Simple Sequence Repeat Genotyping Research Articles

Genetic structure and diversity of Phakopsora pachyrhizi isolates from soyabean

Simple sequence repeat (SSR) markers were used to classify 116 isolates of Phakopsora pachyrhizi, the cause of soyabean rust, collected from infected soyabean leaves in four agroecological zones in Nigeria. A high degree of genetic variation was observed within the sampled populations of P. pachyrhizi. Eighty‐four distinct genotypes were identified among three of the four agroecological zones. Nei’s average genetic diversity across geographical regions was 0·22. Hierarchical analysis of molecular variance showed low genetic differentiation among all populations of P. pachyrhizi. The majority (> 90%) of the genetic diversity was distributed within each soyabean field, while approximately 6% of the genetic diversity was distributed among fields within geographic regions. Low population differentiation was indicated by the low FST values among populations, suggesting a wide dispersal of identical genotypes on a regional scale. Phylogenetic analysis indicated a strictly clonal structure of the populations and five main groups were observed, with group II accounting for 30% of the entire population. Because of the asexual reproduction of P. pachyrhizi, single‐step mutations in SSR genotypes are likely to account for the genetic differences within each group.

Pigeonpea composite collection and identification of germplasm for use in crop improvement programmes

Pigeonpea (Cajanus cajan(L.) Millsp. is one of the most important legume crops as major source for proteins, minerals and vitamins, in addition to its multiple uses as food, feed, fuel, soil enricher, or soil binder, and in fencing, roofing and basket making. ICRISAT's genebank conserves 13,632 accessions of pigeonpea. The extensive use of few parents in crop improvement is contrary to the purpose of collecting a large number of germplasm accessions and has resulted in a narrow base of cultivars. ICRISAT, in collaboration with the Generation Challenge Program, has developed a composite collection of pigeonpea consisting of 1000 accessions representing the diversity of the entire germplasm collection. This included 146 accessions of mini core collection and other materials. Genotyping of the composite collection using 20 microsatellite or simple sequence repeat (SSR) markers separated wild and cultivated types in two broad groups. A reference set comprising 300 most diverse accessions has been selected based on SSR genotyping data. Phenotyping of the composite collection for 16 quantitative and 16 qualitative traits resulted in the identification of promising diverse accessions for the four important agronomic traits: early flowering (96 accessions), high number of pods (28), high 100-seed weight (88) and high seed yield/plant (49). These accessions hold potential for their utilization in pigeonpea breeding programmes to develop improved cultivars with a broad genetic base. Pigeonpea germplasm has provided sources of resistance to abiotic and biotic stresses and cytoplasmic-male sterility for utilization in breeding programmes.

Genetic diversity of Japanese chestnut cultivars assessed by SSR markers

The history of chestnut utilization in Japan is very long. Many cultivars have been preserved without being sufficiently organized and studied with respect to genetic variation. We developed 12 new SSR (simple sequence repeat) markers from Japanese chestnut (Castanea crenata) by using SSR-enriched genomic libraries. All 12 primer pairs selected for SSR analysis successfully amplified fragments in 6 Castanea species. The genetic diversity and relationships among 216 chestnut accessions were assessed by SSR markers, including 142 Japanese chestnuts, 38 Chinese chestnuts (C. mollissima), 2 European chestnuts (C. sativa), 9 American chestnuts (C. dentata), and 23 Japanese–Chinese hybrids. Among the 216 accessions, we identified 21 synonym groups (including 1 bud sport group), and 189 different genotypes. The mean values of observed heterozygosity (HO) and the expected heterozygosity (HE) in the Japanese chestnuts were 0.65 and 0.65, respectively. A phenogram of the 189 genotypes revealed 3 major groups, corresponding to Japanese, Chinese and American chestnuts. Japanese–Chinese hybrid cultivars were mainly included in the Chinese chestnut group. In the present study, genetic backgrounds could be identified for native and cultivated Japanese chestnuts and Japanese–Chinese hybrid cultivars, and many synonyms showing identical SSR genotypes were found. Genetic and geographical relationships of unique characteristics such as “Shidareguri” (weeping) and “Togenashi” (spineless) were also discussed. These results will improve the efficiency of cultivar identification and of chestnut breeding programs.

Virulence Phenotypes and Molecular Genotypes in Collections of Puccinia triticina from Italy.

