Simple Sequence Repeat Data Research Articles

Streamlining of Simple Sequence Repeat Data Mining Methodologies and Pipelines for Crop Scanning.

Genetic markers are powerful tools for understanding genetic diversity and the molecular basis of traits, ushering in a new era of molecular breeding in crops. Over the past 50 years, DNA markers have rapidly changed, moving from hybridization-based and second-generation-based to sequence-based markers. Simple sequence repeats (SSRs) are the ideal markers in plant breeding, and they have numerous desirable properties, including their repeatability, codominance, multi-allelic nature, and locus specificity. They can be generated from any species, which requires prior sequence knowledge. SSRs may serve as evolutionary tuning knobs, allowing for rapid identification and adaptation to new circumstances. The evaluations published thus far have mostly ignored SSR polymorphism and gene evolution due to a lack of data regarding the precise placements of SSRs on chromosomes. However, NGS technologies have made it possible to produce high-throughput SSRs for any species using massive volumes of genomic sequence data that can be generated fast and at a minimal cost. Though SNP markers are gradually replacing the erstwhile DNA marker systems, SSRs remain the markers of choice in orphan crops due to the lack of genomic resources at the reference level and their adaptability to resource-limited labor. Several bioinformatic approaches and tools have evolved to handle genomic sequences to identify SSRs and generate primers for genotyping applications in plant breeding projects. This paper includes the currently available methodologies for producing SSR markers, genomic resource databases, and computational tools/pipelines for SSR data mining and primer generation. This review aims to provide a 'one-stop shop' of information to help each new user carefully select tools for identifying and utilizing SSRs in genetic research and breeding programs.

Comparison of population genetic structure of Pinus mugo Turra forest stands in the Giant Mountains by analysis of nSSR molecular marker data

Abstract Pinus mugo (dwarf mountain pine) communities in the protected area of Krkonoše National Park are extremely valuable. After extensive deforestation during the sixteenth to nineteenth centuries, P. mugo recovered naturally in some parts of the Giant Mountains, and in some places imported seeds were likely used for reforestation. To distinguish between native and possibly introduced local P. mugo populations, genetic variations between 12 populations from the Czech Sudeten part of the Giant Mountains and between the two populations from the Tatra Mountains were analysed using 12 nuclear microsatellite markers (nuclear simple sequence repeats). Based on the simple sequence repeat data, 146 alleles were found, and Shannon’s information index, number of private alleles, and expected heterozygosity (HE) of populations averaged 1.12, 0.16, and 0.53, respectively. Analysis of molecular variance indicated that most of the variation was detected within populations. Low levels of genetic differentiation were also observed (average FST = 0.023). The population structure was examined by Bayesian clustering. The results of STRUCTURE analysis confirmed that the introduced populations were different from the autochthonous populations. Some of the introduced Czech Sudeten populations had similar genetic structure as the more distant native populations from the Tatra Mountains. Our results provide important genetic information for the reconstruction of natural communities in the Giant Mountains.

Curcuma species DNA fingerprinting of wild and cultivated genotypes from different agroclimatic zones

IntroductionCurcuma isolates from different agroclimatic zones in Chhattisgarh have been found to possess a varied spectrum of Curcumin, we set out to learn more about their genetic diversity by molecular fingerprinting in the present study. MethodsUsing SSR primers, the genetic variability of 25 Curcuma genotypes has been analyzed. These included the fingerprints of Curcuma longa, Curcuma aromatica, and Curcuma caesia. 13 inter simple sequence repeat (ISSR) primers amplified 107 unique fragments across all kinds, whereas 11 simple sequence repeat (SSR) primers amplified 51 unique fragments across all varieties, with 97 % and 86 % showing polymorphism, respectively. ResultsA total of 1080 ISSR and 567 SSR loci were amplified across all genotypes; among these, 5 monomorphic and 102 polymorphic alleles were produced in ISSR primers. However, SSR data showed 47 polymorphic and 4 monomorphic alleles. A panel of ISSR and SSR markers calculated DNA fingerprinting, genetic similarity, and distance estimates between Curcuma genotypes. In order to examine the diversity in different Curcuma genotypes, UPGMA data scoring methods were used. DiscussionCurcuma species demonstrate significant genetic variability, which is essential for breeding initiatives focused on creating better cultivars with desirable characteristics such as increased productivity, resistance to diseases, and improved medicinal capabilities.

