

The genetic diversity and relationships among 47 pear cultivars and genotypes (Pyrus spp.), including 4 Japanese pears (Pyrus pyrifolia), 40 European pears (Pyrus communis), 1 Chinese pear (Pyrus bretschneideri) as well as 2 wild relatives (Pyrus salicifolia and Pyrus mazandaranica) were studied using 28 microsatellite primer pairs. A total of 174 alleles were produced at the 28 SSR loci with their sizes ranging from 81 to 290?bp. The number of observed alleles for each locus ranged from 3 (TsuENH014 and TsuENH046) to 12 (NB103a), with an average of 6.21 alleles per locus. In some SSR loci, more than two alleles were amplified in some cultivars and genotypes, suggesting that duplication has occurred in those accessions. This information suggests that at least two genomic regions exist for these loci in the pear genome. The observed heterozygosity (H _o) values of amplified loci ranged from 0.17 (TsuENH006) to 0.97 (NB103a). Shannon's information index (I) value was observed to be highest (2.14) in the NB103a locus, while the TsuENH006 locus had the lowest value with an average of 1.37 among SSR loci. The Dice genetic similarity coefficient ranged from 0.29 (??Nijisseiki?? and P. mazandaranica) to 0.91 (??Chojuro?? and ??Nijisseiki??) among samples. UPGMA cluster analysis showed two major groups corresponding to the Japanese and European pears.     

Somaclonal variation of haploid in vitro tissue culture obtained from Siberian larch (Larix sibirica Ledeb.) megagametophytes for whole genome de novo sequencing

The objective of this study was to obtain a genetically stable haploid in vitro-derived line from Siberian larch (Larix sibirica Ledeb.) using megagametophyte explants, which then could be used for different molecular genetic studies, including whole genome de novo sequencing. However, cytogenetic analysis and genotyping of 11 microsatellite loci showed high levels of genomic instability and a high frequency of mutation in the obtained megagametophyte-derived callus cultures. All cultures contained new mutations in one or more microsatellite loci.     

From the cradle of grapevine domestication: molecular overview and description of Georgian grapevine (Vitis vinifera L.) germplasm

Historical information and archaeological and palaeobotanical findings point Georgia, in the South Caucasus, as a cradle for grapevine (Vitis vinifera L.) domestication from its wild form (V. vinifera silvestris Beck.) and subsequent selection and development of varieties with characters suitable for human consumption. The hypothesis of Georgia being a center of domestication, combined with its distance from western countries and the importance of its viticulture and wine production, make Georgian grape germplasm particularly interesting to be investigated under the genetic point of view. Twenty nuclear microsatellite loci were used to genotype 112 Georgian grapevine accessions (V. vinifera sativa Beck.) from germplasm collections and 18 from spontaneous growing plants (V. vinifera silvestris Beck.) found in wild conditions and to compare them to a large international cultivar collection in France. Data analysis shows that Georgian grapevine germplasm has maintained distinctive traits despite arrival of international, foreign varieties and still conserve characteristics of local breeding linked to traditional wine production regions of the country. Results have identified alleles, overall loci, well represented in the Georgian germplasm (cultivated and wild) and absent or poorly represented in other countries, highlighting uniqueness and originality of traits of this viticulture. Moreover, the search for relationships between Georgian and foreign viticulture has evidenced few interesting cases linking the Georgian varieties with Western European ones and with neighboring Caucasian countries, helping to identify the real place of origin in some doubtful cases. In addition, populations or sparse individuals of wild grapevine still preserved in the Georgian natural environments present smaller genetic distances with local cultivars than in other European regions. Principal component analysis (PCA) has also identified special overlapping of the wild compartment with some cultivated varieties. This work provides a highly significant new contribution to applied aspects of Georgian grapevine genetic resources management and use. Uniqueness of the Georgian cultivated grapevine gene pool together with its close relatedness with the wild compartment makes this country a good candidate to address questions regarding domestication and grapevine genetic resource conservation.     

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.     

Whole Genome Association Mapping of Plant Height in Winter Wheat (Triticum aestivum L.)

