Allelic Diversity Research Articles

Optimizing targeted gene flow to maximize local genetic diversity: when and how to act under various scenarios of environmental change

Targeted gene flow is an emerging conservation approach which involves introducing a cohort of individuals with particular traits to locations where they can produce a conservation benefit. This technique is being proposed to adapt recipient populations to a known threat, but questions remain surrounding how best to maximize conservation outcomes during periods of continuous directional environmental change. Here we introduce a new management objective—to keep the recipient population extant and with maximum diversity of local alleles—and we explore how varying the timing and size of an introduction can maximise this objective. Our results reveal a trade-off between keeping a population extant and maintaining a high level of genetic diversity, but management levers can often optimize this so that nearly 100% of the allelic diversity is preserved. These optimum outcomes sets are highly sensitive to the predicted rate of environmental shift, as well as the level of outbreeding depression in the system.

Altering specificity and auto-activity of plant immune receptors Sr33 and Sr50 via a rational engineering approach.

Many resistance genes deployed against pathogens in crops are intracellular nucleotide-binding leucine-rich repeat receptors (NLRs). The ability to rationally engineer the specificity of NLRs will be crucial in the response to newly emerging crop diseases. Successful attempts to modify NLR recognition have been limited to untargeted approaches or depended on previously available structural information or knowledge of pathogen-effector targets. However, this information is not available for most NLR-effector pairs. Here, we demonstrate the precise prediction and subsequent transfer of residues involved in effector recognition between two closely related NLRs without their experimentally determined structure or detailed knowledge about their pathogen effector targets. By combining phylogenetics, allele diversity analysis, and structural modeling, we successfully predicted residues mediating interaction of Sr50 with its cognate effector AvrSr50 and transferred Sr50's recognition specificity to the closely related NLR Sr33. We created synthetic versions of Sr33 that contain amino acids from Sr50, including Sr33syn that gained the ability to recognize AvrSr50 with 12 amino acid substitutions. Furthermore, we discovered that sites in the leucine rich-repeat domain needed to transfer recognition specificity to Sr33 also influence auto-activity in Sr50. Structural modeling suggests these residues interact with a part of the NB-ARC domain, which we named the NB-ARC latch, to possibly maintain the inactive state of the receptor. Our approach demonstrates rational modifications of NLRs, which could be useful to enhance existing elite crop germplasm.

Divergent allele advantage in the MHC and amphibian emerging infectious disease.

Genetic variation in the major histocompatibility complex (MHC) may be associated with resistance to the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd). The pathogen originated in Asia, then spread worldwide, causing amphibian population declines and species extinctions. We compared the expressed MHC IIβ1 alleles of a Bd-resistant species, Bufo gargarizans, from South Korea with those of a Bd-susceptible Australasian species, Litoria caerulea. We found at least six expressed MHC IIβ1 loci in each of the two species. Amino acid diversity encoded by these MHC alleles was similar between species, but the genetic distance of those alleles known for potential broader pathogen-derived peptide binding was greater in the Bd-resistant species. In addition, we found a potentially rare allele in one resistant individual from the Bd-susceptible species. Deep next-generation sequencing recovered approximately triple the genetic resolution accessible from traditional cloning-based genotyping. Targeting the full MHC IIβ1 enables us to better understand how host MHC may adapt to emerging infectious diseases.

The evolutionary maintenance of ancient recombining sex chromosomes in the ostrich.

Sex chromosomes have evolved repeatedly across the tree of life and often exhibit extreme size dimorphism due to genetic degeneration of the sex-limited chromosome (e.g. the W chromosome of some birds and Y chromosome of mammals). However, in some lineages, ancient sex-limited chromosomes have escaped degeneration. Here, we study the evolutionary maintenance of sex chromosomes in the ostrich (Struthio camelus), where the W remains 65% the size of the Z chromosome, despite being more than 100 million years old. Using genome-wide resequencing data, we show that the population scaled recombination rate of the pseudoautosomal region (PAR) is higher than similar sized autosomes and is correlated with pedigree-based recombination rate in the heterogametic females, but not homogametic males. Genetic variation within the sex-linked region (SLR) (π = 0.001) was significantly lower than in the PAR, consistent with recombination cessation. Conversely, genetic variation across the PAR (π = 0.0016) was similar to that of autosomes and dependent on local recombination rates, GC content and to a lesser extent, gene density. In particular, the region close to the SLR was as genetically diverse as autosomes, likely due to high recombination rates around the PAR boundary restricting genetic linkage with the SLR to only ~50Kb. The potential for alleles with antagonistic fitness effects in males and females to drive chromosome degeneration is therefore limited. While some regions of the PAR had divergent male-female allele frequencies, suggestive of sexually antagonistic alleles, coalescent simulations showed this was broadly consistent with neutral genetic processes. Our results indicate that the degeneration of the large and ancient sex chromosomes of the ostrich may have been slowed by high recombination in the female PAR, reducing the scope for the accumulation of sexually antagonistic variation to generate selection for recombination cessation.