Table Mountain Pine (Pinus pungens): Genetic Diversity and Conservation of an Imperiled Conifer

Abstract Table Mountain pine (Pinus pungens Lamb.) is an imperiled tree species endemic to the southern and central Appalachian Mountains. Generally reliant on fire for regeneration, its fragmented but widespread distribution has declined in recent decades. We quantified the genetic diversity of 26 populations across the range of the species using data from seven highly polymorphic simple sequence repeat (SSR) loci. The species was relatively inbred whereas differentiation among populations was relatively low. Differentiation was significantly but weakly associated with geographic distance among populations. We detected minor genetic differences between northern and southern seed collection zones established based on climate similarity. We conducted a series of simulations using SSR data from 498 seedlings, grown from seed collected from five natural stands of Table Mountain pine, to assess the genetic consequences of different strategies for deploying collected seed in ex situ conservation plantings. Results indicated that reducing the number of families in a planting would not substantially affect the conservation of common alleles but would affect the representation of rare alleles and overall allelic richness. These findings add to our limited knowledge of genetic variation across the distribution of this rare conifer and offer some guidance for its effective genetic conservation.

Genetic patterns of Magnolia in the Lesser Antilles: Stepwise colonisation leading to highly inbred island ‘populations’

AbstractAimIslands are ‘nature's laboratories of evolution’. Most island biogeographical studies have focussed on remote volcanic island chains. Here, we challenge island biogeographical patterns using a slowly evolving recent colonist present on five islands in a nonlinear chronosequence island chain.LocationThe Lesser Antilles (Caribbean).TaxonMagnolia dodecapetala (Magnoliaceae).MethodsGenetic diversity was characterised using Sanger sequencing of 21 individuals amplified for 11 DNA markers, plus microsatellite data of 195 individuals genotyped with 19 simple sequence repeat (SSR) markers. Sanger sequencing data were used to construct a Bayesian phylogenetic hypothesis, while SSR markers were used to run approximate Bayesian computation (ABC) demographic analyses and calculate population statistics.ResultsBoth types of molecular data support stepwise colonisation, decoupled from known island ages. The ABC analyses support a north to south migration while the Sanger sequencing data indicate a mixture of island progression rule and stepping stone dispersal. The SSR data show strong genetic structuring per island and significant inbreeding in all populations except in Saint Lucia. The lowest genetic diversity is found in the population from Saint Vincent. A high amount of genetic linkage occurs in a subpopulation from Dominica.Main ConclusionsBiogeographical patterns in the complex geological setting of the Lesser Antilles are uncovered using a slowly evolving study species. All genetic data support treating each island as distinct Management Units for conservation and call for a re‐evaluation of the species limits. Inbreeding threatens the survival of island populations and the populations of Saint Vincent and Dominica represent conservation priorities.

Genetic Relationship and Diversity Analysis in Arecanut (Areca catechu L.) Genotypes using Molecular Markers

