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A previous study on ovarian and hypothalami transcriptome analysis in white Muscovy duck revealed that MAP3K8 gene participated in MAPK signaling pathway that influence egg production. Additionally, MAP3K8 was predicted as a target gene of miRNA-509-3p that promotes the secretion of oestradiol which is an important hormone in egg ovulation. This suggested that MAP3K8 might have a functional role in the reproductive performance "egg production" of white Muscovy ducks. Herein, we focused on expression level of MAP3K8 in reproductive and non-reproductive tissues of highest (HP) and lowest (LP) egg producing white Muscovy ducks and identified the polymorphism in MAP3K8 and its association with three egg production traits; Age at first egg (AFE), number of eggs at 300 days (N300D) and 59 weeks (N59W). The results of expression level indicated that mRNA of MAP3K8 was significantly (p < 0.01) expressed in the oviduct than in the ovary and hypothalamus. Seven synonymous SNPs were detected, and association analysis showed that g.148303340 G>A and g.148290065 A>G were significantly (p < 0.05) associated with N300D and N59W. The results of this study might serve as molecular marker for marker-assisted selection of white Muscovy ducks for egg production., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Prion diseases, also known as transmissible spongiform encephalopathies (TSEs) remain one of the deleterious disorders, which have affected several animal species. Polymorphism of the prion protein (PRNP) gene majorly determines the susceptibility of animals to TSEs. However, only limited studies have examined the variation in PRNP gene in different Nigerian livestock species. Thus, this study aimed to identify the polymorphism of PRNP gene in Nigerian livestock species (including camel, dog, horse, goat, and sheep). We sequenced the open reading frame (ORF) of 65 camels, 31 village dogs and 12 horses from Nigeria and compared with PRNP sequences of 886 individuals retrieved from public databases., Results: All the 994 individuals were assigned into 162 haplotypes. The sheep had the highest number of haplotypes (n = 54), and the camel had the lowest (n = 7). Phylogenetic tree further confirmed clustering of Nigerian individuals into their various species. We detected five non-synonymous SNPs of PRNP comprising of G9A, G10A, C11G, G12C, and T669C shared by all Nigerian livestock species and were in Hardy-Weinberg Equilibrium (HWE). The amino acid changes in these five non-synonymous SNP were all "benign" via Polyphen-2 program. Three SNPs G34C, T699C, and C738G occurred only in Nigerian dogs while C16G, G502A, G503A, and C681A in Nigerian horse. In addition, C50T was detected only in goats and sheep., Conclusion: Our study serves as the first to simultaneously investigate the polymorphism of PRNP gene in Nigerian livestock species and provides relevant information that could be adopted in programs targeted at breeding for prion diseases resistance., (© 2024. The Author(s).)

Polymorphism of the prion protein gene ( PRNP ) gene determines an animal's susceptibility to scrapie. Three polymorphisms at codons 136, 154, and 171 have been linked to classical scrapie susceptibility, although many variants of PRNP have been reported. However, no study has investigated scrapie susceptibility in Nigerian sheep from the drier agro-climate zones. In this study, we aimed to identify PRNP polymorphism in nucleotide sequences of 126 Nigerian sheep by comparing them with public available studies on scrapie-affected sheep. Further, we deployed Polyphen-2, PROVEAN, and AMYCO analyses to determine the structure changes produced by the non-synonymous SNPs. Nineteen (19) SNPs were found in Nigerian sheep with 14 being non-synonymous. Interestingly, one novel SNP (T718C) was identified. There was a significant difference ( P  < 0.05) in the allele frequencies of PRNP codon 154 between sheep in Italy and Nigeria. Based on the prediction by Polyphen-2, R154H was probably damaging while H171Q was benign. Contrarily, all SNPs were neutral via PROVEAN analysis while two haplotypes (HYKK and HDKK) had similar amyloid propensity of PRNP with resistance haplotype in Nigerian sheep. Our study provides valuable information that could be possibly adopted in programs targeted at breeding for scrapie resistance in sheep from tropical regions.