High functional allelic diversity and copy number in both MHC classes in the common buzzard

BackgroundThe major histocompatibility complex (MHC), which encodes molecules that recognize various pathogens and parasites and initiates the adaptive immune response in vertebrates, is renowned for its exceptional polymorphism and is a model of adaptive gene evolution. In birds, the number of MHC genes and sequence diversity varies greatly among taxa, believed due to evolutionary history and differential selection pressures. Earlier characterization studies and recent comparative studies suggest that non-passerine species have relatively few MHC gene copies compared to passerines. Additionally, comparative studies that have looked at partial MHC sequences have speculated that non-passerines have opposite patterns of selection on MHC class I (MHC-I) and class II (MHC-II) loci than passerines: namely, greater sequence diversity and signals of selection on MHC-II than MHC-I. However, new sequencing technology is revealing much greater MHC variation than previously expected while also facilitating full sequence variant detection directly from genomic data. Our study aims to take advantage of high-throughput sequencing methods to fully characterize both classes and domains of MHC of a non-passerine bird of prey, the common buzzard (Buteo buteo), to test predictions of MHC variation and differential selection on MHC classes.ResultsUsing genetic, genomic, and transcriptomic high-throughput sequencing data, we established common buzzards have at least three loci that produce functional alleles at both MHC classes. In total, we characterize 91 alleles from 113 common buzzard chicks for MHC-I exon 3 and 41 alleles from 125 chicks for MHC-IIB exon 2. Among these alleles, we found greater sequence polymorphism and stronger diversifying selection at MHC-IIB exon 2 than MHC-I exon 3, suggesting differential selection pressures on MHC classes. However, upon further investigation of the entire peptide-binding groove by including genomic data from MHC-I exon 2 and MHC-IIA exon 2, this turned out to be false. MHC-I exon 2 was as polymorphic as MHC-IIB exon 2 and MHC-IIA exon 2 was essentially invariant. Thus, comparisons between MHC-I and MHC-II that included both domains of the peptide-binding groove showed no differences in polymorphism nor diversifying selection between the classes. Nevertheless, selection analysis indicates balancing selection has been acting on common buzzard MHC and phylogenetic inference revealed that trans-species polymorphism is present between common buzzards and species separated for over 33 million years for class I and class II.ConclusionsWe characterize and confirm the functionality of unexpectedly high copy number and allelic diversity in both MHC classes of a bird of prey. While balancing selection is acting on both classes, there is no evidence of differential selection pressure on MHC classes in common buzzards and this result may hold more generally once more data for understudied MHC exons becomes available.

Genetic structure and triazole resistance among Aspergillus fumigatus populations from remote and undeveloped regions in Eastern Himalaya.

