Polymorphic Microsatellite Markers Research Articles

Cryptic sexual reproduction in an emerging Eucalyptus shoot and foliar pathogen

AbstractEucalyptus scab and shoot malformation is an emerging disease and a serious threat to the global plantation forestry industry. The disease appeared in North Sumatra (Indonesia) in the early 2010s and the causal agent was recently described as a novel species, Elsinoe necatrix. Nothing is known regarding its possible origin or why it emerged rapidly to cause a serious local epidemic. To investigate its population biology, we developed 15 polymorphic microsatellite markers as well as mating‐type markers using genome sequences for two E. necatrix isolates. Isolates of the pathogen were collected from different host varieties at four locations in the Lake Toba region of North Sumatra and characterized using these markers. A high level of genotypic diversity was observed for all populations with little to no genetic differentiation between sampling areas. Discriminant analysis of principal components, genotype networks and analysis of molecular variance all showed a lack of population structure and a high level of gene flow among sampling regions. Mating‐type ratios and linkage disequilibrium analyses suggest that sexual recombination is likely to be occurring, although a sexual state has not been found for the pathogen. The results of this study highlight the fact that new genotypes of E. necatrix, probably arising from cryptic sexual recombination, will challenge efforts to manage the disease, and that breeding and selection for tolerance will require substantial host genetic diversity.

Polly: An R package for genotyping microsatellites and detecting highly polymorphic DNA markers from short-read data.

Highly polymorphic markers, such as microsatellites, are invaluable for the study of natural populations. However, contemporary methods for genotyping highly polymorphic variants have serious drawbacks that impede their efficiency. We created Polly, an R package with C++ source code that uses Illumina short-read data to genotype microsatellites, detect highly polymorphic variants and identify clusters of highly polymorphic SNPs, indels and microsatellites. We tested Polly on short-read data from Xiphophorus birchmanni (Teleostei: Poeciliidae) and Arabidopsis thaliana, finding it to be efficient and accurate both for microsatellite genotyping and polymorphic marker detection. This program can be applied to any diploid population for which there exists short-read data and at least one scaffolded reference genome.

Genetic diversity of three consecutive selective breeding generations in Pseudobagrus vachellii (Actinopterygii: Siluriformes: Bagridae)

Pseudobagrus vachellii (Richardson, 1846) is a commercially important freshwater fish species in China. To understand the effects of artificial breeding on the genetic diversity of three consecutive P. vachellii breeding populations (F1, F2, and F3) since 2012, a genetic analysis was conducted using polymorphic microsatellite markers. The mean allele number, expected heterozygosity, observed heterozygosity, and the polymorphic information content from generation F1 to F3 decreased from 7.75 to 5.63, from 0.77 to 0.63, from 0.83 to 0.77, and from 0.72 to 0.58, respectively. Analysis of molecular variance showed greater genetic divergence within the three generations (93.67%) than that among the generations (6.33%), and the overall differentiation level was moderate. Additionally, the lowest genetic differentiation was between F2 and F3 (Fst = 0.0484), and the highest was between F1 and F3 (Fst = 0.12864). Inbreeding occurred in each generation and was the highest in generation F3. Structural analysis showed that the three P. vachellii generations were most likely divided into two different genetic clusters. Although genetic diversity declined slightly in the mass selection lines after three breeding generations, overall genetic diversity was maintained at a relatively high level. To minimize the loss of genetic diversity and inbreeding in the subsequent breeding process, a moderate number of parents can be used for each generation. Information regarding the genetic diversity and structure of the selective P. vachellii breeding generations obtained in this study will be useful for future broodstock management and selective breeding programs.

Population genetic structure of tropical bed bug (Hemiptera: Cimicidae) populations and their breeding pattern in Iraq.