Haemonchus contortus is the most pathogenic and economically restrictive gastrointestinal nematode in the small ruminant industry globally. Morbidity, poor cross-bodily state, and mortality of sheep in Lesotho suggest the presence of H. contortus. The present study investigated the morphological, molecular, and population genetics of H. contortus third-stage larvae infecting sheep in four ecological zones (EZ) of Lesotho. Coprocultures were prepared for larval morphological identification and PCR determination. Larvae were identified morphologically as 100% H. contortus. The Second Internal Transcribed Spacer (ITS-2) gene of the ribosomal DNA of H. contortus isolates in the present study revealed nucleotide homology ranging from 97 to 100% when compared with selected GenBank reference sequences. Pairwise evolutionary divergence among H. contortus isolates was low, with 0.01318 recorded as the highest in the present study. Five haplotypes resulted from 14 Lesotho sequences. Haplotype diversity and nucleotide diversity were 0.76923 and 0.00590, respectively. Genetic differentiation among isolates was low but not statistically significant. An analysis of molecular variance revealed that most molecular variation was distributed within topographic populations at 94.79% (F ST  = 0.05206, p > 0.05) and 5.21% among populations. There was high gene flow and no definite population genetic structure among Lesotho isolates., Competing Interests: Declaration of Competing Interest The authors do not declare any competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Sighthounds, a distinctive group of hounds comprising numerous breeds, have their origins rooted in ancient artificial selection of dogs. In this study, we performed genome sequencing for 123 sighthounds, including one breed from Africa, six breeds from Europe, two breeds from Russia, and four breeds and 12 village dogs from the Middle East. We gathered public genome data of five sighthounds and 98 other dogs as well as 31 gray wolves to pinpoint the origin and genes influencing the morphology of the sighthound genome. Population genomic analysis suggested that sighthounds originated from native dogs independently and were comprehensively admixed among breeds, supporting the multiple origins hypothesis of sighthounds. An additional 67 published ancient wolf genomes were added for gene flow detection. Results showed dramatic admixture of ancient wolves in African sighthounds, even more than with modern wolves. Whole-genome scan analysis identified 17 positively selected genes (PSGs) in the African population, 27 PSGs in the European population, and 54 PSGs in the Middle Eastern population. None of the PSGs overlapped in the three populations. Pooled PSGs of the three populations were significantly enriched in "regulation of release of sequestered calcium ion into cytosol" (gene ontology: 0051279), which is related to blood circulation and heart contraction. In addition, ESR1, JAK2, ADRB1, PRKCE, and CAMK2D were under positive selection in all three selected groups. This suggests that different PSGs in the same pathway contributed to the similar phenotype of sighthounds. We identified an ESR1 mutation (chr1: g.42,177,149 T > C) in the transcription factor (TF) binding site of Stat5a and a JAK2 mutation (chr1: g.93,277,007 T > A) in the TF binding site of Sox5. Functional experiments confirmed that the ESR1 and JAK2 mutation reduced their expression. Our results provide new insights into the domestication history and genomic basis of sighthounds., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Long-read sequencing technology is a powerful approach with application in various genetic and genomic research. Herein, we developed the pipeline for long amplicon high-fidelity (HiFi) sequencing and then applied it for sequencing mitochondrial DNA (mtDNA) genomes from pools of 79 Tibetan Mastiffs. We amplified the mtDNA genome with long-range PCR using two pairs of primers. Two rounds of circular consensus sequencing (CCS) were conducted and their accuracy was evaluated. The results indicate that the second round of CCS can improve the accuracy of HiFi reads. In addition, the analysis of 79 high-quality mtDNA genomes shows the Tibetan Mastiffs from outside of the Tibetan Plateau experienced hybridization with other dogs. The high quality reads generator (HQGR) software is provided to facilitate data analyses, which is publicly accessible on GitHub (https://github.com/Caizf-script/HQGR). Our long amplicon HiFi sequencing pipeline can also be applied in various target enrichment strategies for small genomes and candidate genes., (© 2023 John Wiley & Sons Ltd.)

