Digest Restriction-site Associated DNA Sequencing Research Articles

Genetic diversity and population structure of four Anatolian sheep revealed by genome-wide ddRADseq data

This is the first wide-scale study to reveal next-generation sequencing-based and comprehensive study to reveal genetic diversity, population structure, and phylogenetic relationships of some of Türkiye's indigenous fat-tailed sheep breeds. A total of 80 animals belonging to Akkaraman (AKR), Güney Karaman (GKR), Morkaraman (MKR), and Karakaş (KRK) sheep breeds were genotyped with genome-wide of 296.097 single nucleotide polymorphisms (SNPs) data recovered from double digest restriction site-associated DNA sequencing (ddRADseq) libraries processed by Illumina HiSeq X Ten instrument. Minor allele frequency (MAF) ranged from 0.311 (MKR) to 0.316 (AKR and GKR). Observed heterozygosity (HO) values were higher than expected values (HE) across all populations with a mean of 0.307. Negative inbreeding coefficient (FIS) values were observed in four sheep populations varying between −0.034 (AKR) to −0.060 (MKR). Cross-entropy criterion-based ADMIXTURE analysis demonstrated that four Anatolian sheep breeds were represented by two ancestral populations in which GKR and AKR were distinct, while KRK and MKR were mixed populations. Genetic distance-based tree, discriminant analysis of principal components (DAPC), and ADMIXTURE analyses confirmed that GKR, which is believed to be derived from the AKR breed, has become genetically different from other sheep populations. Moreover, no migration from and into the GKR breed was detected, while a migration edge was drawn from the KRK and MKR clade into the AKR breed by the TreeMix algorithm. This study confirmed that high-density SNP data obtained from advanced next-generation sequencing (NGS) platforms are efficient in genetically distinguishing animal breeds, even varieties. It is recommended that further NGS-based studies could be carried out to better characterize different indigenous livestock populations raised in Türkiye.

Development and utilization of genome-wide InDel markers in Sorghum [Sorghum bicolor (L.) Moench

Sorghum (Sorghum bicolor L. Moench) has become an increasingly valuable crop for food, feed, and especially bioenergy feedstock production, which makes the crop extremely attractive for studying genomics and genetic diversity. Molecular markers and genomics play essential roles in sorghum breeding. The rapid development of next-generation sequencing technology has facilitated the identification of genome-wide insertion-deletion (InDel) polymorphisms, enabling the efficient construction of InDel markers that are suitable for user-friendly PCR. This study was conducted with the objective of discovering and developing InDel markers using double digest restriction site-associated DNA sequencing (ddRAD-Seq) data. A total of 19,226 InDels distributed across 10 chromosomes in the sorghum genome was identified. Of those, deletions constituted 65.7% while the remain was insertions. A comprehensive analysis of all the chromosomes revealed a total of 80 InDel sites with a minimum length of 10 bp. For a good conversion of the InDel regions to beneficial molecular markers, specific primers were designed for the amplification of 47 InDel regions that were selected for further investigation. A diverse panel of sorghum consisting of 16 accessions served a source for the developed InDel markers validation. Of the 47 InDel markers, 14 were tested across 16 sorghum accessions and were demonstrated their helpfulness for marker-assisted selection in sorghum. The polymorphic information content (PIC) values of the 16 markers varied between 0.11 and 0.38, with an average of 0.28. The findings of this study indicated that the identification of InDels and the development of molecular markers for sorghum were accomplished using the ddRAD-Seq data.

Plant kleptomaniacs: geographic genetic patterns in the amphi-apomictic Rubus ser. Glandulosi (Rosaceae) reveal complex reticulate evolution of Eurasian brambles.