Twenty-four isolates of Puccinia triticina from Italy were characterized for virulence to seedlings of 22 common wheat Thatcher isolines, each with a different leaf rust resistance gene, and for molecular genotypes at 15 simple sequence repeat (SSR) loci. The isolates were compared to a set of 13 previously characterized P. triticina isolates from either durum or common wheat. Clustering based on virulence phenotypes and SSR genotypes grouped the Italian P. triticina isolates into three groups. In the first group, the isolates had virulence phenotypes and SSR genotypes that were similar to the isolates collected from durum wheat. Isolates in the second group were unique because they had virulence similar to the isolates from common wheat but were distinct for SSR genotypes compared to the isolates from durum wheat and from common wheat. Isolates in the third group had virulence phenotypes and SSR genotypes closely related to the isolates from common wheat. The isolates were grouped based on the known or assumed host of origin, virulence phenotype, and SSR genotypes. Measures of FST and RST for SSR genotypes, and ΦST for virulence phenotype were significant, which indicated differentiation among the three groups of isolates. Virulence phenotypes and molecular genotypes were highly correlated with r = 0.74.

Genetic Differentiation Within the Puccinia triticina Population in South America and Comparison with the North American Population Suggests Common Ancestry and Intercontinental Migration

Leaf rust, caused by Puccinia triticina, is the most prevalent and widespread disease of wheat in South America. The objective of this study was to determine whether genetically differentiated groups of P. triticina are currently present in South America and to compare the South American population with the previously characterized population in North America. In total, 130 isolates of P. triticina from the wheat-growing regions of Argentina, Brazil, Chile, Peru, and Uruguay, mostly from the 1990s to 2008, were tested for virulence on 20 lines of wheat with single genes for leaf rust resistance and for molecular genotypes with 23 simple-sequence repeat (SSR) markers. After removal of isolates with identical virulence and SSR genotypes, 99 isolates were included for further analysis. Principal coordinate analysis plots indicated five different groups of isolates based on SSR genotypes that also differed for virulence to leaf rust resistance genes. All pairs of groups, except for one pair, were significantly differentiated for SSR genotypes according to R(ST) statistics. All but two pairs of groups were significantly differentiated for virulence phenotype according to Phi(PT) statistics. Isolates in all five groups had high values of fixation index for SSR alleles and linkage disequilibrium was high across all isolates that indicated the clonal reproduction of urediniospores. Only one of the five P. triticina groups from South America was differentiated for SSR genotypes from all of the six P. triticina groups from North America. The high degree of similarity for SSR genotype of isolates from both South America and North America suggested a common European origin of P. triticina that was introduced to both continents. The emergence of the same P. triticina virulence phenotypes with highly related SSR genotypes in the United States in 1996 and in Uruguay in 1999 indicated the likely intercontinental migration of these genotypes from Mexico to both South America and North America.

Insights into the origin and identity of National New Zealand Flax Collection plants from simple sequence repeat (SSR) genotyping

Fifty-five accessions from the National New Zealand Flax Collection were subjected to genotyping using eight nuclear and two plastid simple sequence repeats (SSRs) and compared with a selection of predominantly wild Phormium plants. Several groups of plants with different names but identical SSR genotypes were found and these largely correspond with groups previously identified by analysis of their morphology. Maori cultivars are genetically diverse and include plants with interspecific (Phormium tenax × Phormium cookianum) hybrid origin. Genotyping supports historical and archaeological evidence that Phormium was taken to the subantarctic islands from southern New Zealand by sealers, and probably from the Chatham Islands by Maori and Moriori. As well as such tests of relatedness among Phormium selections, our markers provide opportunities to study the process of speciation, evolution and interspecific hybridization in Phormium.

Characterization of Ugandan Sweetpotato Germplasm Using Fluorescent Labeled Simple Sequence Repeat Markers

The genetic relationships among 192 superior, high–yielding, and disease-resistant sweetpotato [Ipomoea batatas (L.) Lam] accessions from the Ugandan germplasm collection were analyzed using 10 fluorescent labeled simple sequence repeat (SSR) markers. Relatedness among the genotypes was estimated using the Nei and Li genetic distance coefficient, cluster analysis and principle component analysis methods of NTSYS-pc software. The polymorphic information content of the SSR markers used in this study ranged from 0.23 to 0.76 for loci IB-S07 and IB-R12, respectively, with a mean value of 0.62. The number of polymorphic alleles detected per locus ranged from two to six with a mean of four, a confirmation of the effectiveness of microsatellite detection on an automated ABI 3730 sequencer. The mean pairwise genetic distance among the 192 genotypes was 0.57, an indication of moderately high genetic diversity. Cluster analysis divided the accessions into four major groups with no relationship to the district of origin. Two sets of duplicates were identified through SSR genotyping in this study. Up to 190 distinct accessions for use as potential parental genotypes in hybridization schemes for cultivar development in the region were identified.