Aspergillus fumigatus is a ubiquitous mold and a common human fungal pathogen. Recent molecular population genetic and epidemiological analyses have revealed evidence for long-distance gene flow and high genetic diversity within most local populations of A. fumigatus. However, little is known about the impact of regional landscape factors in shaping the population diversity patterns of this species. Here we sampled extensively and investigated the population structure of A. fumigatus from soils in the Three Parallel Rivers (TPR) region in Eastern Himalaya. This region is remote, undeveloped and sparsely populated, bordered by glaciated peaks more than 6,000 m above sea level, and contained three rivers separated by tall mountains over very short horizontal distances. A total of 358 A. fumigatus strains from 19 sites along the three rivers were isolated and analyzed at nine loci containing short tandem repeats. Our analyses revealed that mountain barriers, elevation differences, and drainage systems all contributed low but statistically significant genetic variations to the total A. fumigatus population in this region. We found abundant novel alleles and genotypes in the TPR population of A. fumigatus and significant genetic differentiation between this population and those from other parts of Yunnan and the globe. Surprisingly, despite limited human presence in this region, about 7% of the A. fumigatus isolates were resistant to at least one of the two medical triazoles commonly used for treating aspergillosis. Our results call for greater surveillance of this and other human fungal pathogens in the environment. IMPORTANCE The extreme habitat fragmentation and substantial environmental heterogeneity in the TPR region have long known to contribute to geographically shaped genetic structure and local adaptation in several plant and animal species. However, there have been limited studies of fungi in this region. Aspergillus fumigatus is a ubiquitous pathogen capable of long-distance dispersal and growth in diverse environments. In this study, using A. fumigatus as a model, we investigated how localized landscape features contribute to genetic variations in fungal populations. Our results revealed that elevation and drainage isolation rather than direct physical distances significantly impacted genetic exchange and diversity among the local A. fumigatus populations. Interestingly, within each local population, we found high allelic and genotypic diversities, and with evidence ~7% of all isolates being resistant to two medical triazoles, itraconazole and voriconazole. Given the high frequency of ARAF found in mostly natural soils of sparsely populated sites in the TPR region, close monitoring of their dynamics in nature and their effects on human health is needed.

Deep amplicon sequencing reveals extensive allelic diversity in the erg11/CYP51 promoter and allows multi-population DMI fungicide resistance monitoring in the canola pathogen Leptosphaeria maculans

Continued use of fungicides provides a strong selection pressure towards strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11/CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established.

Selective sweeps identification in distinct groups of cultivated rye (Secale cereale L.) germplasm provides potential candidate genes for crop improvement

BackgroundDuring domestication and subsequent improvement plants were subjected to intensive positive selection for desirable traits. Identification of selection targets is important with respect to the future targeted broadening of diversity in breeding programmes. Rye (Secale cereale L.) is a cereal that is closely related to wheat, and it is an important crop in Central, Eastern and Northern Europe. The aim of the study was (i) to identify diverse groups of rye accessions based on high-density, genome-wide analysis of genetic diversity within a set of 478 rye accessions, covering a full spectrum of diversity within the genus, from wild accessions to inbred lines used in hybrid breeding, and (ii) to identify selective sweeps in the established groups of cultivated rye germplasm and putative candidate genes targeted by selection.ResultsPopulation structure and genetic diversity analyses based on high-quality SNP (DArTseq) markers revealed the presence of three complexes in the Secale genus: S. sylvestre, S. strictum and S. cereale/vavilovii, a relatively narrow diversity of S. sylvestre, very high diversity of S. strictum, and signatures of strong positive selection in S. vavilovii. Within cultivated ryes we detected the presence of genetic clusters and the influence of improvement status on the clustering. Rye landraces represent a reservoir of variation for breeding, and especially a distinct group of landraces from Turkey should be of special interest as a source of untapped variation. Selective sweep detection in cultivated accessions identified 133 outlier positions within 13 sweep regions and 170 putative candidate genes related, among others, to response to various environmental stimuli (such as pathogens, drought, cold), plant fertility and reproduction (pollen sperm cell differentiation, pollen maturation, pollen tube growth), and plant growth and biomass production.ConclusionsOur study provides valuable information for efficient management of rye germplasm collections, which can help to ensure proper safeguarding of their genetic potential and provides numerous novel candidate genes targeted by selection in cultivated rye for further functional characterisation and allelic diversity studies.

Intra-Varietal Diversity and Its Contribution to Wheat Evolution, Domestication, and Improvement in Wheat.

Crop genetic diversity is essential for adaptation and productivity in agriculture. A previous study revealed that poor allele diversity in wheat commercial cultivars is a major barrier to its further improvement. Homologs within a variety, including paralogs and orthologs in polyploid, account for a large part of the total genes of a species. Homolog diversity, intra-varietal diversity (IVD), and their functions have not been elucidated. Common wheat, an important food crop, is a hexaploid species with three subgenomes. This study analyzed the sequence, expression, and functional diversity of homologous genes in common wheat based on high-quality reference genomes of two representative varieties, a modern commercial variety Aikang 58 (AK58) and a landrace Chinese Spring (CS). A total of 85,908 homologous genes, accounting for 71.9% of all wheat genes, including inparalogs (IPs), outparalogs (OPs), and single-copy orthologs (SORs), were identified, suggesting that homologs are an important part of the wheat genome. The levels of sequence, expression, and functional variation in OPs and SORs were higher than that of IPs, which indicates that polyploids have more homologous diversity than diploids. Expansion genes, a specific type of OPs, made a great contribution to crop evolution and adaptation and endowed crop with special characteristics. Almost all agronomically important genes were from OPs and SORs, demonstrating their essential functions for polyploid evolution, domestication, and improvement. Our results suggest that IVD analysis is a novel approach for evaluating intra-genomic variations, and exploitation of IVD might be a new road for plant breeding, especially for polyploid crops, such as wheat.