Rubus ser. Glandulosi represents a unique model of geographic parthenogenesis on a homoploid (2n = 4x) level. We aim to characterize evolutionary and phylogeographic patterns in this taxon and shed light on the geographic differentiation of apomicts and sexuals. Ultimately, we aim to evaluate the importance of phylogeography in the formation of geographic parthenogenesis. R. ser. Glandulosi was sampled across its Eurasian range together with other co-occurring Rubus taxa (587 individuals in total). Double digest restriction-site associated DNA sequencing (ddRADseq) and modelling of suitable climate were employed for evolutionary inferences. Six ancestral species were identified that contributed to the contemporary gene pool of R. ser. Glandulosi. While sexuals were introgressed from R. dolichocarpus and R. moschus in West Asia and from R. ulmifolius agg., R. canescens and R. incanescens in Europe, apomicts were characterized by alleles of R. subsect. Rubus. Gene flow between sexuals and apomicts was also detected, as well as occasional hybridization with other taxa. We hypothesize that sexuals survived the last glacial period in several large southern refugia, whereas apomicts were mostly restricted to southern France from whence they quickly recolonized Central and Western Europe. The secondary contact of sexuals and apomicts was probably the principal factor that established geographic parthenogenesis in R. ser. Glandulosi. Sexual populations are not impoverished in genetic diversity along their borderline with apomicts and maladaptive population genetic processes likely did not shape the geographic patterns.

Comparison of ddRADseq and EUChip60K SNP genotyping systems for population genetics and genomic selection in Eucalyptus dunnii (Maiden).

Eucalyptus dunnii is one of the most important Eucalyptus species for short-fiber pulp production in regions where other species of the genus are affected by poor soil and climatic conditions. In this context, E. dunnii holds promise as a resource to address and adapt to the challenges of climate change. Despite its rapid growth and favorable wood properties for solid wood products, the advancement of its improvement remains in its early stages. In this work, we evaluated the performance of two single nucleotide polymorphism, (SNP), genotyping methods for population genetics analysis and Genomic Selection in E. dunnii. Double digest restriction-site associated DNA sequencing (ddRADseq) was compared with the EUChip60K array in 308 individuals from a provenance-progeny trial. The compared SNP set included 8,011 and 19,008 informative SNPs distributed along the 11 chromosomes, respectively. Although the two datasets differed in the percentage of missing data, genome coverage, minor allele frequency and estimated genetic diversity parameters, they revealed a similar genetic structure, showing two subpopulations with little differentiation between them, and low linkage disequilibrium. GS analyses were performed for eleven traits using Genomic Best Linear Unbiased Prediction (GBLUP) and a conventional pedigree-based model (ABLUP). Regardless of the SNP dataset, the predictive ability (PA) of GBLUP was better than that of ABLUP for six traits (Cellulose content, Total and Ethanolic extractives, Total and Klason lignin content and Syringyl and Guaiacyl lignin monomer ratio). When contrasting the SNP datasets used to estimate PAs, the GBLUP-EUChip60K model gave higher and significant PA values for six traits, meanwhile, the values estimated using ddRADseq gave higher values for three other traits. The PAs correlated positively with narrow sense heritabilities, with the highest correlations shown by the ABLUP and GBLUP-EUChip60K. The two genotyping methods, ddRADseq and EUChip60K, are generally comparable for population genetics and genomic prediction, demonstrating the utility of the former when subjected to rigorous SNP filtering. The results of this study provide a basis for future whole-genome studies using ddRADseq in non-model forest species for which SNP arrays have not yet been developed.