A study was conducted to investigate the population genetic structure and breeding pattern of 140 tropical bed bugs, Cimex hemipterus (F.) (Hemiptera: Cimicidae), collected from 14 infested sites in major cities in Iraq. The samples were genotyped using a set of 7 polymorphic microsatellite markers. High genetic variety was seen among populations, with an average of 2-9 alleles per locus. The number of alleles across 7 microsatellite loci was between 6 and 18. There was a notable disparity in the alleles per loci when comparing the overall population to those within it. The overall population exhibited an average observed heterozygosity of 0.175 and an average expected heterozygosity of 0.730. Among the population, the average observed heterozygosity was 0.173, while the average expected heterozygosity was 0.673. Analysis of molecular variance (AMOVA) revealed that 93% of the genetic variability was within the populations, and 7% was among them. The genetic differentiation coefficient (FST = 0.045), indicates a low degree of genetic differentiation and a high degree of inbreeding (FIS = 0.761), as indicated by notably significant positive inbreeding coefficients. Admixed individuals were revealed using STRUCTURE and neighbor-joining phylogenetic trees, demonstrating moderate gene flow between populations and a lack of genetic structure in the regional groups. Thus, both active dispersion and human-mediated dispersion possess the potential to influence the low population genetic structure of tropical bed bug C. hemipterus populations in Iraq, which can have implications toward tropical bed bug and management strategies.

Population Genetic Structure of the Rubber Tree Powdery Mildew Pathogen (Erysiphe quercicola) from China.

In order to manage agricultural pathogens, it is crucial to understand the population structure underlying epidemics. Rubber tree powdery mildew, caused by Erysiphe quercicola, is a serious threat to rubber plantations worldwide, especially in subtropical environments including all rubber tree-growing regions in China. However, the population structure of the pathogen is uncertain. In this study, 16 polymorphic microsatellite markers were used to genotype powdery mildew samples from the main rubber tree-growing regions including Yunnan (YN), Hainan (HN), western Guangdong (WG), and eastern Guangdong (EG). YN had higher genotypic diversity (Simpson's indices), genotypic evenness, Nei's gene diversity, allelic richness, and private allelic richness than the other regions. Cluster analysis, discriminant analysis of principal components, pairwise divergence, and shared multilocus genotype analyses all showed that YN differed significantly from the other regions. The genetic differentiation was small among the other three regions (HN, WG, and EG). Analysis of molecular variance indicated that the variability among regions accounted for 22.37% of the total variability. Genetic differentiation was significantly positively correlated (Rxy = 0.772, P = 0.001) with geographic distance. Linkage equilibrium analysis suggested possible occurrence of sexual recombination although asexual reproduction predominates in E. quercicola. The results suggested that although significant genetic differentiation of E. quercicola occurred between YN and the other regions, pathogen populations from the other three regions lacked genetic differentiation.

Unfolding genomic regions and identification of novel marker-trait associations for therapeutic traits flavonoid and anthocyanin content in rice (Oryza sativa L.)

The genetic diversity, structure analysis, and marker-trait association for total flavonoid content (TFC) and anthocyanin content (AC) were studied in rice using 79 germplasm lines with 210 polymorphic microsatellite markers. Among the landraces studied, RG83 (RPHP 93) had the highest level of TFC (227.78 QE/100g) and RG126 (Kallimadayan) contained the highest level of AC (0.416 CE/100g). The STRUCTURE analysis grouped the studied landraces into three genetic structure groups which nearly matched the geographical distance. The results of the marker-trait associations study using MLM with PCA and kinship indicated that the SSR markers RM287 and RM19358 were closely associated with AC while another two markers RM452 and RM 2197 were closely linked with TFC and these two markers were found to be in the genomic regions related to the candidate genes for TFC and AC biosynthesis. In addition, the markers RM10961, RM5963, and RM559 were also found to have significant marker-trait associations with both AC and TFC.

Anthropogenic and natural disturbances increase local genetic diversity in an early spring geophyte (Ficaria verna Huds)