Genetic diversity and selection signatures in a gene bank panel of maize inbred lines from Southeast Europe compared with two West European panels

Southeast Europe (SEE) is a very important maize-growing region, comparable to the Corn belt region of the United States, with similar dent germplasm (dent by dent hybrids). Historically, this region has undergone several genetic material swaps, following the trends in the US, with one of the most significant swaps related to US aid programs after WWII. The imported accessions used to make double-cross hybrids were also mixed with previously adapted germplasm originating from several more distant OPVs, supporting the transition to single cross-breeding. Many of these materials were deposited at the Maize Gene Bank of the Maize Research Institute Zemun Polje (MRIZP) between the 1960s and 1980s. A part of this Gene Bank (572 inbreds) was genotyped with Affymetrix Axiom Maize Genotyping Array with 616,201 polymorphic variants. Data were merged with two other genotyping datasets with mostly European flint (TUM dataset) and dent (DROPS dataset) germplasm. The final pan-European dataset consisted of 974 inbreds and 460,243 markers. Admixture analysis showed seven ancestral populations representing European flint, B73/B14, Lancaster, B37, Wf9/Oh07, A374, and Iodent pools. Subpanel of inbreds with SEE origin showed a lack of Iodent germplasm, marking its historical context. Several signatures of selection were identified at chromosomes 1, 3, 6, 7, 8, 9, and 10. The regions under selection were mined for protein-coding genes and were used for gene ontology (GO) analysis, showing a highly significant overrepresentation of genes involved in response to stress. Our results suggest the accumulation of favorable allelic diversity, especially in the context of changing climate in the genetic resources of SEE.

Микросателлитные локусы в генетической оценке плюсовых деревьев Pinus sylvestris L.

In this study, a comparative analysis was performed to estimate the genetic diversity levels of Pinus sylvestris L. in 49 seed orchards of I generation and progeny test plantings using 4-nucleus microsatellite loci. The seed orchards are located in the Petrozavodsk and Zaonezhye regions of the Republic of Karelia. The progeny trials were created by half-sibling seed progenies of the clones in the Petrozavodsk seed orchard. Micro-Checker software was used to identify null alleles and exclude genotyping errors. Coefficients of genetic originality were determined by examining the genetic structure of breeding objects in relation to the proportion between the rare and the dominant alleles for each selected tree. A major part of the rarest alleles (27.3–37.0 %) and a small fraction of the most common alleles (3.3–14.8 %) were detected for the distinct range of the pine trees in both seed orchards and test cultures. The largest number (40) of all identified alleles (51) was found in the Petrozavodsk seed orchard. The Hardy-Weinberg test comparing allele rates to expectational values showed a lack of heterozygotes in each category of the empirical substances. The selected material also had high levels of allelic and genetic diversity. The average number of alleles per locus ranged from 7.75 to 10.50. The average effective number was from 5.00 to 6.54. The observed heterozygosity varied from 0.60 to 0.70. The expected heterozygosity was 0.63–0.71. The numerical deviations were statistically insignificant. The AMOVA molecular dispersion result was 5 %, indicating the absence of significant genetic differentiation between the breeding objects. The heterozygote deficiency was caused not only by the presence of null alleles, but also by the selection. Namely, the set of alleles in the experimental material, randomly selected from a limited number of genotypes (clones), may differ from the natural population. The high level of the rarest alleles in the given range of the pine trees can also be explained as a selection effect. The results obtained in this study are important for creating breeding objects with higher genetic value. For citation: Ilinov A.A., Raevsky B.V. Genetic Evaluation by Microsatellite Loci of Pinus sylvestris L. Plus Trees. Lesnoy Zhurnal = Russian Forestry Journal, 2023, no. 3, pp. 48–68. (In Russ.). https://doi.org/10.37482/0536-1036-2023-3-48-68