A complex biosystematic approach to reveal evolutionary and diversity patterns in West Asian brambles (Rubus subgen. Rubus, Rosaceae)

West Asia, with its high mountain ranges and glacial refugia, stands out as a biodiversity hotspot for various plant taxa, including the taxonomically complex Rubus subgen. Rubus. Despite this significance, our understanding of the evolutionary processes shaping this group in the region has been limited. We employed an integrative approach combining flow cytometry, Sanger sequencing of two plastid regions, microsatellite genotyping, and double digest restriction-site associated DNA sequencing to characterize evolutionary and diversity patterns in West Asian brambles. We identified four diploid and two sexual tetraploid species, with the remaining diversity comprising apomictic tetra- and triploids, mostly belonging to R. ser. Discolores, and sexual pentaploid hybrids/hybridogens derived from R. caesius. Rubus dolichocarpus, newly reported here as diploid, emerged as a significant ancestor for many polyploids. Most apomictic genotypes are locally distributed and only three genotypes exhibit extremely large ranges from Armenia to Kyrgyzstan and from Georgia to Iran. These genotypes are probably of an ancient (Pleistocene) European origin and likely laid the foundations for the evolution of apomicts in West Asia, whose diversification was subsequently boosted by the genetic contribution of sexual taxa. DNA markers confirmed the West Asian origin of Rubus armeniacus, a globally significant invasive species, yet rare in its native range. We report on the first indigenous occurrence of this species based on microsatellite genotyping. Our findings contribute to filling existing gaps in understanding the evolution and diversity of Rubus subgen. Rubus in West Asia.

New linkage maps for the Cupressaceae species Chamaecyparis obtusa and Thujopsis dolabrata var. hondae and comparison with the linkage map of Cryptomeria japonica

ABSTRACT Comparative mapping studies, which can determine whether the genome structure differs among genera, have yet to be conducted in Cupressaceae. This study analyzed three important forestry species in Japan: Cryptomeria japonica, Chamaecyparis obtusa, and Thujopsis dolabrata var. hondae. To date, linkage maps have only been constructed for C. japonica. We constructed the first linkage maps for C. obtusa and T. dolabrata var. hondae using double digest restriction-site associated DNA sequencing (ddRAD-Seq) data, expressed sequence tag-derived simple sequence repeat (EST-SSR) markers, and single-nucleotide polymorphism (SNP) markers designed from homologous unigenes obtained from RNA-seq data. The lengths of the linkage maps were 1,835 cM (1,981 markers) for C. obtusa and 1,833 cM (1,295 markers) for T. dolabrata var. hondae. A total of 11 distinct linkage groups were identified in these maps, corresponding to the haploid number of Cupressaceae. Using four published linkage maps for C. japonica, we then constructed a composite linkage map with 7,784 markers. Finally, the three linkage maps were compared using 207 SNP markers designed from homologous unigenes. Although most markers were mapped to the same LG despite representing different species, indicating strong synteny. The high level of interspecific and intergeneric synteny and macro-collinearity detected in Cupressaceae were similar to those of other trees. We also detected three major chromosomal rearrangements between but not within subfamilies, which may have been caused by the taxonomic distance. This is the first example of comparative mapping for Cupressaceae species.

Development and parentage analysis of SNP markers for Chestnut-vented Nuthatch (Sitta nagaensis) based on ddRAD-seq data

Extra-pair paternity (EPP) is commonly found in socially monogamous birds, especially in small passerine birds, and there are interspecific and intraspecific variations in the extent of EPP. The Chestnut-vented Nuthatch (Sitta nagaensis) is a socially monogamous passerine bird, and verifying whether this species has EPP relies on parentage testing—S. nagaensis is not known to have EPP. In this study, we developed SNP markers of this species that are informative for parentage analysis from double digest restriction site-associated DNA sequencing (ddRAD-seq) data. A panel consisting of 50 SNP markers, with a mean heterozygosity of 0.343, was used to resolve 95% of nestlings to fathers. The combined exclusion probabilities for the first parent and second parent were 0.991 and 0.9999, respectively. This panel of SNP markers is a powerful tool for parentage assignments in S. nagaensis. In addition, we found that three offspring (7.9%) from three nests (23.1%) were the result of extra-pair fertilization out of 38 offspring in 13 nests. Our study provided information on parentage analysis that has not been reported before in S. nagaensis. It also supplemented the understudied EPP behavior of birds in Asia, contributing to a general understanding of the EPP behaviors of birds.