AbstractThe tetraploid Ficaria verna is a common spring geophyte in central Europe and is considered invasive in the USA and Canada. It is considered an almost seed‐sterile taxon, relying on vegetative reproduction by underground tubers and aerial bulbils. Recent studies have revealed high levels of population genetic diversity in F. verna, raising the question of how genetic diversity is maintained and which factors may be responsible for the observed patterns. Polymorphic nuclear microsatellite markers were established to define multi‐locus genotypes (MLGs), to analyze fine‐scale spatial genetic structure (SGS) using grid and cross‐sampling schemes, and to quantify genetic diversity within and between nine populations with different disturbance regimes in central Germany. In total, 115 MLGs were identified among a total of 347 samples. The G/N ratio varied between 0.16 and 0.70 among populations, and in each population several unique MLGs occurred. Genotypes were highly intermingled within populations, suggesting a “guerrilla” dispersal strategy. Significant SGS (negative regression slope of kinship coefficients against inter‐individual distances) was found in four out of nine populations in fine‐scale cross‐sampling (up to 4 m) and in only one population in grid sampling (up to 14.6 m). No single MLG was found in more than one population, while many alleles were shared between populations. Within‐population clonal and allelic diversity increased with greater exposure to both anthropogenic and natural disturbances. Regular gap openings, facilitated propagule establishment, and propagule dispersal by water and mowing machines are likely important factors explaining the positive effects of disturbance on local genetic diversity of F. verna.

Does the geographic distance affect the genetic differentiation among bilberry populations sampled in Bosnia and Herzegovina?

Isolation-by-distance (IBD) pattern among bilberry (Vaccinium myrtillus L.) populations has previously been reported for this species in northern Europe. However, the number of molecular studies conducted on bilberry, using everything from isoenzymes, RAPDs to microsatellite markers, are very few and far between. Considering that Bosnia and Herzegovina (B&H) is a country rich with diverse fruit genetic resources, conducting a genetic characterization of the naturally occurring V. myrtillus populations could yield valuable data for the conservation and utilization of this resource. This study entailed genotyping samples collected from three bilberry populations located in Fojnica, Kladanj, and Srebrenica municipalities using seven polymorphic microsatellite or SSR (simple sequence repeats) markers. The obtained molecular data was used to calculate the correlation between the physical distance of the individual B&H populations and a parameter of the genetic differentiation (pairwise Fst). The results of the correlation analyses revealed an absence of a significant isolation-by-distance pattern among the three examined B&H bilberry populations. In addition, the most pronounced genetic differentiation was detected between the Srebrenica and each of the two remaining B&H populations. At the same time, the values for pFst were significant, albeit much lower, between the Fojnica and Kladanj populations. Bilberries from the sampled Srebrenica population appear to be distinct from the other B&H populations, possibly due to the different genetic origin of this population.

Major Histocompatibility Complex Region and Diversity of the Local Chicken Populations In Niger

Introduction: The major histocompatibility complex (MHC) of chicken is highly polymorphic, and it is linked to several disease resistance or susceptibility traits. Therefore, the current study aimed to analyze the genetic diversity in the MHC region of Nigerien local chicken (Dourgou, Goggori, Kolonto, Tchagara, Gouzou-gouzou, and Popular) using a high polymorphic microsatellite marker named LEI0258 to determine the diversity of chickens kept at the four agroecological zones in Niger. Materials and methods: A total of 601 chickens from six local Nigerien chickens were sampled. By capillary electrophoresis using LEI0258 marker, 403 samples with different fragment sizes were randomly chosen and sequenced. Results: The findings indicated 80 different alleles ranging in size from 181 to 474 bp. A total of 22 new alleles and 39 private alleles (that existed in only one breed) were detected. The alleles 309, 295, and 193 were the most predominant in the Nigerien local chicken population. Nine polymorphisms were observed along the LEI0258 sequence, including three in the upstream (one indel and two Single Nucleotide Polymorphism [SNP]), one in the repeat region at the last R12 (SNP), and five in the downstream (two indels and three SNPs). Conclusion: The chickens are not clustering according to their agroecological zone of origin. They are randomly distributed across the four investigated agroecological zones. The information found in this study is invaluable in breeding and conservation programs associated with several disease resistance or susceptibility traits.

Genotyping blastoderms of avian eggs.