Background: Arecanut (Areca catechu L.) is an important plantation crop of India belongs to the family Arecaceae. As it is a highly cross-pollinated crop there is a significant difference among the genotypes. Morphological traits used for determining diversity and relationships among genotypes or variety are prone to environmental influences. Thus, the usefulness of molecular markers has been investigated as a means of characterizing and discriminating difference more precisely. Therefore, the present study was carried out to assess the genetic relationship and diversity among the arecanut genotypes by employing RAPD, ISSR and SSR markers. Methods: A total of 11 genotypes are being grown in University of Agricultural and Horticultural Sciences (UAHS), Shivamogga, Karnataka, India, which includes both local and exotic collections. These 11 genotypes were evaluated for genetic variations using molecular markers viz., random amplified polymorphic DNA (RAPD), inter-simple sequence repeats (ISSR) and simple sequence repeats (SSR). Result: All the markers studied showed significant polymorphism among the genotypes. Jaccard’s genetic similarity coefficient values of RAPD, ISSR and SSR markers were found in the range of 0.56-0.76, 0.55-0.74 and 0.52-0.91. The higher similarity was observed between the genotypes Mohitnagar and Mangala, as well as Theerthahalli Local and Hirehalli Dwarf. The genotypes Maidan Local and Cameroon were placed in very distinct clusters, showing that they are having very distinct traits. The dendrogram prepared using SSR data separately and combining data of RAPD, ISSR, SSR markers showed similar results. Therefore, employing SSR markers for further studies will be more useful for identifying germplasm for varietal improvement programmes.

Population Genetic Structure of the Bean Leaf Beetle Ootheca mutabilis (Coleoptera: Chrysomelidae) in Uganda.

Bean leaf beetle (BLB) (Ootheca mutabilis) has emerged as an important bean pest in Uganda, leading to devastating crop losses. There is limited information on the population genetic structure of BLB despite its importance. In this study, novel microsatellite DNA markers and the partial mitochondrial cytochrome oxidase subunit I (mtCOI) gene sequences were used to analyze the spatial population genetic structure, genetic differentiation and haplotype diversity of 86 O. mutabilis samples from 16 (districts) populations. We identified 19,356 simple sequence repeats (SSRs) (mono, di-, tri-, tetra-, penta-, and hexa-nucleotides) of which 81 di, tri and tetra-nucleotides were selected for primer synthesis. Five highly polymorphic SSR markers (4-21 alleles, heterozygosity 0.59-0.84, polymorphic information content (PIC) 50.13-83.14%) were used for this study. Analyses of the 16 O. mutabilis populations with these five novel SSRs found nearly all the genetic variation occurring within populations and there was no evidence of genetic differentiation detected for both types of markers. Also, there was no evidence of isolation by distance between geographical and genetic distances for SSR data and mtCOI data except in one agro-ecological zone for mtCOI data. Bayesian clustering identified a signature of admixture that suggests genetic contributions from two hypothetical ancestral genetic lineages for both types of markers, and the minimum-spanning haplotype network showed low differentiation in minor haplotypes from the most common haplotype with the most common haplotype occurring in all the 16 districts. A lack of genetic differentiation indicates unrestricted migrations between populations. This information will contribute to the design of BLB control strategies.

Genetic Diversity Study on Geographical Populations of the Multipurpose Species Elsholtzia stauntonii Using Transferable Microsatellite Markers.

Elsholtzia stauntonii Benth. (Lamiaceae) is an economically important ornamental, medicinal and aromatic plant species. To meet the increasing market demand for E. stauntonii, it is necessary to assess genetic diversity within the species to accelerate the process of genetic improvement. Analysis of the transferability of simple sequence repeat (SSR) markers from related species or genera is a fast and economical method to evaluate diversity, and can ensure the availability of molecular markers in crops with limited genomic resources. In this study, the cross-genera transferability of 497 SSR markers selected from other members of the Lamiaceae (Salvia L., Perilla L., Mentha L., Hyptis Jacq., Leonurus L., Pogostemon Desf., Rosmarinus L., and Scutella L.) to E. stauntonii was 9.05% (45 primers). Among the 45 transferable markers, 10 markers revealed relatively high polymorphism in E. stauntonii. The genetic variation among 825 individuals from 18 natural populations of E. stauntonii in Hebei Province of China was analyzed using the 10 polymorphic SSR markers. On the basis of the SSR data, the average number of alleles (NA), expected heterozygosity (HE), and Shannon’s information index (I) of the 10 primers pairs were 7.000, 0.478, and 0.688, respectively. Lower gene flow (Nm = 1.252) and high genetic differentiation (Fst = 0.181) were detected in the populations. Analysis of molecular variance (AMOVA) revealed that most of the variation (81.47%) was within the populations. Integrating the results of STRUCTURE, UPGMA (Unweighted Pair Group Method with Arithmetic Mean) clustering, and principal coordinate analysis, the 825 samples were grouped into two clusters associated with geographical provenance (southwestern and northeastern regions), which was consistent with the results of a Mantel test (r = 0.56, p < 0.001). Overall, SSR markers developed in related genera were effective to study the genetic structure and genetic diversity in geographical populations of E. stauntonii. The results provide a theoretical basis for conservation of genetic resources, genetic improvement, and construction of a core collection for E. stauntonii.