Genome-wide selection signatures address trait specific candidate genes in cattle indigenous to arid regions of India

The peculiarity of Indian cattle lies in milk quality, resistance to diseases and stressors as well as adaptability. The investigation addressed selection signatures in Gir and Tharparkar cattle, belonging to arid ecotypes of India. Double digest restriction-site associated DNA sequencing (ddRAD-seq) yielded nearly 26 million high-quality reads from unrelated seven Gir and seven Tharparkar cows. In all, 19,127 high-quality SNPs were processed for selection signature analysis. An approach involving within-population composite likelihood ratio (CLR) statistics and between-population F ST statistics was used to capture selection signatures within and between the breeds, respectively. A total of 191 selection signatures were addressed using CLR and F ST approaches. Selection signatures overlapping 86 and 73 genes were detected as Gir- and Tharparkar-specific, respectively. Notably, genes related to production (CACNA1D, GHRHR), reproduction (ESR1, RBMS3), immunity (NOSTRIN, IL12B) and adaptation (ADAM22, ASL) were annotated to selection signatures. Gene pathway analysis revealed genes in insulin/IGF pathway for milk production, gonadotropin releasing hormone pathway for reproduction, Wnt signalling pathway and chemokine and cytokine signalling pathway for adaptation. This is the first study where selection signatures are identified using ddRAD-seq in indicine cattle breeds. The study shall help in conservation and leveraging genetic improvements in Gir and Tharparkar cattle.

Implications of methodologies for integrating empirical kinships into ex situ population management using PMx: A case study of Baer's Pochard (Aythya baeri) in North America.

The application of molecular tools to population management can improve the long-term genetic viability of ex situ populations. In this study, we aimed to understand the implications of integrating empirical kinships into the genetic management of an ex situ population of the endangered waterfowl, Baer's pochard (Aythya baeri), in North America. Single nucleotide polymorphism data were generated for 141 Baer's pochard using double digest restriction site-associated DNA sequencing and empirical kinships were derived and integrated into the population management software PMx. Analyses suggested 37.7% of pairwise relationships previously assumed to be unrelated were first, second, or third-order relatives. We determined that most genetic summary statistics were impacted through the calculation of the population's mean kinship, which increased from MK¯=0.0772 to MK¯=0.2074 after empirical kinships were integrated into our analyses. Our results also revealed the importance of understanding how molecular kinships derived from a particular estimator are scaled, if the scale differs significantly from pedigree-based kinships. We describe the theory behind the genetic metrics impacted and provide general guidance on incorporating empirical kinships into ex situ population management as well as provide suggestions for sampling strategies to minimize the biases inherent in merging two types of kinship estimators.

Origins of cryophilous lineages and the role of Beringia: evidence from the cosmopolitan angiosperm genus Stellaria L. (Caryophyllaceae)

Abstract The cosmopolitan angiosperm genus Stellaria L. (Caryophyllaceae) occurs across the Arctic and is most diverse in the high southern Asian mountains, including presence at the most extreme latitudinal and elevational limits of vascular plants. Using the primary fossil record and double digest restriction site-associated DNA sequencing data, we estimated divergence times of Stellaria and relatives across the Caryophyllaceae. We then used ancestral area and ancestral state reconstructions to interpret the biogeographical history of the group. We found that Stellaria originated in the Miocene and gave rise to repeated New World lineages, mostly from temperate Old World regions and probably via Bering Land Bridges. Circumboreal lineages were recovered of recent, Pleistocene origin and several might have originated in southerly mountains of both the Old World and the New World before subsequently colonizing the Arctic. Ancestral state reconstruction of inhabitance of cold vs. temperate climates and of wet vs. dry habitats revealed repeated evolutionary transitions across these extremes by members of the genus worldwide. Our study, which samples nearly all species within a diverse and cosmopolitan lineage of flowering plants, recovers a group characterized by niche lability and helps to support prior findings of temperate origins of many cryophilous plant lineages. Pre-adaptation to cold might have been a prerequisite for colonization of the Arctic.