Differentiation of Molecular Genotypes and Virulence Phenotypes of Puccinia triticina from Common Wheat in North America

Wheat leaf rust caused by Puccinia triticina is widely distributed in the wheat growing regions of the United States and Canada, and is subject to selection for virulence phenotype by leaf rust resistance genes in wheat cultivars. The objective of this study was to determine the number of genetically differentiated groups of P. triticina that are currently present in North America. In total, 148 isolates of P. triticina from the 1980s to 2005 were collected from wheat-growing regions of the United States and Canada and tested for virulence on 20 lines of wheat with single genes for leaf rust resistance and for molecular genotype with 23 simple sequence repeat (SSR) markers. In total, 91 virulence phenotypes and 65 SSR genotypes were found. After removal of isolates with identical virulence and SSR genotypes, 125 isolates were included for further analysis. Bayesian cluster analysis indicated five different groups of isolates based on SSR genotypes that also differed for virulence to leaf rust resistance genes Lr2a, Lr2c, Lr3bg, Lr17, and Lr28. Isolates avirulent to Lr14a and Lr20 that have increased since 2003 had SSR genotypes identical or similar to older isolates in one of the five groups, indicating that these isolates were derived by mutation from the previously existing population of P. triticina. The representative collection of P. triticina isolates had characteristics consistent with an asexual dikaryotic population of genetically differentiated groups of SSR genotypes with high levels of heterozygosity and disequilibrium within which stepwise mutation at avirulence or virulence loci regularly occurs.

Detection of Virulence to Resistance Gene Sr36 Within the TTKS Race Lineage of Puccinia graminis f. sp. tritici.

The stem rust resistance gene Sr36 confers a near-immune resistance reaction to many races of Puccinia graminis f. sp. tritici and is highly effective against race TTKSK (syn. Ug99), which possesses unusually broad virulence combinations. Because this gene is widely used in United States soft winter wheat germplasm and cultivars, it has been considered to be an important source of resistance to TTKSK. In 2007, moderately susceptible infection responses were observed on wheat lines and cultivars carrying Sr36 in a field screening nursery for stem rust at Njoro, Kenya. We derived 18 single-pustule isolates from stem rust samples collected from the 2007 Njoro nursery. The isolates were evaluated for virulence on 20 North American stem rust differential lines and on wheat lines and cultivars carrying Sr36, Sr31+Sr36, and Sr24+Sr31. Of the 18 isolates, 10 produced infection types 3+ to 4 on line W2691SrTt-1 (monogenic for Sr36) and other lines that carry Sr36 and belonged to a new virulence phenotype that was not detected in previous years. These isolates were identified as race TTTSK. The remaining eight isolates were identified as races TTKSK (five isolates) and TTKST (three isolates), with avirulence and virulence, respectively, to Sr24. Thirteen simple sequence repeat (SSR) markers were used to examine the genetic relationships among the three races in the TTKS lineage. All isolates in the lineage shared an identical SSR genotype and were clearly different from North American races. In all, 16 wheat cultivars and 60 elite breeding lines, postulated to possess Sr36, were susceptible to race TTTSK. The occurrence of race TTTSK with combined virulence on Sr31 and Sr36 has further broadened the virulence spectrum of the TTKS lineage and rendered an important source of resistance ineffective.