Undeveloped eggs occur frequently in birds and are often considered infertile, and discarded. However, the majority of undeveloped eggs may in fact have been fertilised and embryos might have died at an early stage. Such eggs contain valuable information, for example about offspring sex and paternity, and level of inbreeding. Obtaining such information may also give insight into the patterns and causes of early embryo mortality. Here we describe a simple technique for removing embryo cells from the blastoderm to obtain DNA to genotype the offspring and unequivocally ascertain fertilisation status, while retaining the overlying perivitelline layer (PVL) for sperm counts over the entire membrane. We tested this method on freshly collected eggs (high-quality material), as well as on eggs from abandoned clutches and unhatched eggs (potentially deteriorated material) of blue tits (Cyanistes caeruleus). We sampled a total of 707 eggs from a wild population of blue tits, extracted DNA from the eggs' blastoderm using a Qiagen kit, and genotyped the samples with 14 polymorphic microsatellite markers, plus one sexing marker. Overall, we successfully genotyped 97% of all eggs. Our study is the most extensive dataset of genotyped undeveloped eggs to date and demonstrates that one can reliably genotype undeveloped fertile eggs as well as retain the PVL for observations of sperm and embryo cells.

Population Genetic Structure of a Viviparous Sand Lizard, the Phrynocephalus forsythii in the Tarim Basin, Xinjiang of China

The desert ecosystem occupies an important position in the composition of global biodiversity. The Tarim Basin is located in south Xinjiang of China and has the world’s second largest mobile desert, the Taklamakan Desert. As an endemic species in this region, Phrynocephalus forsythii has been demonstrated to have a potentially high extinction risk due to climate change. In order to understand the overall genetic status and provide accordant conservation strategies for the species, we investigated the genetic diversity and population structure of P. forsythii from 15 sites in the Tarim Basin using 21 highly polymorphic microsatellite markers. We found significant genetic structure across the study region. We also revealed generally low levels of gene flow between the 25 sites, suggesting individual dispersal and migration may be restricted within populations. In addition, geographical distance and ambient temperature might be important factors in explaining the observed genetic structure. Our results will provide a scientific basis for the future protection of P. forsythii in this area, as well as an important reference for the conservation and management of biodiversity in desert ecosystems.

Evaluation for Parental Polymorphism and Identification of Microsatellites Linked to Drought Tolerance in Rice (Oryza sativa L.)

Aims: To identify the ready-to-use polymorphic microsatellite markers associated with drought tolerance for marker-assisted backcross breeding through a polymorphism survey between Jyothi and Chuvannamodan rice varieties.
 Place and Duration of Study: Centre for plant biotechnology and molecular biology, Thrissur, Kerala, India, during January to May 2023.
 Methodology: The genomic DNA of Jyothi and Chuvannamodan was isolated by following CTAB (Cetyltrimethyl Ammonium Bromide) method with modification. Isolated DNA from both varieties was subjected to PCR amplification using 208 Simple Sequence Repeats (SSRs) primer pairs distributed in 12 chromosomes. The amplified PCR products were electrophoresed in 3% agarose gel and separated fragments were visualized and documented in the Gel Documentation System. Different allele sizes produced by the same SSRs primers between two varieties are identified as polymorphic markers. Polymorphism per-cent was calculated, and frequency distribution and chromosome distribution of polymorphic markers were analyzed.
 Results: A total of 208 SSR primers were surveyed for the parental polymorphism. Out of which, 85 SSR primers exhibited clear polymorphism between Jyothi and Chuvannamodan and the remaining 123 were monomorphic primers. The amplicon size ranged from 83bp (RM430) to 495bp (RM18919) among the different primers. The survey revealed maximum parental polymorphism on chromosome 4 (69.23%), followed by chromosome 5 (64.28%), and minimum polymorphism on chromosome 8 (21.73%). The average per cent of polymorphism between the parents was 40.86%. Among the 85 polymorphic markers, 66 had dinucleotide repeats, 17 had trinucleotide repeats, and 1 had tetranucleotide repeats.
 Markers with dinucleotide repeats showed higher level of polymorphism. A group of 28 polymorphic markers were identified to be linked with traits including root-related traits, grain yield, leaf rolling and leaf drying under drought conditions.
 Conclusion: The polymorphic markers identified in the present study form the basis for tagging drought-tolerant QTLs/genes, fine mapping of those genes, and subsequently in marker-assisted breeding programs. The polymorphic markers linked with the QTLs for grain yield under drought can be pyramided in Jyothi through marker-assisted backcross breeding.

Evidence of High Genetic Diversity and Differences in the Population Diversity of the Eucalyptus Leaf Blight Pathogen Calonectria pseudoreteaudii from Diseased Leaves and Soil in a Plantation in Guangxi, China.