Assessment of Genetic Diversity among Intra- and Interspecific Lowland Rice using Morpho-agronomic Traits and SSR Markers

Rice is staple food in many countries of Africa and a major part of the diet in many others. However, Africa’s demand for rice exceeds production with the deficit of 40% being imported. One way to improve Africa’s rice production is through breeding high yielding varieties suitable for the different environment conditions. This study was conducted to assess the genetic variability and stability performance of 48 lowland rice genotypes including 37 interspecific (Oryza glaberrima × Oryza sativa ssp. indica) and 11 intraspecific (O. sativa ssp. indica × O. sativa ssp. indica) in 12 environments in Nigeria, Benin Republic and Togo using Additive Main Effect and Multiplicative Interaction (AMMI) and Genotype+ Genotype x Environment (GGE) biplot models. The combined analysis of variance revealed significant differences (P&lt;0.01) among the genotypes, environments, and genotypes x environment interaction. Both the AMMI and GGE models identified NERICA-L8 and NERICA-LI2 as the best genotypes for cultivation across environments. Ouedeme environments in Benin Republic were the most stable and ideal for rice cultivation, while Ibadan sites were the most unstable. TOG 5681 had the least yield and was the most unstable across seasons. Genetic diversity was analyzed using 22 important morpho-agronomic traits and 50 simple sequence repeat (SSR) markers and the results were subjected to principal components analysis (PCA). The results revealed that the first eight PC axes (PC1–8) accounted for 75.13% of the total variation, while PC1–4 accounted for 50.39% of the total variation among rice genotypes. However, 10 of the 50 SSR markers were polymorphic and generated 49 alleles (average = 4.9 alleles per locus), suggesting moderate to substantial genetic diversity among the rice genotypes. The polymorphic information content (PIC) ranged from 0.24 to 0.65, with an average PIC value of 0.45. Two structured populations were observed which clustered into five heterotic groups and an outgroup, respectively. This suggests that heterosis could be exploited in the next hybridization program by crossing one of the genotypes in any SSR marker-defined cluster, with the rice accession TOG 5681 in cluster I. The results of this study suggest that morpho-agronomic traits should be used to compliment SSR data in rice diversity studies, especially if a few polymorphic SSR markers are to be used.

Assessment of Genetic Diversity among Intra- and Interspecific Lowland Rice using Morpho-agronomic Traits and SSR Markers

Rice is staple food in many countries of Africa and a major part of the diet in many others. However, Africa’s demand for rice exceeds production with the deficit of 40% being imported. One way to improve Africa’s rice production is through breeding high yielding varieties suitable for the different environment conditions. This study was conducted to assess the genetic variability and stability performance of 48 lowland rice genotypes including 37 interspecific (Oryza glaberrima × Oryza sativa ssp. indica) and 11 intraspecific (O. sativa ssp. indica × O. sativa ssp. indica) in 12 environments in Nigeria, Benin Republic and Togo using Additive Main Effect and Multiplicative Interaction (AMMI) and Genotype+ Genotype x Environment (GGE) biplot models. The combined analysis of variance revealed significant differences (P&lt;0.01) among the genotypes, environments, and genotypes x environment interaction. Both the AMMI and GGE models identified NERICA-L8 and NERICA-LI2 as the best genotypes for cultivation across environments. Ouedeme environments in Benin Republic were the most stable and ideal for rice cultivation, while Ibadan sites were the most unstable. TOG 5681 had the least yield and was the most unstable across seasons. Genetic diversity was analyzed using 22 important morpho-agronomic traits and 50 simple sequence repeat (SSR) markers and the results were subjected to principal components analysis (PCA). The results revealed that the first eight PC axes (PC1–8) accounted for 75.13% of the total variation, while PC1–4 accounted for 50.39% of the total variation among rice genotypes. However, 10 of the 50 SSR markers were polymorphic and generated 49 alleles (average = 4.9 alleles per locus), suggesting moderate to substantial genetic diversity among the rice genotypes. The polymorphic information content (PIC) ranged from 0.24 to 0.65, with an average PIC value of 0.45. Two structured populations were observed which clustered into five heterotic groups and an outgroup, respectively. This suggests that heterosis could be exploited in the next hybridization program by crossing one of the genotypes in any SSR marker-defined cluster, with the rice accession TOG 5681 in cluster I. The results of this study suggest that morpho-agronomic traits should be used to compliment SSR data in rice diversity studies, especially if a few polymorphic SSR markers are to be used.