Agronomic performance and genetic characterization of sugarcane transformed for resistance to sugarcane yellow leaf virus

Sugarcane yellow leaf virus (SCYLV, a Poleovirus of the Luteoviridae family) is already widespread in Florida, and resistance in the Canal Point (CP) sugarcane population is limited. Genetic transformation of sugarcane for disease resistance holds promise but tissue culture and transformation processes produce undesirable agronomic characteristics necessitating thorough field evaluation. A 3-year sugarcane (a complex hybrid of Saccharum species) field study was conducted in Belle Glade, FL with the following objectives: (1) thoroughly evaluate the agronomic performance of two transgenic lines transformed for SCYLV resistance (6-1, 6-2) compared with parental cultivar control CP 92-1666, (2) determine level of SCYLV resistance in the transgenic lines, and (3) characterize genetic differences in the transgenic lines compared with CP 92-1666 using simple sequence repeat (SSR) genotyping. Sugarcane yields of CP 92-1666 were superior to both transgenic lines, as well as tissue culture (C-1) and nptII marker gene (20-1) controls, in the plant cane, first ratoon and second ratoon crops. CP 92-1666 recorded an average of 6.5–8.7 tons sucrose ha −1 yr −1 more than genotypes subjected to tissue culture and biolistic transformation. However, SCYLV infection rates in transgenic lines were only 0–5%, compared with 98% in CP 92-1666. Kanamycin field assays indicated that selectable marker gene nptII was stably expressed in all co-transformed lines. SSR genotyping showed 35 additional fragments to be present and 25 existing fragments absent among 6-1, 6-2, C-1 and 20-1 compared with CP 92-1666. Although all clones had unique genotypes, the four regenerated clones showed a greater genetic distance from the donor clone CP 92-1666 (mean GD 0.4) than to one another (mean GD 0.03). This study reports the first successful gene transfer of SCYLV resistance in sugarcane and the first report of variations in microsatellite repeat number associated with regeneration from embryogenic callus. Our results highlight the potential of genetic transformation methods to incorporate desirable traits into sugarcane, combined with the necessity of thorough agronomic evaluation of transgenic genotypes. Transgenic lines 6-1 and 6-2 are being used as parents in crosses designed to combine SCYLV resistance from these genotypes with agronomic characteristics of high-yielding materials.

Phytophthora infestans isolates from Northern China show high virulence diversity but low genotypic diversity

Phenotypic and genotypic characteristics of 48 Phytophthora infestans isolates, collected in five provinces in Northern China between 1997 and 2003, were determined and compared with reference isolates. Characterisation included mating type, virulence, mitochondrial DNA (mtDNA) haplotype and DNA fingerprinting patterns based on simple sequence repeats (SSR) and amplified fragment length polymorphisms (AFLP). All isolates had the A1 mating type, mtDNA haplotype IIa and an identical SSR genotype (designated as SG-01-01) that differed from SSR genotypes found in the reference isolates, including those representing the 'old' US-1 lineage that dominated the P. infestans population worldwide prior to 1980. In contrast, the virulence spectra were highly variable and virulence to all resistance genes present in the standard differential set (R1 to R11) was found. AFLP analysis revealed some diversity; eight different AFLP genotypes were found that could be grouped into two major clusters. This study shows that there is very little genotypic diversity in the P. infestans population in Northern China. The occurrence of many different races within this rather uniform population is discussed in the framework of recent insights into the molecular determinants of avirulence in potato-P. infestans'gene-for-gene' interactions.

Detection of Virulence to Resistance Gene Sr24 Within Race TTKS of Puccinia graminis f. sp. tritici.

The stem rust resistance gene Sr24 is effective against most races of Puccinia graminis f. sp. tritici, including race TTKS (syn. Ug99), and is used widely in commercial wheat cultivars worldwide. In 2006, susceptible infection responses were observed on wheat lines and cultivars carrying Sr24 in a field stem rust screening nursery at Njoro, Kenya. We derived 28 single-pustule isolates from stem rust samples collected from the 2006 Njoro nursery. The isolates were evaluated for virulence on 16 North American stem rust differential lines; on wheat lines carrying Sr24, Sr31, Sr38, and SrMcN; and on a wheat cultivar with a combination of Sr24 and Sr31. All isolates were identified as race TTKS with additional virulence on Sr31 and Sr38. These isolates were divided into two groups: group A (seven isolates and the two control isolates), producing a low infection type, and group B (21 isolates), producing a high infection type on Sr24, respectively. Isolates of group B represented a new variant of race TTKS with virulence to Sr24. Eighteen simple sequence repeat (SSR) markers were used to examine the genetic relationship between these two groups of isolates in race TTKS and five North American races (MCCF, QCCQ, RCRS, RTHS, and TPMK) that are representative of distinct lineage groups. All isolates of race TTKS shared an identical SSR genotype and were clearly different from North American races. The virulence and SSR data indicated that the new variant of race TTKS with Sr24 virulence likely has arisen via mutation within the TTKS genetic lineage. We propose to revise the North American stem rust nomenclature system by the addition of four genes (Sr24, Sr31, Sr38, and SrMcN) as the fifth set. This revision recognizes the virulence on Sr31 and differentiates isolates within race TTKS into two separate races: TTKSK and TTKST, with avirulence and virulence on Sr24, respectively. The occurrence of race TTKST with combined virulence on Sr24 and Sr31 has substantially increased the vulnerability of wheat to stem rust worldwide.