The genetic architecture of plant height was investigated in a set of 358 recent European winter wheat varieties plus 14 spring wheat varieties based on field data in eight environments. Genotyping of diagnostic markers revealed the Rht-D1b mutant allele in 58% of the investigated varieties, while the Rht-B1b mutant was only present in 7% of the varieties. Rht-D1 was significantly associated with plant height by using a mixed linear model and employing a kinship matrix to correct for population stratification. Further genotyping data included 732 microsatellite markers, resulting in 770 loci, of which 635 markers were placed on the ITMI map plus a set of 7769 mapped SNP markers genotyped with the 90 k iSELECT chip. When Bonferroni correction was applied, a total of 153 significant marker-trait associations (MTAs) were observed for plant height and the SSR markers (−log₁₀ (P-value) ≥4.82) and 280 (−log₁₀ (P-value) ≥5.89) for the SNPs. Linear regression between the most effective markers and the BLUEs for plant height indicated additive effects for the MTAs of different chromosomal regions. Analysis of syntenic regions in the rice genome revealed closely linked rice genes related to gibberellin acid (GA) metabolism and perception, i.e. GA20 and GA2 oxidases orthologous to wheat chromosomes 1A, 2A, 3A, 3B, 5B, 5D and 7B, ent-kaurenoic acid oxidase orthologous to wheat chromosome 7A, ent-kaurene synthase on wheat chromosome 2B, as well as GA-receptors like DELLA genes orthologous to wheat chromosomes 4B, 4D and 7A and genes of the GID family orthologous to chromosomes 2B and 5B. The data indicated that besides the widely used GA-insensitive dwarfing genes Rht-B1 and Rht-D1 there is a wide spectrum of loci available that could be used for modulating plant height in variety development.     

Polymorphic microsatellite markers in the guava fruit fly, Bactrocera correcta (Diptera: Tephritidae)

Bactrocera correcta (Bezzi) is one of the most destructive insect pests of fruits and vegetables in tropical and subtropical regions. At present, this fly is primarily distributed in Southeast Asia. Twelve microsatellite loci were isolated from an enriched genomic library based on a biotin/streptavidin capture protocol. The polymorphism of these loci was tested on 74 individual flies from two natural populations. Allele number ranged from 6 to 14 and 10 loci demonstrated a polymorphic information content (PIC) greater than 0.5. The pairwise F _ST value between the two populations was 0.0048 (P > 0.05). These microsatellite loci have potential utility for studies of population genetic structure in this species.     

Extensive Microsatellite Variation in Rice Induced by Introgression from Wild Rice (Zizania latifolia Griseb.)

Background

It is widely accepted that interspecific hybridization may induce genomic instability in the resultant hybrids. However, few studies have been performed on the genomic analysis of homoploid hybrids and introgression lines. We have reported previously that by introgressive hybridization, a set of introgression lines between rice (Oryza sativa L.) and wild rice (Zizania latifolia Griseb.) was successfully generated, and which have led to the release of several cultivars.

Methodology

Using 96 microsatellite markers located in the nuclear and organelle genomes of rice, we investigated microsatellite stability in three typical introgression lines. Expression of a set of mismatch repair (MMR) genes and microsatellite-containing genes was also analyzed.

Results/Conclusions

Compared with the recipient rice cultivar (Matsumae), 55 of the 96 microsatellite loci revealed variation in one or more of the introgression lines, and 58.2% of the altered alleles were shared by at least two lines, indicating that most of the alterations had occurred in the early stages of introgression before their further differentiation. 73.9% of the non-shared variations were detected only in one introgression line, i.e. RZ2. Sequence alignment showed that the variations included substitutions and indels that occurred both within the repeat tracts and in the flanking regions. Interestingly, expression of a set of MMR genes altered dramatically in the introgression lines relative to their rice parent, suggesting participation of the MMR system in the generation of microsatellite variants. Some of the altered microsatellite loci are concordant with changed expression of the genes harboring them, suggesting their possible cis-regulatory roles in controlling gene expression. Because these genes bear meaningful homology to known-functional proteins, we conclude that the introgression-induced extensive variation of microsatellites may have contributed to the novel phenotypes in the introgression lines.     

Polymorphism of microsatellite markers in Russian common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.) breeds