HLA-II immunopeptidome profiling and deep learning reveal features of antigenicity to inform antigen discovery

CD4+ Tcell responses are exquisitely antigen specific and directed toward peptide epitopes displayed by human leukocyte antigen class II (HLA-II) on antigen-presenting cells. Underrepresentation of diverse alleles in ligand databases and an incomplete understanding of factors affecting antigen presentation invivo havelimited progress in defining principles of peptide immunogenicity. Here, we employed monoallelic immunopeptidomics to identify 358,024 HLA-II binders, with a particular focus on HLA-DQ and HLA-DP. We uncovered peptide-binding patterns across a spectrum of binding affinities and enrichment of structural antigen features. These aspects underpinned the development of context-aware predictor of Tcell antigens (CAPTAn), a deep learning model that predicts peptide antigens based on their affinity to HLA-II and full sequence of their source proteins. CAPTAn was instrumental in discovering prevalent Tcell epitopes from bacteria in the human microbiome and a pan-variant epitope from SARS-CoV-2. Together CAPTAn and associated datasets present a resource for antigen discovery and the unraveling genetic associations of HLA alleles with immunopathologies.

Increased genetic diversity and immigration after West Nile virus emergence in American crows: No evidence for a genetic bottleneck.

Infectious diseases can cause steep declines in wildlife populations, leading to changes in genetic diversity that may affect the susceptibility of individuals to infection and the overall resilience of populations to pathogen outbreaks. Here, we examine evidence for a genetic bottleneck in a population of American crows (Corvus brachyrhynchos) before and after the emergence of West Nile virus (WNV). More than 50% of marked birds in this population were lost over the 2-year period of the epizootic, representing a 10-fold increase in adult mortality. Using analyses of single-nucleotide polymorphisms (SNPs) and microsatellite markers, we tested for evidence of a genetic bottleneck and compared levels of inbreeding and immigration in the pre- and post-WNV populations. Counter to expectations, genetic diversity (allelic diversity and the number of new alleles) increased after WNV emergence. This was likely due to increases in immigration, as the estimated membership coefficients were lower in the post-WNV population. Simultaneously, however, the frequency of inbreeding appeared to increase: Mean inbreeding coefficients were higher among SNP markers, and heterozygosity-heterozygosity correlations were stronger among microsatellite markers, in the post-WNV population. These results indicate that loss of genetic diversity at the population level is not an inevitable consequence of a population decline, particularly in the presence of gene flow. The changes observed in post-WNV crows could have very different implications for their response to future pathogen risks, potentially making the population as a whole more resilient to a changing pathogen community, while increasing the frequency of inbred individuals with elevated susceptibility to disease.

WED-368 - Impact of allelic HLA divergence on the risk of bacterial infections in cirrhotic patients awaiting liver transplantation

WED-368 - Impact of allelic HLA divergence on the risk of bacterial infections in cirrhotic patients awaiting liver transplantation

Dynamics of Rye Translocations Frequency in Russian Common Wheat <i>Triticum aestivum</i> L. Cultivars

The widespread using 1RS/1BL and 1RS/1AL translocation cultivars in wheat breeding was due to their high complex resistance provided by genes on 1RS chromosomes. Numerous data on the emergence of new virulent pathogen races that caused the loss of protective properties by genes on 1RS make it relevant to analyse the prospects for using both rye translocations in modern breeding programs. The use of various genetic sources of the 1RS rye chromosome in introgressive hybridization poses the problem of studying the diversity of 1RS linkage groups. We have studied the rye translocations dynamics in 240 genotypes of the wheat cultivars from 3 breeding centers of the Russian Federation. Multiple alleles of storage protein genes specific for 1RS were used to mark 1RS. The effectiveness of 1RS resistance genes to a number of pathogens, in particular to powdery mildew and stem rust, has been established. The grain quality dynamics of the 1RS/1BL cultivars has been traced. A statistically significant grain quality improvement of winter wheat cultivars since the 2000s has been confirmed. The spring cultivars quality does not depend on the presence of rye translocations in the genotypes. Accumulation of cultivars with TR:1RS/1BL observed in all three centers. A high allelic diversity of the secalin coding locus on 1RS chromosome was shown. However, all cultivars with rye translocations, regardless of 1RS origin, carried the same allele. Possible reasons for this uniformity are discussed. Translocation 1RS/1AL was not detected in the studied cultivars. Possible reasons are discussed.