Revelation of genetic diversity and genomic footprints of adaptation in Indian pig breeds

In the present study, the genetic diversity measures among four Indian domestic breeds of pig namely Agonda Goan, Ghurrah, Ghungroo, and Nicobari, of different agro-climatic regions of country were explored and compared with European commercial breeds, European wild boar and Chinese domestic breeds. The double digest restriction site-associated DNA sequencing (ddRADseq) data of Indian pigs (102) and Landrace (10 animals) were generated and whole genome sequencing data of exotic pigs (60 animals) from public data repository were used in the study. The principal component analysis (PCA), admixture analysis and phylogenetic analysis revealed that Indian breeds were closer in ancestry to Chinese breeds than European breeds. European breeds exhibited highest genetic diversity measures among all the considered breeds. Among Indian breeds, Agonda Goan and Ghurrah were found to be more genetically diverse than Nicobari and Ghungroo. The selection signature regions in Indian pigs were explored using iHS and XP-EHH, and during iHS analysis, it was observed that genes related to growth, reproduction, health, meat quality, sensory perception and behavior were found to be under selection pressure in Indian pig breeds. Strong selection signatures were recorded in 24.25–25.25 Mb region of SSC18, 123.25–124 Mb region of SSC15 and 118.75–119.5 Mb region of SSC2 in most of the Indian breeds upon pairwise comparison with European commercial breeds using XP-EHH. These regions were harboring some important genes such as EPHA4 for thermotolerance, TAS2R16, FEZF1, CADPS2 and PTPRZ1 for adaptability to scavenging system of rearing, TRIM36 and PGGT1B for disease resistance and CCDC112, PIAS1, FEM1B and ITGA11 for reproduction.

Phylogenomic data reveal cryptic diversity and deep phylogeographical structure within the common chuckwalla, Sauromalus ater (Squamata: Iguanidae)

Abstract Understanding how historical geological processes drive diversification and shape the contemporary distribution of species is fundamental to phylogeography. We take a genomic approach in order to elucidate the deep phylogeographical history and species limits of chuckwallas (Sauromalus), a conspicuous group of lizards of the arid lands of south-western North America. Phylogenetic and population genomic analyses of double digest restriction site-associated DNA sequencing data confirm the presence of at least two major lineages, peninsular and continental groups, within the widespread and morphologically variable common chuckwalla (Sauromalus ater). These lineages diversified in the vicinity of the head of the Gulf of California in north-eastern Baja California in the early Pliocene to late Miocene, during the formation of the northern gulf. The peninsular lineage of S. ater subsequently gave rise to the four insular endemic species of Sauromalus associated with the Baja California peninsula. Genomic analyses strongly support the continued recognition of the insular gigantics Sauromalus varius and Sauromalus hispidus as distinct species, although their relationship as sister species remains unresolved. Weaker phylogenetic signal for the insular species Sauromalus slevini and Sauromalus klauberi is provided by the genomic data; thus, it is advocated to continue recognizing these species until additional data can be analysed to evaluate their distinctiveness.

Limited population genetic structure in the littoral gastropod Nerita tessellata (Neritimorpha, Neritidae) suggests high dispersal across the Caribbean Sea