Using five microsatellite loci, genotyping and genetic diversity estimates were obtained for nine samples representing seven common carp breeds most widespread in Russia. For comparison, the samples of Amur wild common carp (Cyprinus carpio haematopterus) and a sample of European Hungarian carp were used. In the samples examined (n = 148) a total of 78 alleles were revealed. The highest mean allele number per locus (7.3) was identified in Amur wild common carp, while the lowest number was found in Cherepets carps (4.0). In different breeds, the observed heterozygosities varied from 0.819 (Altai carp) to 0.651 (Cherepets scaly carp). Three out of five microsatellite loci (MFW-24, MFW-28, and MFW-19) revealed a high level of population differentiation. In the dendrogram of genetic differences, all breeds clustered into two groups. One of these groups was composed of the two strains of Ropsha carp, Stavropol carp, Amur wild common carp, and the two samples of Cherepets carp. The second cluster included Altai carp (Priobskii and Chumysh populations), two Angelinskii carp breeds (mirror and scaly), and Hungarian carp. The pairs of breeds/populations/strains, having common origin, were differentiated. Specifically, these were two populations of Altai carp, two strains of Ropsha carp, as well as the breeds of Angelinskii and Cherepets carps. The reasons for genetic differentiation of Russian common carp breeds, as well as the concordance of the evolutionary histories of these breeds, some of which originated from the European breeds, while the others contain substantial admixture of the Amur wild common carp, are discussed.     

Characterization and mapping of sequence-tagged microsatellite sites in the chickpea (Cicer arietinum L.) genome

A size-selected genomic library comprising 280,000 colonies and representing ≈18% of the chickpea genome, was screened for (GA)_n, (GAA)_n and (TAA)_n microsatellite-containing clones, of which 389 were sequenced. The majority (～75%) contained perfect repeats; interrupted, interrupted compound and compound repeats were only present in 6%–9% of cases. (TAA)-microsatellites contained the longest repeats, with unit numbers from 9 to 131. For 218 loci primers could be designed and used for the detection of microsatellite length polymorphisms in six chickpea breeding cultivars, as well as in C. reticulatum and C. echinospermum, wild, intercrossable relatives of chickpea. A total of 174 primer pairs gave interpretable banding patterns, 137 (79%) of which revealed at least two alleles on native polyacrylamide gels. A total of 120 sequence-tagged microsatellite site (STMS) markers were genetically mapped in 90 recombinant inbred lines from an inter-species cross between C. reticulatum and the chickpea cultivar ICC 4958. Markers could be arranged in 11 linkage groups (at a LOD score of 4) covering 613?cM. Clustering as well as random distribution of loci was observed. Segregation of 46 markers (39%) deviated significantly (P?≥?0.05) from the expected 1:1 ratio. The majority of these loci (73%) were located in three distinct regions of the genome. The present STMS marker map represents the most advanced co-dominant DNA marker map of the chickpea genome.     

Estimation of the Genetic Diversity of Willow Warbler Populations of the Subspecies <Emphasis Type="Italic">Phylloscopus trochilus acredula</Emphasis> (L.) in Different Parts of Its Nesting Area in the European Part of Russia

The genetic diversity (GD) of the Phylloscopus trochilus acredula subspecies populations was studied in different parts of the breeding area in the European part of Russia using microsatellite analysis. It was detected that the level of GD sufficient for the preservation of population genetic structure of the species with optimal ratio of intra- and interpopulation components of the gene diversity and minimal deviations from panmixia is maintained in the studied populations. The maximal values of GD indices were registered in an ecologically suboptimum zone on the periphery of the breeding range.     

Siberian Roe Deer (<Emphasis Type="Italic">Capreolus pygargus</Emphasis> Pallas, 1771) in Ukraine: Analysis of the Mitochondrial and Nuclear DNA

A molecular-genetic analysis of the nucleotide sequences of the cytochrome b gene (1140 base pairs) of the mitochondrial DNA and 17 microsatellite loci of eight samples of roe deer from the Samara forest of Dnipropetrovsk oblast (Ukraine) was carried out. For comparison, 212 corresponding mtDNA sequences of the Siberian and European roe deer and data on the variability of microsatellite markers in 49 representatives of these species were included in the study. It was noted that all the analyzed mitochondrial sequences of individuals from the Samara forest are characteristic of the Siberian roe Capreolus pygargus Pallas, 1771. Four haplotypes were described, all of which belonged to the haplogroup typical for the western part of the range of C. pygargus. A fragment analysis of the microsatellite loci of nuclear DNA confirmed the identification of the investigated group with the Siberian species.     

Mapping of the loose smut resistance gene Ut6 in wheat (Triticum aestivum L.)