Characterization of Phytophthora infestans Populations in Cyprus, the Southernmost Potato-Producing European Country.

Cyprus is the southernmost island country of Europe, located in the Mediterranean. Despite its limited area, potato production is considered an integral source of the national agricultural revenue. During 2010-2012, a late blight epidemic period for the country, the population structure of Phytophthora infestans was analyzed via a sample of 539 isolates collected from all of the main potato-cultivating regions of Cyprus. We determined mating type, mefenoxam sensitivity, and genetic polymorphism at 12 simple sequence repeat (SSRs) loci. Although both mating types were detected in the country, a gradual but dynamic shift toward A2 dominance was manifested over time. The pathogen population also demonstrated reduced sensitivity to the phenylamide fungicide, since 96.2% of the tested isolates had high (70.3%) and intermediate (25.9%) resistance to mefenoxam, which suggests that it should be replaced with other active ingredients in local disease management strategies. The genotypic analysis also revealed the predominance of the highly aggressive mefenoxam-insensitive EU_13_A2 lineage across the country, with a frequency of 79.2%. Other samples comprised an older lineage EU_2_A1 (19.5%), a very low proportion of EU_23_A1 (0.37%), and others that did not match any known lineage (0.92%). SSRs data supported triploid genomes among the dominant lineages, and patterns of their asexual population history were also apparent. A high subclonal variation of the 13_A2 population was detected, which suggested introduction events of this widespread genotype to Cyprus from major tuber-exporting countries. Present data indicate the severe impact of inoculum migration to the structure of the local population; thus, current phytosanitary procedures should be reconsidered and possibly attuned. This is the first comprehensive study to elucidate the diversity of P. infestans in Cyprus and could serve as a baseline for future monitoring of this highly adaptive plant pathogen, given that late blight management strategies should be constantly refined according to the traits of the dominant genotypes of P. infestans.

Pathogenic and genetic diversity ofPuccinia triticinafrom triticale in Poland between 2012 and 2015

AbstractVariation amongPuccinia triticinaisolates collected from triticale in Poland between 2012 and 2015 was studied based on virulence and simple sequence repeat (SSR) markers. Two hundred and forty‐two single‐uredinial isolates from four geographically separated locations were tested for virulence against 33 near‐isogenic Thatcher lines containing knownLrresistance genes, and their molecular genotypes were characterized with 34 SSR markers. Structure and relationships of the regional and annual populations ofP.triticinawere analysed using an assignment‐based approach for both the virulence and SSR data. The molecular marker analysis was based on two different models of SSR evolution: the stepwise mutation model with a variable mutation rate (SMMv) and the infinite alleles model (IAM). A highly significant deviation from Hardy–Weinberg equilibrium among SSR genotypes, a high proportion of heterozygotes, and a moderate association of relationships between virulence phenotypes and SSR genotypes were consistent with the occurrence of clonal lineages of related races within the population. While the results suggest that genetic drift and mutation affect variation within the pathogen population, it seems that migration has the most significant role in shaping the population structure ofP.triticinaoccurring on triticale in Poland.