Calonectria pseudoreteaudii is an important causal agent of Eucalyptus leaf blight in southern China. This pathogen causes Eucalyptus tree disease across numerous regions in southern China. In addition to diseased leaves, C. pseudoreteaudii has occasionally been isolated from soil in Eucalyptus plantations. The aim of this study was to clarify whether C. pseudoreteaudii causing Eucalyptus leaf blight in China is mainly clonally reproduced and to determine the potential spreading mechanism of C. pseudoreteaudii between diseased leaves and soil. To this end, 10 polymorphic microsatellite markers were analyzed to detect the genetic diversity of 97 C. pseudoreteaudii isolates from diseased leaves and soil in a Eucalyptus plantation in Guangxi Zhuang Autonomous Region, southern China. The analysis showed that the genetic diversity of the isolates from both the diseased leaves and soil was high. However, the gene and genotype diversity of the C. pseudoreteaudii isolates from diseased leaves were higher than those of the isolates from the soil. Moreover, all genotypes detected in the isolates from the soil were also found in the isolates from the diseased leaves. Structural analyses did not show clear population structures related to the population substrates of the diseased leaves or soil, and molecular variance analyses indicated that no significant genetic differentiation existed between the diseased leaf and soil populations. These results suggest that C. pseudoreteaudii in soil spreads from diseased leaves, and that an asexual cycle is the primary reproductive mode in both diseased leaf and soil populations. This is the first study on the genetic diversity and population structure of C. pseudoreteaudii. The high genetic diversity and spread pathways of this pathogen may pose challenges in controlling the disease. C. pseudoreteaudii from both diseased leaves and soils in Eucalyptus plantations needs to be carefully monitored for disease control and management.

Molecular evidence for multiple origins and high genetic differentiation of non-native winter crane fly, Trichocera maculipennis (Diptera: Trichoceridae), in the maritime Antarctic

Native biodiversity and ecosystems of Antarctica safeguarded from biological invasion face recent threats from non-native species, accelerated by increasing human activities and climate changes. Over two decades ago, the winter crane fly, Trichocera maculipennis, was first detected on King George Island. It has now successfully colonized several research stations across King George Island. To understand the origin, genetic diversity, and population structure of this Holarctic species, we conducted mitochondrial DNA cytochrome c oxidase subunit I (COI) sequence analysis across both its native and invasive ranges. In parallel, we performed microsatellite loci analysis within the invasive ranges, utilizing 12 polymorphic microsatellite markers. Furthermore, we compared body sizes among adult males and females collected from three different locations of King George Island. Our COI sequence analysis exhibited two different lineages present on King George Island. Lineage I was linked to Arctic Svalbard and Polish cave populations and Lineage II was related to Canadian Terra Nova National Park populations, implying multiple origins. Microsatellite analysis further exhibited high levels of genetic diversity and significant levels of genetic differentiation among invasive populations. Body sizes of adult T. maculipennis were significantly different among invasive populations but were not attributed to genetics. This significant genetic diversity likely facilitated the rapid colonization and establishment of T. maculipennis on King George Island, contributing to their successful invasion. Molecular analysis results revealed a substantial amount of genetic variation within invasive populations, which can serve as management units for invasive species control. Furthermore, the genetic markers we developed in the study will be invaluable tools for tracking impending invasion events and the travel routes of new individuals. Taken together, these findings illustrate the highly invasive and adaptable characteristics of T. maculipennis. Therefore, immediate action is necessary to mitigate their ongoing invasion and facilitate their eradication.