Apple Proliferation Resistance in Apomictic Rootstocks and Its Relationship to Phytoplasma Concentration and Simple Sequence Repeat Genotypes

In an effort to select and characterize apple rootstock resistant to apple proliferation (AP), progenies from seven apomictic rootstock selections and their parental apomictic species, Malus sieboldii and M. sargentii, were compared to standard stocks M 9 and M 11. Seedlings derived from open pollinated mother plants were grafted with cv. Golden Delicious and grown under natural infection conditions. The progenies differed greatly in resistance to the AP agent 'Candidatus Phytoplasma mali'. Progenies of M. sieboldii and its descendent rootstock selections D2212, 4608, 4551, and D1131 showed a high level of resistance, whereas progenies of M. sargentii and its descendent selections D1111 and C1828 proved susceptible. M 9 and M 11 showed an intermediate level of resistance. Phytoplasma titer in roots of the M. sieboldii and M. sargentii progeny groups was similarly low, whereas the concentration in the standard stocks was 100 to 5,000 times higher. In trees on most of the resistant stocks, only a minority was colonized in the scion, while in trees on susceptible and standard stocks, infection rate was often higher. Also, the titer in the top of trees on resistant stocks was usually lower than in trees on susceptible and standard stocks. Four progenies derived from open pollinated M. sieboldii and M. sieboldii descendents were subjected to DNA typing using simple sequence repeat (SSR) markers. This study revealed that the selected groups consisted mainly of mother-like plants (apomicts) and type I hybrids (unreduced mother genotype plus one male allele at each locus). Type II hybrids (full recombinants) and autopollinated offspring were rare. In the 4608 progeny, trees grown on type I hybrid rootstocks were significantly less affected than trees on mother-like stocks. In other progenies with fewer or no type I hybrids, trees on type II hybrids and autopollinated offspring suffered considerably more from disease than trees on mother-like stocks.

Cultivar Differentiation Identified by SSR markers and the Application for Polyploid Loquat Plants

Here we established a DNA marker-based method to identify cultivars of loquat (Eriobotrya japonica Lindl.) using simple sequence repeat (SSR) markers derived from pears and apples. A total of 24 loquat cultivars commercially grown in Japan were used for genetic identification, including 15 diploid-, 6 triploid-, and 3 tetraploid cultivars. Analysis using the 88 SSR markers derived from pears and apples, which belong to the same subfamily (family Rosaceae, subfamily Maloideae) as the loquat, indicated that 26 SSR markers were applicable to the identification of loquat cultivars. A total of 82 putative SSR alleles were obtained by the 26 SSR markers. SSR analysis enabled the identification of all loquat varieties tested, except for ‘Tomihusa’ and ‘4N-Tomihusa’. In addition, the parentages of several cultivars, including ‘Oohusa’ and ‘Mizuho’, were confirmed from the SSR genotype data obtained. Triploid and tetraploid varieties could be identified from the SSR genotypes because some SSR loci generated 2 or more alleles for polyploids. The relationship between ‘Kibou’ and its parents was confirmed since SSR alleles of the seedless triploid offspring ‘Kibou’ were clearly inherited from the female parent ‘4N-Tanaka 1’ and the male parent ‘Nagasakiwase’. The phenogram obtained by cluster analysis showed no distinctive separation of Japanese commercial cultivars from Chinese cultivars. This result was consistent with the hypothesis that Japanese commercial cultivars were derived from introduced Chinese loquat.