The taxonomic relationship between Elaeagnus moorcroftii and E. angustifolia (Elaeagnaceae) based on morphological similarities and simple‐sequence repeat markers

Elaeagnus L. (Elaeagnaceae) is an important plant genus widely used in erosion control due to its high wind breaking and soil binding capacity, but also possessing medicinal and ornamental values. The phenotypic similarity of fruits and flowers of E. moorcroftii and E. angustifolia has often led researchers to speculate on their close relationship and they have sometimes been considered conspecific. In this study, 16 morphological characters were selected and assigned for cluster analysis which divided the samples (n = 49) into six groups: three of E. moorcroftii and three of E. angustifolia. Simple‐sequence repeat (SSR) markers were used to discern the genetic diversity of the 49 individuals. Genetic diversity was found to be high (He = 288, I = 0.938) and there was a strong correlation between the morphological and SSR data. Thus, when combined, the phenotypic classification and SSR marker based evidence confirmed E. moorcroftii as a distinct species independent from E. angustifolia, a conclusion not in agreement with the treatment in recent floras.

Identification and characterization of SSR markers in taro [Colocasia esculenta (L.) Schott] by RAD sequencing

Taro [Colocasia esculenta (L.) Schott] is an old crop with high genetic diversity. However, the breeding of taro is limited by the lack of well-developed genetic markers. To help further research on taro germplasm resource management, we developed a set of SSR marker primers. Restriction site-associated DNA sequencing (RAD-seq) was used to identify SSRs from a taro variety. PCR and capillary electrophoresis were applied to evaluate the efficiency of the primers. A phylogenetic tree was constructed to analyse the evolutionary relationship of 30 taro varieties. A total of 4438 SSRs (simple sequence repeats)were identified using RAD sequencing. A total of 3919 pairs of primers were designed based on the SSR data. Trinucleotide repeats (49.1%) were the most abundant type of SSR, while AT was the most common motif type (327 instances). Among 50 randomly selected primer pairs, 46 primer pairs were able to amplify the corresponding bands. A phylogenetic tree cluster analysis using SSR markers mainly supported the classification of taro varieties according to their phenotypes and ecological distribution. The SSR markers identified in this study provide a useful resource for taro genetic and germplasm research and genetic breeding.

Genetic diversity and population structure of the medicinal plant Docynia delavayi (Franch.) Schneid revealed by transcriptome-based SSR markers

Docynia delavayi is a wild fruit tree with high medicinal value that grows in southwest China. The exploitation and utilization of D. delavayi as a wild resource is constrained by a relative lack of genetic information. In the present study, 18 transcriptome-based simple sequence repeat (SSR) markers were developed using RNA-seq technology. Based on the SSR data, the D. delavayi populations showed high genetic diversity with mean observed heterozygosity (Ho), expected heterozygosity (He), Shannon’s Information Index (I) and polymorphism information content (PIC) values of 0.697, 0.522 0.884, and 0.587, respectively. Lower gene flow (Nm = 1.461) and higher genetic differentiation (Fst = 0.180) were detected among the populations. The results of clustered heatmap, Structure, and PCoA analyses showed that the 12 populations were divided into two clusters (Southwest and Northwest Prefecture populations), which was consistent with the Mantel test revealing a positive correlation between geographic and genetic distance (r = 0.342, p < 0.01). Overall, the genetic diversity of the D. delavayi natural populations was successfully investigated based on SSR markers. This study will provide a theoretical basis for conservation management, genetic improvement, and construction of the core germplasm.

