Simple sequence repeat markers distinguish among morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5' end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49 degrees C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.     

Isolation and characterization of eight microsatellite loci in two morphotypes of the Southeast Asian army ant,Aenictus laeviceps

Eight polymorphic microsatellite markers were developed from morphotype L1 of the Southeast Asian army ant, Aenictus laeviceps in Borneo, and characterized for morphotypes L1 and L2. The number of alleles per locus ranged from nine to 24 (average 15) in morphotype L1 and two to 16 (average 9.8) in L2. Observed heterozygosity ranged from 0.75 to 0.96 in morphotype L1 and from 0.08 to 0.96 in L2. All loci were in Hardy–Weinberg equilibrium. One pair of loci showed linkage disequilibrium in morphotype L1. All markers successfully amplified in morphotype S, and some markers amplified in other Aenictus species.     

Genome-Wide Analysis of Simple Sequence Repeats and Efficient Development of Polymorphic SSR Markers Based on Whole Genome Re-Sequencing of Multiple Isolates of the Wheat Stripe Rust Fungus

The biotrophic parasitic fungus Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, a devastating disease of wheat, endangering global food security. Because the Pst population is highly dynamic, it is difficult to develop wheat cultivars with durable and highly effective resistance. Simple sequence repeats (SSRs) are widely used as molecular markers in genetic studies to determine population structure in many organisms. However, only a small number of SSR markers have been developed for Pst. In this study, a total of 4,792 SSR loci were identified using the whole genome sequences of six isolates from different regions of the world, with a marker density of one SSR per 22.95 kb. The majority of the SSRs were di- and tri-nucleotide repeats. A database containing 1,113 SSR markers were established. Through in silico comparison, the previously reported SSR markers were found mainly in exons, whereas the SSR markers in the database were mostly in intergenic regions. Furthermore, 105 polymorphic SSR markers were confirmed in silico by their identical positions and nucleotide variations with INDELs identified among the six isolates. When 104 in silico polymorphic SSR markers were used to genotype 21 Pst isolates, 84 produced the target bands, and 82 of them were polymorphic and revealed the genetic relationships among the isolates. The results show that whole genome re-sequencing of multiple isolates provides an ideal resource for developing SSR markers, and the newly developed SSR markers are useful for genetic and population studies of the wheat stripe rust fungus.     

Difference in antifungal activity of morphotypes of clavicipitaceous endophytes within and between species

In a previous study, a total of 484 endophytic fungi were isolated and purified from seven populations of Achnatherum sibiricum (L.) Keng collected at six geographical locations in Inner Mongolia, China. Based on growth rates as well as morphological characteristics, the isolates were classified into five morphotypes. Among them, morphotypes A, B and C were ascribed to the same species, Neotyphodium chisosum, based on ITS sequences. Morphotype E was identified as Epichloë amarillans. In the present study, four morphotypes, A, B, C and E, belonging to two species, were chosen for an in vitro pathogen trial. The results showed that both endophyte colonies and endophyte filtrate of all morphotypes could inhibit the mycelia growth and spore germination of the pathogen fungi tested. The magnitude of inhibition varied not only between species, but also among morphotypes of the same species. Overall, the antifungal ability of E. amarillans (morphotype E) was higher than that of N. chisosum. Within N. chisosum, the antifungal ability was highest in morphotype C, followed by morphotype A, and lowest in morphotype B. This variability suggests that different morphotypes might represent different genotypes of endophyte. The effect of endophyte infection on the host grass should be examined not only on the species level but also on the morphotype level to determine the possible interactions.     