Identification of Parent-offspring Relationships in 55 Japanese Pear Cultivars Using S-RNase Allele and SSR markers

S-RNase alleles and 18 simple sequence repeats (SSR) markers were used to analyze 55 Japanese pear cultivars, including 47 hybrid cultivars, 6 bud sport mutants, and 2 derived from open pollinations. Fifteen cultivars used in the breeding of some of the hybrid cultivars were also examined. Each cultivar was genotyped at the S-locus and SSR loci. Parent-offspring relationships were analyzed by comparing genotypes among the cultivars. Of the 47 crossbreeding cultivars and selections, parent-offspring relationships of 37 were re-confirmed because the hybrids inherited S-RNase and SSR alleles from both parents without any discrepancies. Discrepancies at 3 or more SSR loci were found between 10 hybrid cultivars (‘Kisui’, ‘Tanzawa’, ‘Niitaka’, ‘Seiryu’, ‘Akemizu’, ‘Atago’, ‘Echigonishiki’, ‘Ishiiwase’, ‘Shusui’, and ‘Yachiyo’) and their previously reported parents, suggesting that these hybrid cultivars have different parents than reported. Furthermore, ‘Momoeduki’ and ‘Sanko’, which were previously reported as bud sports of ‘Niitaka’ and ‘Kosui’, respectively, exhibited SSR genotypes that were different from their reported parents. Paternal cultivars were previously unknown in ‘Shinko’ and ‘Yoshikaori’, which are open-pollinated seedlings of ‘Nijisseiki’. SSR genotypes revealed that their paternal cultivars are ‘Amanogawa’ and ‘Chojuro’, respectively.

Identification and validation of a core set of informative genic SSR and SNP markers for assaying functional diversity in barley

A ‘core set’ of 28 simple sequence repeat (SSR) and 28 single nucleotide polymorphism (SNP) markers for barley was developed after screening six diverse genotypes (DGs) representing six countries (Afghanistan, Pakistan, Algeria, Egypt, Jordan and Syria) with 50 SSR and 50 SNP markers derived from expressed sequence tags (ESTs). The markers of the core set are single locus with very high quality amplifications, high polymorphism information content (PIC) and are distributed across the barley genome. PIC values for the selected SSR and SNP markers ranged between 0.32–0.72 (average 0.58) and 0.28–0.50 (average 0.42), respectively. To make the SNP genotyping cost effective, CAPS (cleaved amplified polymorphic sequence) and indel assays were developed for 23 markers and the remaining 5 SNP markers were optimized for pyrosequencing. A high coefficient of correlations (r = 0.96, P < 0.005) between the genetic similarity matrices of SSR and SNP genotyping data of the core set on diverse genotypes (DGs) and their similar groupings according to the geographical distribution in both SSR and SNP phenograms with high bootstrap values underline the utility and reliability of the core set. A comparative allelic and sequence diversity for SSR and SNP markers between the DGs and six elite parental genotypes (PGs) of mapping populations showed comparable diverse nature of two germplasm sets. However, unique SNPs and indels were observed in both germplasm sets providing more datapoints for analysing haplotypes in a better way for the corresponding SNP marker.

Extensive simple sequence repeat genotyping of potato landraces supports a major reevaluation of their gene pool structure and classification

Contrasting taxonomic treatments of potato landraces have continued over the last century, with the recognition of anywhere from 1 to 21 distinct Linnean species, or of Cultivar Groups within the single species Solanum tuberosum. We provide one of the largest molecular marker studies of any crop landraces to date, to include an extensive study of 742 landraces of all cultivated species (or Cultivar Groups) and 8 closely related wild species progenitors, with 50 nuclear simple sequence repeat (SSR) (also known as microsatellite) primer pairs and a plastid DNA deletion marker that distinguishes most lowland Chilean from upland Andean landraces. Neighbor-joining results highlight a tendency to separate three groups: (i) putative diploids, (ii) putative tetraploids, and (iii) the hybrid cultivated species S. ajanhuiri (diploid), S. juzepczukii (triploid), and S. curtilobum (pentaploid). However, there are many exceptions to grouping by ploidy. Strong statistical support occurs only for S. ajanhuiri, S. juzepczukii, and S. curtilobum. In combination with recent morphological analyses and an examination of the identification history of these collections, we support the reclassification of the cultivated potatoes into four species: (i) S. tuberosum, with two Cultivar Groups (Andigenum Group of upland Andean genotypes containing diploids, triploids, and tetraploids, and the Chilotanum Group of lowland tetraploid Chilean landraces); (ii) S. ajanhuiri (diploid); (iii) S. juzepczukii (triploid); and (iv) S. curtilobum (pentaploid). For other classifications, consistent and stable identifications are impossible, and their classification as species is artificial and only maintains the confusion of users of the gene banks and literature.