Founder effect of a pathogenic TNNT2 variant in the northwest of Spain responsible for a non-severe phenotype of hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder, with a prevalence between 1/250-500 individuals. The yield of genetic testing for this condition stands at 40%. The most prevalent genes are MYBPC3 and MYH7, with small contributions of other genes, such as TNNT2, TNNI3, TPM1, MYL2 or FHOD3. At our center, we have identified a recurrent variant in TNNT2, p.Asn271Ile, associated with HCM. Most probands came from the northwest of Spain (Galicia), which suggested that they may share a common origin. Purpose We aimed to determine whether carriers of the p.Asn271Ile variant in TNNT2 share a common haplotype, in order to estimate the age of the mutation and describe the associated phenotype. Methods We analyzed probands who had TNNT2 sequenced by NGS at our center. Enrichment of the variant was observed in the HCM cohort. Eight highly polymorphic microsatellite markers flanking the variant in 18 Galician HCM probands were also analyzed, compromising a region of approximately 7,300 kb. Custom PCR assays were designed, and fragment length analysis was performed by capillary electrophoresis. Results TNNT2 p.Asn271Ile was identified in 49/28,802 probands (0.17%). The variant was significantly enriched in HCM probands (47/11,870; 0.39%) compared to internal non-HCM controls (2/14,804; 0.02%), with an OR=29.3 (IC95%=7.1-120.7). The analysis revealed that the 18 HCM Galician probands shared a common haplotype of 500 kb, estimating that p.Asn271Ile has arisen approximately 26.5 generations ago (650 years). Regarding genotype-phenotype correlation, our data (more than 180 carriers from 64 families) shows a relatively benign behavior, with an advance mean age at diagnosis [(49.8 (±17.7)], non-severe left ventricular hypertrophy [15.3 (±5.3)], and a lower rate of major events among carriers of p.Asn271Ile in TNNT2, compared to both other genetic variants in TNNT2 and the main genetic cause of HCM (i.e., null variants in MYBPC3) (p=0.0001 for both comparisons). The prevalence of left ventricular systolic dysfunction and left ventricular tract obstruction was also smaller than that described for other sarcomeric genes (3.54% and 12%, respectively). Conclusions A founder effect of the pathogenic variant p.Asn271Ile has been demonstrated, with the variant arising approximately 650 years ago. The clinical behavior in carriers of this cohort showed to be relatively benign compared to the behavior of other genetic variants in this gene and null variants in MYBPC3.

Genetic diversity and population structure of Mastacembelus armatus in the river systems of southern China revealed by microsatellites

The Zig-zag eel (Mastacembelus armatus) is an economically important species in southern China. Its natural resources have declined year by year due to overfishing. Understanding its genetic diversity and population structure is very important for resource conservations. Here, we first successfully developed 28 polymorphic microsatellite markers for zig-zag eels and ten of them were used to examine the genetic diversity and differentiation of 7 populations collected from the major river systems of south China. In total, 224 alleles were found with the 10 microsatellite loci in 7 populations, ranging from 4.6 (Nandujiang: NDJ) to 11.1 (Xijiang, XJ), with an average of 8.871 alleles. The average observed and expected heterozygosity ranged from 0.550 (NDJ) to 0.964 (Yuangjiang, YJ) and from 0.537 (NDJ) to 0.775 (Tanjiang, TJ), respectively. The average polymorphism-information content ranged from 0.472 (NDJ) to 0.757 (TJ). Hardy–Weinberg equilibrium test results revealed the loci showed differing deviation in different populations. In total, low level of genetic diversity was only found in HJ (Hanjiang) and NDJ populations. Besides, evidence of recent bottleneck was found in the HJ populations. Analysis of molecular variation showed that the percent variation within individuals (75.00%) was higher than that among populations (25%). In addition, population structure and the pairwise FST revealed that there was low differentiation among XJ, TJ and YJ populations. These data provide important genetic resources for understanding the population differentiation and facilitating genetic conservation and utilization of this species.

Characterization and development of 14 microsatellite markers for mountain stewartia (Stewartia ovata): an uncommon, Appalachian-endemic member of the tea family

ABSTRACT Mountain stewartia [Stewartia ovata (Cav.) Weatherby] is a rare, endemic understory tree native to the southeastern U.S.A. While found only in microsites across southeastern Appalachia, the species distribution spans from northern Alabama and Georgia to southern Kentucky and Virginia. However, most occurrences have been recorded in Tennessee. While recent distribution surveys and habitat suitability modeling conducted for this species have improved our understanding of the environmental parameters that constitute its fundamental niche, the ecological profile of S. ovata remains poorly understood. Consequently, its rarity has raised concerns as to the long-term resilience of this species in the wild. Faced with these challenges, the assessment of existing genetic diversity in S. ovata through genetically informative molecular resources is critical to understanding the adaptive potential and ecological resilience of the species. Microsatellites are cost-efficient molecular tools capable of addressing these concerns by elucidating trends in population structure, population demography, and inbreeding. To increase the available genetic resources for S. ovata, we screened 105 microsatellite loci for their suitability to assess these population features using capillary gel electrophoresis. The utility of these markers was evaluated by assessing polymorphism information content (PIC) and population statistics, which yielded 14 highly polymorphic microsatellite markers. These markers displayed an average PIC of 0.695 (PIC range of 0.538–0.851), which supports their ability to capture fine-scale genetic diversity within the species. We also report a low average observed heterozygosity of 0.375 (versus an expected heterozygosity of 0.645) among screened loci, which reinforces the need for broader genetic diversity assessments within this species.