Microsatellite markers to monitor a commercialized isolate of the entomopathogenic fungus Beauveria bassiana in different environments: Technical validation and first applications

Here, we report on the application of five previously developed microsatellite markers (simple sequence repeats, SSRs) to monitor an isolate of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. in different environments. Discriminatory power of these SSR markers was assessed in two commercialized B. bassiana isolates as well as in 16 B. bassiana isolates from a world-wide collection, and three of the five SSR markers were estimated to allow a confident discrimination among the given isolates. Sensitivity thresholds of 0.1 pg DNA were subsequently determined for all SSR markers in case pure genomic fungal B. bassiana DNA was used as a template for PCR assays, but threshold levels varied depending on the environment (soil, plant) of the PCR assay. Furthermore, presence of a commercialized B. bassiana isolate was monitored via these SSR markers in three different types of potting substrates over a period of 14 weeks. With two SSR markers, strain-specific products were detected up to 14 weeks after application of B. bassiana to the substrate. Infectivity of B. bassiana conidia in the respective soil samples was confirmed by the Galleria baiting technique. Together these results indicate that molecular markers like SSRs specific for commercialized strains of entomopathogenic fungi are important tools to monitor a particular fungal strain in complex environmental samples such as bulk soil or plant DNA.     

Development and characterization of microsatellite loci for the tropical tree pathogen Botryosphaeria rhodina

Nineteen simple sequence repeat (SSR) markers were developed for the pathogen and blue stain fungus Botryosphaeria rhodina. Eight pairs were found to be polymorphic among isolates collected from Pinus spp., whereas a further five pairs were polymorphic when isolates from Pinus spp. were compared with those from Eucalyptus spp. Nine isolates of B. rhodina collected from pines and eucalypts in South Africa, Mexico and Indonesia were used to demonstrate the range of the markers. Testing the markers yielded preliminary evidence that relationships among isolates are more closely linked to host than to geographical origin.     

Spirotrichonymphea (Parabasalia) symbionts of the termite Paraneotermes simplicicornis

The desert dampwood termite Paraneotermes simplicicornis harbors several species of obligately symbiotic protists that support its nutrition by fermenting lignocellulose. Among them are three morphotypes with the dexiotropic spiraling flagellar bands characteristic of Spirotrichonymphea (Parabasalia). The largest morphotype, characterized by an elongated cell apex with axial columella and internally positioned spiraling flagellar bands, was previously described as Spirotrichonympha polygyra. A smaller morphotype, with similarly internalized flagellar bands but a more rounded posterior without a protruding axostyle, was previously reported but not named. The smallest morphotype has surface flagellar bands and can attach to other protist cells by its apex. In this study, we combine light microscopy of live specimens and 18S rRNA gene sequencing of individually isolated cells to better understand the diversity of symbionts in P. simplicicornis. We found that S. polygyra branches distantly from true Spirotrichonympha, which are associated with Reticulitermes termites. Thus, we propose the new genus Cuppa to accommodate C. polygyra n. comb. (type species) and the similar but smaller morphotype Cuppa taenia n. sp. The undescribed smallest morphotype can be excluded from all previously described Spirotrichonymphea genera by molecular and behavioral evidence, so we propose Fraterculus simplicicornis n. gen., n. sp., to accommodate this organism.     

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.     

Genetic structure of the Russian populations of <Emphasis Type="Italic">Pyrenophora tritici-repentis</Emphasis>, determined by using microsatellite markers

The population genetic structure of plant pathogenic fungus Pyrenophora tritici-repentis was examined using microsatellite (SSR) markers. According to the geographical origin of the pathogen populations, they were designated as North Caucasian (S, 33 isolates), northwest (Nw, 39), and Omsk (Om, 43). The populations were analyzed at the nine most polymorphic SSR loci, at which 75 alleles were identified. To characterize the genetic variation within and between populations, the AMOVA algorithm as implemented in the Arlequin v. 3.5 software program was used. The number of alleles per locus ranged from 5 to 12 and their sizes varied within the range from 180 to 400 bp. The mean gene diversity at SSR loci was high for all populations (H = 0.58–0.75). The populations were considerably different in the frequencies of individual alleles of the SSR loci. Most isolates in the populations were represented by unique haplotypes. The within-population variation of the isolates at molecular markers was 86.4%; among the populations, 13.6%. Substantial interpopulation differences were found between the Om and S (F_st = 0.16) and between the Om and Nw (F_st = 0.20) populations, whereas between the S and Nw populations, these differences were small (F_st = 0.05). Thus, it was demonstrated that the population of P. tritici-repentis from Omsk oblast had the independent status of the geographical population; northwest and North Caucasian populations differed in the allelic diversity of SSR loci, and despite the low F_st value (0.05), they also belonged to independent geographical populations.     