The Caribbean Sea is characterized by an incredible biodiversity, including several endemic mollusk species, most of which have larval dispersal as their main mechanism of gene flow. It is known that the present-day population structure of a species reflects the combination of oceanographic currents, life-history traits, and historical events. Nerita tessellata is a common gastropod species in coastal, rocky, and exposed shores across the Caribbean Sea; however, little is known about the mechanisms that have shaped its distribution. Here, we tested potential barriers to gene flow and the associated genetic structure of N. tessellata in 12 populations across the Caribbean using digest restriction-site associated DNA sequencing (ddRAD-seq). Likewise, we evaluated demographic history and migration patterns through two coalescence approaches using Approximate Bayesian Computation (ABC) combined with supervised machine learning. The global analysis, based on 26,421 single nucleotide polymorphism (SNP) markers derived from ddRAD-seq, revealed low nucleotide diversity with high levels of gene flow and a lack of significant population differentiation within the survey area, indicating that N. tessellata is panmictic across the Caribbean Sea. Our results reveal that a pattern of north to south movement along Caribbean currents could potentially explain the limited genetic structure and high gene flow among N. tessellata populations. The scenarios simulated with ABC showed a constant effective population size for the ancestral population. Additionally, the four populations tested experienced demographic expansions that date to the Pleistocene. An extended pelagic larval stage could explain the large-scale genetic uniformity of this species across its distribution. Our findings contribute to understanding genetic diversity and population structure in a common gastropod within the Caribbean Sea.

Using genomics and morphometrics to monitor data-poor and commercially exploited octopod populations

Over 150 species of benthic octopods have been described within the ‘catch-all’ Octopus genus (Family: Octopodidae) and yet, many Octopus species harvested by fisheries remain unidentified to species-level due to a lack of distinguishing traits. Within species, there is also limited information on how populations differ genetically and the level of connectivity between populations. Therefore, we sampled octopods from commercial fisheries in southeast Australia, in order to identify the species, examine the phylogeographic relationships among species and the level of population genetic structuring within species, as well as to look for any adaptive genetic variation. The mitochondrial gene, cytochrome oxidase subunit III (COIII), was sequenced in 346 octopods along with single nucleotide polymorphisms using double digest restriction site-associated DNA sequencing (ddRADseq). Morphometric traits were also measured in mature specimens. The southern keeled octopus (‘Octopus’berrima) and pale octopus (‘Octopus’pallidus) were identified using COIII data. For ‘Octopus’berrima, we found that some populations whilst being morphologically similar were genetically distinct. In contrast, ‘Octopus’pallidus populations were both morphologically and genetically distinct across the studied regions. Our results provide key information to better inform conservation and management decisions for developing octopod fisheries in southeast Australia and highlight the importance of genomics tools in the conservation management of commercially and recreationally important species.

Genetic analysis of heat tolerance in hot pepper: insights from comprehensive phenotyping and QTL mapping.

High temperatures present a formidable challenge to the cultivation of hot pepper, profoundly impacting not only vegetative growth but also leading to flower and fruit abscission, thereby causing a significant reduction in yield. To unravel the intricate genetic mechanisms governing heat tolerance in hot pepper, an F2 population was developed through the crossing of two distinct genotypes exhibiting contrasting heat tolerance characteristics: DLS-161-1 (heat tolerant) and DChBL-240 (heat susceptible). The F2 population, along with the parental lines, was subjected to comprehensive phenotyping encompassing diverse morphological, physiological, and biochemical heat-related traits under high temperature conditions (with maximum temperature ranging from 31 to 46.5°C and minimum temperature from 15.4 to 30.5°C). Leveraging the Illumina Nova Seq-6000 platform, Double digest restriction-site associated DNA sequencing (ddRAD-seq) was employed to generate 67.215 Gb data, with subsequent alignment of 218.93 million processed reads against the reference genome of Capsicum annuum. Subsequent variant calling and ordering resulted in 5806 polymorphic SNP markers grouped into 12 LGs. Further QTL analysis identified 64 QTLs with LOD values ranging from 2.517 to 11.170 and explained phenotypic variance ranging from 4.05 to 19.39%. Among them, 21 QTLs, explaining more than 10% phenotypic variance, were identified as major QTLs controlling 9 morphological, 3 physiological, and 2 biochemical traits. Interestingly, several QTLs governing distinct parameters were found to be colocalized, suggesting either a profound correlation between the QTLs regulating these traits or their significant genomic proximity. In addition to the QTLs, we also identified 368380 SSR loci within the identified QTL regions, dinucleotides being the most abundant type (211,381). These findings provide valuable insights into the genetics of heat tolerance in hot peppers. The identified QTLs and SSR markers offer opportunities to develop heat-tolerant varieties, ensuring better crop performance under high-temperature conditions.