The hops (Humulus lupulus) genome contains a mid-sized terpene synthase family that shows wide functional and allelic diversity

BackgroundHops (Humulus lupulus L.) are a dioecious climbing perennial, with the dried mature “cones” (strobili) of the pistillate/female inflorescences being widely used as both a bittering agent and to enhance the flavour of beer. The glandular trichomes of the bract and bracteole flowering structures of the cones produce an abundance of secondary metabolites, such as terpenoids, bitter acids and prenylated phenolics depending on plant genetics, developmental stage and environment. More knowledge is required on the functional and allelic diversity of terpene synthase (TPS) genes responsible for the biosynthesis of volatile terpenes to assist in flavour-directed hop breeding.ResultsMajor volatile terpene compounds were identified using gas chromatography–mass spectrometry (GC–MS) in the ripe cones of twenty-one hop cultivars grown in New Zealand. All cultivars produced the monoterpene β-myrcene and the sesquiterpenes α-humulene and β-caryophyllene, but the quantities varied broadly. Other terpenes were found in large quantities in only a smaller subset of cultivars, e.g. β-farnesene (in seven cultivars) and α-pinene (in four). In four contrasting cultivars (Wakatu™, Wai-iti™, Nelson Sauvin™, and ‘Nugget’), terpene production during cone development was investigated in detail, with concentrations of some of the major terpenes increasing up to 1000-fold during development and reaching maximal levels from 50–60 days after flowering. Utilising the published H. lupulus genome, 87 putative full-length and partial terpene synthase genes were identified. Alleles corresponding to seven TPS genes were amplified from ripe cone cDNA from multiple cultivars and subsequently functionally characterised by transient expression in planta. Alleles of the previously characterised HlSTS1 produced humulene/caryophyllene as the major terpenes. HlRLS alleles produced (R)-(-)-linalool, whilst alleles of two sesquiterpene synthase genes, HlAFS1 and HlAFS2 produced α-farnesene. Alleles of HlMTS1, HlMTS2 and HlTPS1 were inactive in all the hop cultivars studied.ConclusionsAlleles of four TPS genes were identified and shown to produce key aroma volatiles in ripe hop cones. Multiple expressed but inactive TPS alleles were also identified, suggesting that extensive loss-of-function has occurred during domestication and breeding of hops. Our results can be used to develop hop cultivars with novel/improved terpene profiles using marker-assisted breeding strategies to select for, or against, specific TPS alleles.

Morphological to Molecular Markers: Plant Genetic Diversity Studies in Walnut (Juglans regia L.)—A Review

Persian walnut populations have tremendous morphological and allelic diversity in their germplasm due to heavy outcrossing and years of seed multiplication. These variations are assessed by morphological, cytological, biochemical, and molecular markers. Various researchers have used different tree, foliage, flower, nut, and kernel traits to evaluate morphological/phenotypic diversity. In walnut, morphological indices are first taken into consideration to describe and classify the germplasm, but the environment influences them. In comparison, DNA-based markers can detect genetic diversity at any stage of plant development and have been shown to be a potential tool for assessing variation at the DNA level and deciphering genetic relationships within and between species. Microsatellites are very powerful and informative among DNA-based markers in studying genetic relationships and genetic identity at different levels. They are neutral, highly frequent, uniformly distributed, hypervariable, codominant, highly reproducible, produce many alleles per locus, and require a small amount of DNA for analysis. Current breeding objectives can be achieved by selecting superior genotypes from the germplasm, supplemented by molecular characterization in the selection of parents for each breeding program. Therefore, the use of morphological and molecular markers is recommended for efficient exploration and utilization of germplasm resources and to improve diversity among genetic resources. The published literature on morphological and molecular markers, especially simple sequence repeats (SSRs), is presented in this review to provide current insights into the level of genetic diversity in walnut.