Comparative assessment of EST-SSR, EST-SNP and AFLP markers for evaluation of genetic diversity and conservation of genetic resources using wild, cultivated and elite barleys

A set of 16 expressed sequence tag (EST)-derived simple sequence repeat (SSR) and 15 EST-derived single nucleotide polymorphism (SNP) markers together with 4 amplified fragment length polymorphism (AFLP) primer combinations were analyzed on 43 wild ( Hordeum vulgare ssp. s pontaneum – HS), 35 cultivated ( H. vulgare ssp. vulgare – HV) and 12 elite ( H. vulgare ssp. vulgare – from EU) barley lines. SSR markers were found most polymorphic with an average PIC value of 0.593 and eight alleles per marker, while AFLP markers showed the highest effective multiplex ratio (26.4) and marker index (5.042). The effective marker index (EMI) was recorded highest (0.468) for AFLP markers and lowest (0.341) for the SNP markers while the SSR markers had an intermediate EMI (0.442). Cluster analysis on combined set of SSR, SNP and AFLP genotyping data classified wild, cultivated and elite barley lines in three distinct groups. The present study suggests the SNP markers as the best class of markers for characterizing and conserving the genebank materials and the AFLP and SSR markers more suitable for diversity analysis and fingerprinting.

Genetic Differentiation of Puccinia triticina Populations in Central Asia and the Caucasus

ABSTRACT Isolates of Puccinia triticina collected from common wheat in the Central Asia countries of Kazakhstan, Uzbekistan, Tajikistan, and Kyrgyzstan and the Caucasus countries of Azerbaijan, Georgia, and Armenia were tested for virulence to 20 isolines of Thatcher wheat with different leaf rust resistance genes and molecular genotype at 23 simple sequence repeat (SSR) loci. After clone correction within each country, 99 isolates were analyzed for measures of population diversity, variation at single SSR loci, and for genetic differentiation of virulence phenotypes and SSR genotypes. Isolates from Central Asia and the Caucasus were also compared with 16 P. triticina isolates collected from common wheat in North America that were representative of the virulence and molecular variation in this region and two isolates collected from durum wheat in France and the United States. Populations from the Caucasus, Uzbekistan, Tajikistan, and Kyrgyzstan were not significantly (P > 0.05) differentiated for SSR variation with F(st) and R(st) statistics. Populations from the Caucasus, Uzbekistan, Tajikistan, and Kyrgyzstan were significantly (P < 0.05) differentiated from the populations in South and North Kazakhstan for SSR variation. All populations from Central Asia and the Caucasus were significantly differentiated from the North American isolates and isolates from durum wheat for SSR variation and virulence phenotypes. There was a correlation between virulence phenotype and SSR genotype among individual isolates and at the population level. Mountain barriers may account for the differentiation of P. triticina geographic populations in Central Asia and the Caucasus.

Cultivation-Independent Analysis of Fungal Genotypes in Soil by Using Simple Sequence Repeat Markers

Cultivation-independent analyses of fungi are used for community profiling as well as identification of specific strains in environmental samples. The objective of the present study was to adapt genotyping based on simple sequence repeat (SSR) marker detection for use in cultivation-independent monitoring of fungal species or strains in bulk soil DNA. As a model system, a fungal biocontrol agent (BCA) based on Beauveria brongniartii, for which six SSR markers have been developed, was used. Species specificity of SSR detection was verified with 15 fungal species. Real-time PCR was used to adjust for different detection sensitivities of the six SSR markers as well as for different template quantities. The limit for reliable detection per PCR assay was below 2 pg target DNA, corresponding to an estimated 45 genome copies of B. brongniartii. The cultivation-independent approach was compared to cultivation-dependent SSR analysis with soil samples from a B. brongniartii BCA-treated field plot. Results of the cultivation-independent method were consistent with cultivation-dependent genotyping and allowed for unambiguous identification and differentiation of the applied as well as indigenous strains in the samples. Due to the larger quantities of soil used for cultivation-dependent analysis, its sensitivity was higher, but cultivation-independent SSR genotyping was much faster. Therefore, cultivation-independent monitoring of B. brongniartii based on multiple SSR markers represents a rapid and strain-specific approach. This strategy may also be applicable to other fungal species or strains for which SSR markers have been developed.