Loose smut of wheat (Triticum aestivum L.) caused by Ustilago tritici (Pers.) Rostr. can cause considerable yield losses in the absence of appropriate management practices. The use of wheat varieties with loose smut resistance is an efficient and effective control technique. However, the development of commercial wheat lines with resistance to loose smut is time- and labour-consuming. DNA markers linked to loose smut resistance gene(s) would assist the development of loose smut resistant genotypes. The genetics of loose smut resistance was studied in an F5‐derived recombinant inbred line (RIL) population of 94 lines from the cross BW278/AC Foremost. The line AC Foremost is resistant and line BW278 is susceptible to U. tritici race T10. Phenotypic assessment revealed that a single gene, designated Ut6, segregated for resistance to race T10 in the RIL population. A modified bulked segregant analysis identified a microsatellite marker linked to Ut6. A linkage map was developed consisting of linked microsatellite loci and the resistance gene. The loose smut resistance gene Ut6 mapped to the long arm of chromosome 5B. Five microsatellite markers mapped within 6.7 cM of Ut6. The microsatellite markers gpw5029 and barc232 flanked Ut6 at distances of 1.3 and 2.8 cM on the distal and proximal sides, respectively. A diverse set of wheat lines was haplotyped for Ut6 using the linked microsatellite markers gpw5029 and barc232. The haplotype analysis suggested that the microsatellite markers associated with Ut6 will be useful for marker-assisted selection of loose smut resistant wheat lines.     

Development and application of 15 novel polymorphic microsatellite markers for sect. Paeonia (Paeonia L.)

Herbaceous peony, which belongs to sect. Paeonia, Paeonia L., Paeoniaceae, is not only a famous traditional flower in China; it is also highly regarded as an ornamental in Europe and the USA. On account of the abundance in germplasm resources and wide distribution ranges, herbaceous peony can be divided into three cultivar groups, the Chinese Peony Cultivar Group, the European Peony Cultivar Group and the Hybrid Peony Cultivar Group. However, most studies on genetic relationships in Paeonia are limited to the first group. This study used 15 polymorphic microsatellite markers that were developed by magnetic bead enrichment to explore the genetic diversity of 89 genotypes collected from Beijing, the USA and Canada and introduced to China. From 145 allelic loci that were detected from 15 primers, 140 (96.6%) were polymorphic. The number of allelic loci ranged from 4 to 16 with an average of 9.3, and the polymorphic index content ranged from 0.362 to 0.825 with an average of 0.678. Both cluster analysis and principal component analysis based on the 15 polymorphic primer pairs could distinguish the species from cultivars although the cluster analysis was better able to reflect the genetic relationships between all individuals. Some new insights about Paeonia L. are suggested. Furthermore, the 15 SSR markers have great significance as they will allow for furthering the studies on genetic diversity, genetic relationships and even the molecular marker-assisted breeding of Paeonia L.     

Characterization of 28 microsatellite loci in the European roller Coracias garrulus (Coracidae,AVES)

One hundred and forty-four existing microsatellite avian loci were tested for polymorphism in two to four unrelated European rollers. This allowed the identification of 28 unique polymorphic loci that we characterized in 34 unrelated individuals from a population in Guadix, Spain. The putative chromosomal locations of all the polymorphic loci in European rollers could be assigned based on the location of their orthologues in the assembled zebra finch and chicken genomes. All loci were assigned to autosomes based on predictive mapping and/or the presence of female heterozygotes, except CAM-11 that was assigned to the Z chromosome. By using a selected subset of 15 autosomal loci, the combined non-exclusion probability for identity was 4.7E-09.     

Characterization of polymorphic microsatellite markers and genetic diversity in wild bronze featherback, Notopterus notopterus (Pallas, 1769)

Six polymorphic microsatellite DNA loci were identified in the primitive fish, bronze featherback, Notopterus notopterus for the first time and demonstrated significant population genetic structure. Out of the six primers, one primer (NN90) was specific to N. notopterus (microsatellite sequence within the RAG1 gene) and five primers were product of successful cross-species amplification. Sixty-four primers available from 3 fish species of order Osteoglossiformes and families Notopteridae and Osteoglossidae were tested to amplify homologous microsatellite loci in N. notopterus. Fifteen primer pairs exhibited successful cross-priming PCR product. However, polymorphism was detected only at five loci. To assess the significance of these six loci (including NN90) in population genetic study, 215 samples of N. notopterus from five rivers, viz Satluj, Gomti, Yamuna, Brahmaputra and Mahanadi were analyzed. The five sample sets displayed different diversity levels and observed heterozygosity ranged from 0.6036 to 0.7373. Significant genotype heterogeneity (P < 0.0001) and high F_ST (0.2205) over all loci indicated that the samples are not drawn from the same genepool. The identified microsatellite loci are promising for use in fine-scale population structure analysis of N. notopterus.