Assessment of Genetic Diversity Among Rice Genotypes with Differential Adaptations to Salinity Using Physio-morphological and Molecular Markers

Breeding for salt tolerance using traditional screening and selection methods have been limited by the complex and polygenic nature of salt tolerance trait. This study was designed to evaluate some of the premium Basmati rice varieties for salt tolerance and to characterize genetic diversity among the rice varieties with different adaptations to saline soils using microsatellite (SSR) and ISSR markers. Plants of nine rice varieties including salt tolerant, salt sensitive and traditional Basmati, were grown in hydroponics using Yoshida solution containing 0 (control, pH 5.0) and 30 mM NaCl (Electrical conductivity 4.8 d/S, pH 5.0) and assessed for salinity tolerance on 1–9 scale as per IRRI standard evaluation system using seedling growth parameters, visual salt injuries and Na-K ratio. Physio-morphological studies showed that traditional Basmati rice varieties (Basmati 370 and HBC19) were more sensitive than the salt sensitive control variety, MI-48. SSR as well as ISSR marker systems generated higher levels of polymorphism and could distinguish between all the 9 rice cultivars. A total of 299 (225 polymorphic) and 437 (430 polymorphic) bands were detected using 28 UBC ISSR primers and 100 welldistributed mapped SSR markers, respectively. ISSR and SSR marker data-sets showed moderate levels of positive correlation (Mantel test, r = 0.43). The ISSR and SSR marker data analyzed using clustering algorithms showed two distinct clusters separating the Basmati (Basmati 370, HBC19 and CSR-30) from other non-aromatic indica (IR36, Pokkali, CSR10 and MI-48) rice varieties indicating greater divergence between Basmati and non-aromatic indica rice genotypes. Marker analysis showed a close relationship among the two traditional (Basmati 370 and HBC19) and cross-bred (CSR30) Basmati rice varieties and greater diversity between the two salt-tolerant genotypes, Pokkali and BR4-10.     

High-resolution mapping of the dull fruit skin gene D in cucumber (Cucumis sativus L.)

Dull/glossy fruit skin is a highly valuable external quality trait that affects the market value of cucumbers. In this study, genetic analysis showed that one single dominant gene, D (dull fruit skin), determines the dull fruit skin trait in cucumber. By combining bulked segregant analysis with 11 published polymorphic molecular markers on chromosome 5, the D/d gene was preliminarily mapped between markers SCZ69 and SSR16203, at genetic distances of 0.3 and 0.6 cM, respectively. Subsequently, a larger F2 (S06 × S94) population (842 individuals in total) was used for high-resolution mapping of the D/d gene. Finally, the D/d gene was fine-mapped between markers SSR37 and SSR112, at a physical distance of 244.9 kb (containing 31 candidate genes), using eight newly developed polymorphic simple sequence repeat (SSR) markers between SCZ69 and SSR16203. Based on semi-quantitative RT-PCR analysis, the possible candidate gene D was identified as Csa016880 or Csa016887. Meanwhile, validity analysis of the markers SSR37 and SSR112 was performed with 72 dull/glossy fruit lines, and showed that the two co-dominant SSR markers could be used for marker-assisted selection of the dull/glossy fruit trait in cucumber breeding. Moreover, this study will be helpful for cloning of the D gene in cucumber.     