Source tracing of Anthonomus grandis captured in areas of the USA where the species had previously been eradicated

Anthonomus grandis Boheman (Coleoptera: Curculionidae) is comprised of two subspecies: the boll weevil (A. g. grandis) and the thurberia weevil (A. g. thurberiae). The thurberia weevil is generally regarded as a non-pest variant of the species, whereas the boll weevil is an important economic pest of commercial cotton. In the USA, a nationwide eradication program has been largely successful against the boll weevil, but reintroductions of eradicated zones remain a threat. In 2015, A. grandis was trapped in the functionally eradicated Winter Garden growing region in Texas, where it had not been found since 2012. Weevils established in the area and triggered a multi-year, multi-million-dollar mitigation effort. In 2017, the species was trapped in Hidalgo County, New Mexico, another area where it had been previously eradicated. In this study, we obtained specimens from both trapping events and used double digest restriction site-associated DNA sequencing to obtain genetic data in homology with previously published A. grandis population genetic data. We conducted source tracing to match the captured weevils to previously described populations to determine a likely source population. We also compared the accuracy of the approach to other diagnostic methods currently used by the United States Department of Agriculture. The results suggested that a small founder population of weevils dispersed from the Lower Rio Grande Valley population in southern Texas and northern Tamaulipas and established in the Winter Garden. In Hidalgo Co., NM, the weevils were found to be genetically indistinct from non-pest populations of thurberia weevil associated with Arizona wild cotton (Gossypium thurberi). Thurberia weevils in Arizona remain unlikely to threaten commercial growers. Improved rapid diagnostic tools may be desirable to enable better assessment of trap captures and allow for a reduction in the application of mitigation efforts when the non-pest variant is identified.

No evidence of inbreeding depression despite a historical severe bottleneck in the endangered Bermuda petrel (Pterodroma cahow).

The Bermuda petrel Pterodroma cahow is an island endemic seabird that belongs to the Procellariiformes, one of the most endangered orders of birds. Historical records suggest a significant population size decline following human settlement in Bermuda, bringing the species to near extinction. Since the 1950's the population has been recovering aided by the implementation of an ongoing conservation plan. However, it still faces several threats, and negative genetic effects resulting from that drastic decline are to be expected, including inbreeding and genetic drift. We studied genetic diversity and levels of inbreeding, and their effects on individual fitness and mating choice. We also tested for a genetic signature of the recent demographic bottleneck. For this, we analysed variation in thousands of nuclear single nucleotide polymorphisms derived from double digest restriction site associated DNA sequencing and one mitochondrial gene (cytochrome oxidase I). The results revealed that the Bermuda petrel suffered a recent genetic bottleneck and shows low mitochondrial diversity compared to other petrel species. Conversely, nuclear diversity was similar to that of other endangered petrels. Inbreeding levels were not high overall, although some individuals were highly inbred. However, we found no evidence that individual inbreeding or relatedness between mates affected hatching success, or that mate choice is influenced by kinship in this very small population.