English

Cape gooseberry (Physalis peruviana L.) is a neglected high potential crop, knowledge of the genetic diversity of the genotypes domesticated in Kenya is limited. To understand the genetic diversity and structure within and between Cape gooseberry germplasm, 70 accessions from six selected counties were analyzed using 15 pairs of highly polymorphic SSR primers. In this study, a total of 61 polymorphic SSR alleles were identified with mean polymorphic information content (PIC) of 0.43. Analysis of Molecular Variance (AMOVA) revealed that 92.8% of the total genetic variation was within accessions whereas variation among accessions accounted for 7.2% of the total genetic variation. Genetic diversity parameters among the 70 accessions revealed that Cape gooseberry was more diverse than previously recorded. Based on the SSR data, the 70 accessions were classified into five main phylogenetic groups, which corresponded to the county of origin through factorial analysis, principal component analysis (PCA), and phylogenetic analysis. Seven core SSR primer pairs, namely SSR1, SSR2, SSR10, SSR11, SSR123, SSR138, and SSR146 were found to have a wide applicability in genotype identification of cape gooseberry, and thus they are recommended for use in genetic characterization of germplasm collected from other counties not covered by the present study. This study demonstrated the existence of considerable genetic diversity in Cape gooseberry accessions growing in selected counties in Kenya and can therefore be used as a basis for future breeding programs in the development of hybrids with desirable traits. This wider genetic diversity is vital for posterity as it will help cope with unpredictable climatic changes and human needs. &nbsp; Key words: Simple sequence repeats (SSRs), genetic diversity, germplasm, Physalis peruviana L., polymorphic information content (PIC).

Genetic diversity and genetic structure of Acer monspessulanum L. across Zagros forests of Iran using molecular markers

Acer monspessulanum L. is an important tree species found in the temperate Zagros forests of Iran. Despite its importance, the long-term persistence of its small and fragmented populations is jeopardised by genetic erosion and hence, monitoring its genetic resource and variability is practically required for providing conservation measures of the species germplasm in Zagros woodland ecosystem. The present study aimed to provide the first data on genetic diversity and genetic differentiation pattern of 19 natural populations comprising 240 individuals of A. monspessulanum across its growing area in Zagros forests using three molecular tools including inter-simple sequence repeats (ISSR), start codon targeted (SCoT), and simple sequence repeat (SSR). In total, ISSR and SCoT primers generated a total of 141 and 121 clear and scorable bands for analysis with the polymorphism rate of 90.50 and 90.02% and a mean of 10.85 and 11 fragments per marker, respectively. In addition, 73 alleles were achieved using 10 polymorphic SSR loci from the studied accessions with 100% polymorphism, ranging between 5 and 10 alleles per locus. Average percentage of polymorphic alleles per population for ISSR, SCoT, and SSR data was 84.02%, 83%, and 100%, respectively, and generally, Nei's gene diversity (H) and Shannon's index of diversity (I) values for all populations demonstrated moderate to high levels of genetic diversity (H=0.267-0.707; I=0.38-1.38). The AMOVA results of the three marker systems attributed higher genetic variation to individuals within in each population than among populations. Furthermore, overall GST value for all populations detected the moderate to high levels of genetic differentiation, indicating a limited gene flow occurrence among the populations. STRUCTURE analysis (K=5) clustered the populations into four to five distinct groups, in accordance with geographical distances. These results could represent an important contribution for effective germplasm characterization and could be eventually used in in situ or ex situ conservation of A. monspessulanum genetic resources.

Genetic diversity and population structure of bread wheat genotypes determined via phenotypic and SSR marker analyses under drought-stress conditions

ABSTRACT Genetic diversity is fundamental for conducting successful crop improvement programs. The objectives of this study were to assess the genetic diversity and to deduce the population structure among bread wheat (Triticum aestivum L.) genotypes using phenotypic traits and simple sequence repeat (SSR) markers to identify divergent and complementary parental genotypes for drought-tolerance breeding. Fifty-two selected bread wheat genotypes were evaluated at five test sites in the 2018/19 cropping season using a 4 × 13 alpha lattice design with two replications. The study was carried out under two sowing dates, i.e., early and late planting, corresponding to non-stress and drought-stress conditions, respectively. The test genotypes were assessed using eight phenotypic traits and 20 SSR markers. Analysis of variance showed significant differences among the genotypes for all phenotypic traits studied. SSR analysis identified a total of 181 alleles, with a mean of 10.1 alleles per locus. Population structure analysis grouped the test genotypes into three main populations. Analysis of molecular variance revealed that 85% of the variance was attributable to intra-population differences. Cluster analysis based on phenotypic and SSR data grouped the test genotypes into three major groups. Three genotypes [10 (ETW17-295), 37 (ETW17-385) and 38 (ETW17-386)] were identified as the most divergent, and were recommended for drought-tolerance breeding. Overall, the SSR markers were useful and provided complementary data for selecting agronomically suitable parental lines for drought-tolerance breeding.