Demographic history of the Punjab urial and implications for its management

AbstractThe Punjab urial (Ovis vignei punjabiensis) is endemic to Northern Punjab, Pakistan, and is categorized as vulnerable by the International Union for Conservation of Nature Red List of Threatened Species. The urial population has declined by 30% over the last 3 generations. We used non‐invasive fecal samples to identify individuals and estimate population size of Punjab urial in the Kalabagh Game Reserve, Pakistan. We genotyped samples using 12 microsatellite markers to assess genetic variation, population structure, and demographic changes. Microsatellite analysis revealed high levels of genetic variation in urials in terms of expected and observed heterozygosity and allelic diversity. The population structure of the Punjab urial in the Kalabagh Game Reserve, based solely on microsatellite variation using Bayesian clustering, indicated 3 different clusters in the reserve. Results revealed that the urial population may be facing inbreeding pressure because its ancestral effective population size has declined from between 20,000 and 50,000 to ≤1,000 animals today. This reduction has partly occurred because of a bottleneck that occurred about 10,000 years ago. Results also indicate that 1 urial population cluster has the signature of a bottleneck, which may be due to population isolation. The 3 urial clusters are small and broadly dispersed in a large territory, meaning they could be extirpated without any opportunity for natural re‐population through dispersion. The results of our study support a management strategy that encourages maintaining connectivity between urial localities within the Kalabagh Game Reserve, increased diversity so the effective population size may recover from the historical decline, and the use of data generated here as a baseline of urial genetic diversity in the reserve for monitoring diversity over the long term.

Variable Region Sequences Influence 16S rRNA Performance.

16S rRNA gene sequences are commonly analyzed for taxonomic and phylogenetic studies because they contain variable regions that can help distinguish different genera. However, intra-genus distinction using variable region homology is often impossible due to the high overall sequence identities among closely related species, even though some residues may be conserved within respective species. Using a computational method that included the allelic diversity within individual genomes, we discovered that certain Escherichia and Shigella species can be distinguished by a multi-allelic 16S rRNA variable region single nucleotide polymorphism (SNP). To evaluate the performance of 16S rRNAs with altered variable regions, we developed an in vivo system that measures the acceptance and distribution of variant 16S rRNAs into a large pool of natural versions supporting normal translation and growth. We found that 16S rRNAs containing evolutionarily disparate variable regions were underpopulated both in ribosomes and in active translation pools, even for an SNP. Overall, this study revealed that variable region sequences can substantially influence the performance of 16S rRNAs and that this biological constraint can be leveraged to justify refining taxonomic assignments of variable region sequence data. IMPORTANCE This study reevaluates the notion that 16S rRNA gene variable region sequences are uninformative for intra-genus classification and that single nucleotide variations within them have no consequence to strains that bear them. We demonstrated that the performance of 16S rRNAs in Escherichia coli can be negatively impacted by sequence changes in variable regions, even for single nucleotide changes that are native to closely related Escherichia and Shigella species; thus, biological performance is likely constraining the evolution of variable regions in bacteria. Further, the native nucleotide variations we tested occur in all strains of their respective species and across their multiple 16S rRNA gene copies, suggesting that these species evolved beyond what would be discerned from a consensus sequence comparison. Therefore, this work also reveals that the multiple 16S rRNA gene alleles found in most bacteria can provide more informative phylogenetic and taxonomic detail than a single reference allele.

Genetic monitoring of sterlet juveniles (Acipenser ruthenus) in the Volga river

The aim of the work was to carry out genetic monitoring of the natural reproduction of juveniles (larvae and underyearlings) of sterlet (Acipenser ruthenus Linnaeus, 1958), caught in the middle and lower spawning zones of the Volga River for the period 2021–2022, which is necessary and relevant in the context of a decrease in the number of sturgeon species of fish. The study of fish was carried out according to five nuclear markers and a fragment of the control region of mitochondrial DNA.According to the results of microsatellite analysis, sterlet individuals were characterized by species-specific dominant alleles and high values of the main indicators of genetic diversity: the number of alleles, allelic diversity and heterozygosity, and a slight deficit of heterozygotes was noted. By analyzing of the nucleotide sequences of the mtDNA D-loop region a high level of diversity of maternally inherited haplotypes was revealed, reflecting the number of females involved in natural reproduction. According to the monitoring data, no differences in the distribution and number of identified variants of the D-loop region were registered in the interannual aspect. During the study period, 48 sterlet mitochondrial haplotypes were recorded, of which 18 mtDNA variations were unique, occurring in the Volga-Caspian fishery basin. In the study of sterlet larvae by molecular genetic methods in the Volga River was revealed the process of hybridization between stellate sturgeon and sterlet (Acipenser stellatus × Acipenser ruthenus).