Genetic diversity analysis of Cynodon dactylon (bermudagrass) accessions and cultivars from different countries based on ISSR and SSR markers

ISSR and SSR markers were used to evaluate genetic diversity among 33 Cynodon dactylon accessions and 22 cultivars from four different countries in order to provide information on how to improve the utilization of bermudagrass germplasms. Eighty eight bands were amplified by nine SSR primer combinations and 236 bands were observed from 23 ISSR primers. The results showed that 97.7% of the SSR primers and 86.9% of the ISSR primers were polymorphic. The genetic similarity coefficients (GSC), gene diversity (He) and Shannon index (I) were 0.58–0.97, 0.27 and 0.41, respectively, for ISSR and 0.52–0.97, 0.29, and 0.43 for SSR. The UPGMA analysis clustered the 55 accessions (cultivars) into three groups. The cluster results produced by the ISSR data were close to the SSR data results. Analysis based on the combined ISSR and SSR data was more closely related to the geographical distribution of the tested germplasm.     

Development and characterization of microsatellite markers for a mangrove tree species Sonneratia caseolaris (L.) Engler (Lythraceae sensu lato)

Sonneratia caseolaris is a typical non-viviparous mangrove species and a key component of mangrove community in the Indo-West Pacific region. Here we isolated nine microsatellite simple sequence repeat (SSR) loci from the genome of S. caseolaris. Our isolated loci provided SSR markers with polymorphism of 2–6 alleles per locus. The expected and observed heterozygosities ranged from 0.242 to 0.745 and from 0.083 to 0.417, respectively. Cross-species amplification in S. alba and S. ovata showed that a subset of these markers holds promise for these congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on S. caseolaris as well as other congeneric species.     

Cross-species amplification and characterization of microsatellite loci in Pinus mugo Turra

Pinus mugo (dwarf mountain pine) is an important component of European mountain ecosystems. However, little is known about the present genetic structure and population differentiation of this species at the DNA level, possibly due to a lack of nuclear microsatellite markers (SSR) developed for Pinus mugo. Therefore in this study we transferred microsatellite markers originally developed for Pinus sylvestris and Pinus taeda to Pinus mugo. This cross-species amplification approach is much faster and less expensive than isolation and characterization of new microsatellite markers. The transfer rates from the source species to Pinus mugo were moderately low (26%). There were no differences in microsatellite repeat motifs between the source species and Pinus mugo. Nuclear microsatellite markers successfully transferred to Pinus mugo can be applied to various genetic studies on this species, due to the high level of their polymorphism and high value of polymorphic information content.     

Identification and genetic mapping of highly polymorphic microsatellite loci from an EST database of the septoria tritici blotch pathogen Mycosphaerella graminicola

A database of 30,137 EST sequences from Mycosphaerella graminicola, the septoria tritici blotch fungus of wheat, was scanned with a custom software pipeline for di- and trinucleotide units repeated tandemly six or more times. The bioinformatics analysis identified 109 putative SSR loci, and for 99 of them, flanking primers were developed successfully and tested for amplification and polymorphism by PCR on five field isolates of diverse origin, including the parents of the standard M. graminicola mapping population. Seventy-seven of the 99 primer pairs generated an easily scored banding pattern and 51 were polymorphic, with up to four alleles per locus, among the isolates tested. Among these 51 loci, 23 were polymorphic between the parents of the mapping population. Twenty-one of these as well as two previously published microsatellite loci were positioned on the existing genetic linkage map of M. graminicola on 13 of the 24 linkage groups. Most (66%) of the primer pairs also amplified bands in the closely related barley pathogen Septoria passerinii, but only six were polymorphic among four isolates tested. A subset of the primer pairs also revealed polymorphisms when tested with DNA from the related banana black leaf streak (Black Sigatoka) pathogen, M. fijiensis. The EST database provided an excellent source of new, highly polymorphic microsatellite markers that can be multiplexed for high-throughput genetic analyses of M. graminicola and related species.     