Genetic Status of the Swedish Central collection of heirloom apple cultivars

Cultivated apple is one of the most widely grown fruit crops worldwide. With the introduction of modern apple cultivars, from foreign and national breeding programs, the use of local cultivars decreased during the 20th century. In order to minimize genetic erosion and avoid loss of special genotypes, a number of local clonal archives were established across Sweden, with the goal of retaining old and local cultivars. About 220 apple cultivars, appointed for preservation, obtained the status of mandate cultivars. Initially, they were identified based on pomological traits, but prior to the establishment of the Swedish Central Collection they were genotyped with simple sequence repeat (SSR) markers. SSR markers helped to evaluate the status of the preserved material, as well as to find the best possible true-to-type source for propagation, thus guiding the establishment of the Central Collection. Recently, 215 accessions from this collection were genotyped using the 20 K apple Infinium® single nucleotide polymorphism (SNP) array, in order to gain insight into its genetic structure. The initial SSR analysis confirmed the identity of multiple samples with the same cultivar name grown in different locations and identified several mislabeled samples. In the subsequent SNP analysis we identified 30 clonal relationships and a number of parent-offspring relationships, including 18 trios. We also identified five cultivar samples with inconsistent ploidy levels between the SNP and SSR data, in some cases indicating problematic samples preserved in either the Central Collection or some of the local clonal archives. These cultivars need further investigation to ensure their true-to-typeness. Furthermore, the Swedish Central Collection has continued to grow since the onset of this work and now contains additional cultivars, which should be included in future studies. The results indicate that a number of the preserved mandate cultivars holds high potential value for modern breeding programs.

Flower Color Variation in Jones' Penstemon, Penstemon ×jonesii Pennell (P. eatonii A. Gray × P. laevis Pennell) (Plantaginaceae)

Penstemon × jonesii is described as having flowers with the colors of “Tyrian rose,” “amaranth purple,” or “red-purple to maroon.” It has been recorded only in localized areas of southwestern Utah and just over the border of Arizona, where both putative parents commonly occur in sandy soils. Penstemon × jonesii has been reported and widely accepted as a natural hybrid of P. laevis × P. eatonii, though no research has been conducted to verify this assumption. We examined claims of its hybrid origin by making interspecific reciprocal first-generation hybrid plants from the 2 suspected parental species (P. eatonii and P. laevis) as well as by making second-generation hybrids through backcrossing to both parental species. Using 9 Penstemon simple sequence repeat (SSR), or microsatellite, markers, we examined the allelic variation among natural populations of P. × jonesii, P. eatonii, and P. laevis in southwestern Utah. These SSR data, in conjunction with our controlled crosses, support claims that P. × jonesii likely descends from hybridization events between P. eatonii and P. laevis. Flower color of the typical P. × jonesii reported in the literature and found in herbarium samples does not resemble the flower color of F1P. eatonii × P. laevis hybrids from our controlled crosses. However, in subsequent controlled backcrossing of the F1 hybrids to P. eatonii, we found blossom morphotypes and corolla colors matching previous descriptions of P. × jonesii. We also observed many hybrids with lighter corolla colors, such as light pinks, pinkish yellows, and lavender, which are not recorded in the literature or found in herbarium specimens. Field surveys for natural color variation in P. × jonesii populations also revealed greater flower color variation than previously reported, which should be considered as part of this hybrid taxa as well, though the predominant floral colors of P. × jonesii are “Tyrian rose,” “amaranth purple,” and “red-purple to maroon,” which suggests some selective bias. We suggest that pollinator preference for dark red to purple blooms may be responsible for this phenomenon.