Development of Polymorphic Microsatellite Markers and Identification of Applications for Wild Walnut (Juglans regia L.) in Middle Asia

The common walnut (Juglans regia L.), a species of significant economic and ecological importance, along with its variant, holds developmental value. Simple sequence repeat (SSR) markers are used as an effective method for material identification due to their co-dominant inheritance. In this work, a total of 357629 SSR loci were searched based on previously published walnut genome sequences, with a density of 662.28 (SSRs/Mb). The majority of all these loci were found to be single nucleotide A/T (58.49%), followed by the dinucleotide AT (19.48%). Based on the results of genome-wide SSR site design, 22 effective primer pairs were designed and screened to analyze the genetic diversity of 48 wild walnut samples from three countries. Each locus had an average of 5.17 effective alleles (Ne), with an average polymorphism information content (PIC) of 0.71, average heterozygosity (He) of 0.52, and average genetic differentiation index (Fst) of 0.09. The analysis of 48 wild walnut samples from three countries (Kyrgyzstan, Tajikistan, and China) using the unweighted pairwise clustering method and arithmetic mean (UPGMA) along with principal coordinate analysis revealed the division of all samples into three groups. In summary, these novel SSR markers serve as a reference for the identification of wild walnut germplasm in Central Asia and for the development and utilization of wild walnut seed resources.

Development of polymorphic microsatellite markers for Fagus pashanica (Fagaceae) using next-generation sequencing.

Fagus pashanica is an endangered and endemic tree species in China. To understand its genetic diversity and structure for effective conservation, we used next-generation sequencing data to develop a set of microsatellite markers. Twenty-three of the 68 designed loci were successfully amplified. Fifteen polymorphic loci with clear peaks were selected for further analyses in three F. pashanica populations sampled from Nanjiang, Wangcang and Pingwu counties in Sichuan Province, China. The number of alleles per locus ranged from two to 11. The levels of observed and expected heterozygosity ranged from 0.033-0.852 and 0.033-0.787, respectively. All 23 loci were also successfully amplified in F. longipetiolata and F. lucida, and 19 were successfully amplified in F. engleriana. These microsatellite markers will be useful for population genetic studies of F. pashanica and other Fagus species.

Microsatellite Markers and Genetic Diversity Analysis in Grape (Vitis vinifera L)

Abstract Grape (Vitis vinifera L), commonly cultivated in India, shows a wide range of ripening periods and fruit quality and is an unexploited resource for breeding programs. The main purpose of this study was to fingerprint these accessions and to construct a molecular database including the cultivars commonly grown in India. A total of 42 genotypes were analyzed using seven microsatellite simple sequence repeat (SSR) markers and their main morphological and agronomic characteristics were com-pared. A total of 45 alleles in all 42 genotypes were obtained with 7 primers with an average number of 6.4 alleles per locus. The dis-similarity matrix showed that a maximum of 110 units was obtained between the genotypes “Convent Large Black” and “Arka Hans,” while a minimum dissimilarity if 37 units were obtained between the genotypes “Anab-e-Shahi” and “Dilkhush.” In the dendrogram the microsatellite segregated the genotypes into two clusters (A and B) at 476 units with two subclusters each. The sub-clusters grouped genotypes predominantly as A1 with seeded fruits, A2 with seedless fruits, B1 with pigmented and seedless fruits, and B2 with colorless and seeded fruits. The use of seven polymorphicmicrosatellite markers and the level of genetic variability detected within Indian grapevine germplasm suggested that this is a reliable, efficient, and effective marker system that can be used for diversity analysis and subsequently in crop improvement programs