Attack of the clones: Population genetics reveals clonality of Colletotrichum lupini, the causal agent of lupin anthracnose

Colletotrichum lupini, the causative agent of lupin anthracnose, affects lupin cultivation worldwide. Understanding its population structure and evolutionary potential is crucial to design successful disease management strategies. The objective of this study was to employ population genetics to investigate the diversity, evolutionary dynamics, and molecular basis of the interaction of this notorious lupin pathogen with its host. A collection of globally representative C. lupini isolates was genotyped through triple digest restriction site-associated DNA sequencing, resulting in a data set of unparalleled resolution. Phylogenetic and structural analysis could distinguish four independent lineages (I-IV). The strong population structure and high overall standardized index of association (r̅d ) indicates that C. lupini reproduces clonally. Different morphologies and virulence patterns on white lupin (Lupinus albus) and Andean lupin (Lupinus mutabilis) were observed between and within clonal lineages. Isolates belonging to lineage II were shown to have a minichromosome that was also partly present in lineage III and IV, but not in lineage I isolates. Variation in the presence of this minichromosome could imply a role in host-pathogen interaction. All four lineages were present in the South American Andes region, which is suggested to be the centre of origin of this species. Only members of lineage II have been found outside South America since the 1990s, indicating it as the current pandemic population. As a seedborne pathogen, C. lupini has mainly spread through infected but symptomless seeds, stressing the importance of phytosanitary measures to prevent future outbreaks of strains that are yet confined to South America.

Species-Level Recognition and Redescription of the Kentucky Arrow Darter, Etheostoma spilotum Gilbert (Percidae: litocara)

The Kentucky Arrow Darter, Etheostoma spilotum, endemic to the upper Kentucky River basin of eastern Kentucky, is redescribed and recognized as a distinct species closely related to E. sagitta in the upper Cumberland River basin and E. nianguae in the Osage River drainage (Missouri River basin). Originally described as a subspecies of E. nianguae, it was later considered a full species and then a subspecies of E. sagitta, based on close geographic proximity to Cumberland basin populations and overlapping meristic variation interpreted as character intergradation. We present meristic, morphometric, and genetic data that support species-level recognition of E. spilotum. It differs from E. sagitta by lower counts of total and pored lateral scales, lower counts of caudal peduncle scales, fewer second dorsal-fin rays, and fewer pectoral-fin rays. Interspecific divergence of E. spilotum and E. sagitta is further demonstrated through analyses of variation in the mitochondrial nd2 gene and species delimitation using genome-wide double digest restriction-site associated DNA sequencing. Although allopatrically distributed, both species inhabit upland headwater streams on the Cumberland Plateau and have similar life history characteristics. Endemism, fragmented distributions, and low densities and genetic diversity within populations make these species extremely vulnerable to anthropogenic activities. Etheostoma spilotum was federally listed as threatened in 2016 due to degradation of stream habitat and water quality in the upper Kentucky basin that has eliminated the species from a significant portion of its range.

Genome-wide identification and annotation of SNPs and their mapping in candidate genes related to milk production and fertility traits in Badri cattle

This study was conducted in Badri cattle using a double digest restriction-site associated DNA sequencing approach. The study aimed to identify and annotate high confidence single nucleotide polymorphisms (SNPs) and their mapping in candidate genes related to production and fertility in dairy cattle. A total of 7,168,552 genome-wide SNPs were initially identified in Badri cattle by alignment with theBos indicusreference genome. After filtration of SNPs, 65,483 high confidence SNPs were retained and further used for downstream analysis. Annotation of high confidence SNPs revealed 99.197% SNPs had modifier impact, 0.326% SNPs were low impact, 0.036% were high impact, and 0.441% were moderate impact SNPs. Most SNPs in Badri cattle were found in intergenic, transcript and intronic regions. The candidate genes for milk production PRKCE, ABCG2, GHR, EPS8, CAST and NRXN1 were found to harbour maximum high confidence variants. Among candidate genes for fertility in cattle, ATP2B1, SOX5, WDR27, ARHGAP12, CACNA1D, ANKRD6, GRIA3, ZNF521 and CAST822 have maximum high confidence variants mapped in them. The SNPs found mapped in the candidate genes will be important genetic tools in the search for phenotype-modifying nucleotide changes and will aid in formulating relevant genetic improvement programmes for dairy cattle.