Pathogenic and molecular characterisations of pigeonpea wilt pathogen Fusarium udum

Thirty two pathogenic isolates of Fusarium udum from different pigeonpea growing areas in India were studied for pathogenic and molecular variability. Pathogenic variability was tested on 12 pigeonpea differential genotypes, which revealed prevalence of five variants in F. udum. The amount of genetic variation was evaluated by Polymerase Chain Reaction (PCR) amplification with 20 random amplified polymorphic DNA (RAPD) markers and nine microsatellite markers. All amplifications revealed scorable polymorphisms among the isolates, and a total of 137 polymorphic fragments were scored for the RAPD markers and 16 alleles for the simple sequence repeat (SSR) markers. RAPD primers showed 86% polymorphism. Genetic similarity was calculated using Jaccard's similarity coefficient and cluster analysis was used to generate a dendrogram showing relationships between them. Isolates could be grouped into three subpopulations based on molecular analysis. Results indicated that there is high genetic variability among a subpopulation of F. udum as identified by RAPD and SSR markers and pathogenicity on differential genotypes.     

Signatures of speciation? Distribution and diversity of Hypoplectrus (Teleostei: Serranidae) colour morphotypes

Aim To test historical and current influences on the distributions of sympatric colour morphotypes in the coral reef fish genus Hypoplectrus. Location The Caribbean and surrounding tropical waters. These areas cover the entire distribution of the genus. Methods A large and extensive database of Hypoplectrus sightings was used to establish the distribution of colour morphotypes and test a long‐standing hypothesis regarding their origin. First, we considered the evidence for the previously proposed ‘population centre’ hypothesis, which suggests that current morphotype distributions reflect past conditions where these colour forms evolved in allopatry. Using morphotype sighting data, the existence of clusters in occurrence and density was tested. Second, we examined whether the observed patterns of morphotype co‐occurrence deviate from random expectations using null model simulations, within subregions of the distribution of the genus, to infer ecological influences on distribution. Results There is considerable variation in morphotype distribution, with even widespread morphotypes showing geographical clustering. There is also little evidence to suggest past or current geographical isolation, with only one of the 11 morphotypes (Hypoplectrus chlorurus) showing a density distribution that is consistent with the population centre hypothesis. Null model analyses show that variation in local morphotype co‐occurrence is typically significantly lower than expected under random dispersal conditions. Main conclusions Our results strongly suggest that morphotype co‐occurrence is not random, but there is no evidence to suggest a past allopatric radiation in Hypoplectrus colour. Current distributions are likely to be driven by competitive interactions and/or habitat preferences. Our study highlights the value of the Hypoplectrus species complex as a system for the study of speciation in the marine environment, and implies that these closely related morphotypes have ecological relevance rather than being simple colour variants of a single polymorphic species.     

Highly Informative Single-Copy Nuclear Microsatellite DNA Markers Developed Using an AFLP-SSR Approach in Black Spruce (Picea mariana) and Red Spruce (P. rubens)

Background

Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies.

Principal Findings

A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean = 0.67) in black spruce and from 0.161 to 0.851 (mean = 0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.

Significance

The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.     

Assessment of genetic diversity and phylogenetic relationship among grouper species Epinephelus spp. from the Saudi waters of the Arabian Gulf

Understanding of fish genetic characterization plays a vital role in the conservation and utilization of fish genetic resources of grouper species. The present study was carried out to assess the genetic diversity and phylogenetic relationships in five grouper species, Epinephelus spp. from eastern Saudi Arabian coast using two molecular marker systems, inter simple sequence repeat (ISSR) and microsatellite (SSR) markers. In total, 219 individuals grouper specimens (Epinephelus tauvina, E. coioides, E. bleekeri, E. malabaricus, and E. areolatus) were genotyped with 10 ISSR and 11 SSR selected primers. The ISSR produced 94 DNA fragments, of which 44 were polymorphic with an average of 2.13 fragment per primer. While SSR primers generated 107 alleles, all of them were polymorphic with an average 9.72 per primer. ISSR and SSR techniques demonstrated a high level of gene diversity and genetic distances illustrated by UPGMA dendrograms among the grouper species. The results proved that the SSR markers were highly informative and efficient in detecting genetic variability and relationships of the Epinephelus spp.