African swine fever (ASF) is a contagious viral disease that affects domestic pigs and wild boars, causing significant economic losses globally. After the first Nigerian outbreak in 1997, there have been frequent reports of ASF in pig-producing regions in the country. To facilitate control, it is important to understand the genotype and phylogenetic relationship of ASF viruses (ASFVs). Recent genetic analysis of Nigerian ASFV isolates has revealed the presence of both genotypes I and II; this is based on analysis of a few selected genes. Phylogenetic analysis of ASFV whole genomes highlights virus origins and evolution in greater depth. However, there is currently no information on the ASFV genome from Nigerian isolates. Two ASFV-positive samples were detected during a random survey of 150 Nigerian indigenous pig samples collected in 2016. We assembled near-complete genomes of the two ASFV-positive samples using in-solution hybrid capture sequencing. The genome-wide phylogenetic tree assigned these two genomes into p72 genotype I, particularly close to the virulent Benin 97/1 strain. The two ASFVs share 99.94 and 99.92 % genomic sequence identity to Benin97/1. This provides insight into the origin and relationship of ASFV strains from Nigeria and Italy. The study reports for the first time the determination of near-complete genomes of ASFV using in-solution hybrid capture sequencing, which represents an important advance in understanding the global evolutionary landscape of ASFVs.

Chicken is a major source of protein for the increasing human population and is useful for research purposes. There are almost 1,600 distinct regional breeds of chicken across the globe, among which a large body of genetic and phenotypic variations has been accumulated due to extensive natural and artificial selection. Moreover, natural selection is a crucial force for animal domestication. Several approaches have been adopted to detect selection signatures in different breeds of chicken using whole genome sequencing (WGS) data including integrated haplotype score (iHS), cross-populated extend haplotype homozygosity test (XP-EHH), fixation index (F ST ), cross-population composite likelihood ratio (XP-CLR), nucleotide diversity (Pi), and others. In addition, gene enrichment analyses are utilized to determine KEGG pathways and gene ontology (GO) terms related to traits of interest in chicken. Herein, we review different studies that have adopted diverse approaches to detect selection signatures in different breeds of chicken. This review systematically summarizes different findings on selection signatures and related candidate genes in chickens. Future studies could combine different selection signatures approaches to strengthen the quality of the results thereby providing more affirmative inference. This would further aid in deciphering the importance of selection in chicken conservation for the increasing human population., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Scrapie is a fatal prion protein disease stiffly associated with single nucleotide polymorphism (SNPs) of the prion protein gene (PRNP). The prevalence of this deadly disease has been reported in small ruminants, including goats. The Nigerian goats are hardy, trypano-tolerant, and contribute to the protein intake of the increasing population. Although scrapie has been reported in Nigerian goats, there is no study on the polymorphism of the PRNP gene. Herein, we evaluated the genetic and allele distributions of PRNP polymorphism in 132 Nigerian goats and compared them with publicly available studies on scrapie-affected goats. We utilized Polyphen-2, PROVEAN and AMYCO programs to examine structural variations produced by the non-synonymous SNPs. Our study revealed 29 SNPs in Nigerian goats, of which 14 were non-synonymous, and 23 were novel. There were significant differences (P < 0.001) in the allele frequencies of PRNP codons 139, 146, 154 and 193 in Nigerian goats compared with scrapie-affected goats, except for Northern Italian goats at codon 154. Based on the prediction by Polyphen-2, R139S and N146S were 'benign', R154H was 'probably damaging', and T193I was 'possibly damaging'. In contrast, PROVEAN predicted 'neutral' for all non-synonymous SNPs, while AMYCO showed a similar amyloid propensity of PRNP for resistant haplotype and two haplotypes of Nigerian goats. Our study is the first to investigate the polymorphism of scrapie-related genes in Nigerian goats., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Grasscutter ( Thryonomys swinderianus ) is a large-body old world rodent found in sub-Saharan Africa. The body size and the unique taste of the meat of this major crop pest have made it a target of intense hunting and a potential consideration as a micro-livestock. However, there is insufficient knowledge on the genetic diversity of its populations across African Guinean forests. Herein, we investigated the genetic diversity, population structures and evolutionary history of seven Nigerian wild grasscutter populations together with individuals from Cameroon, Republic of Benin, and Ghana, using five mitochondrial fragments, including D-loop and cytochrome b ( CYTB ). D-loop haplotype diversity ranged from 0.571 (± 0.149) in Republic of Benin to 0.921 (± 0.013) in Ghana. Within Nigeria, the haplotype diversity ranged from 0.659 (± 0.059) in Cross River to 0.837 (± 0.075) in Ondo subpopulation. The fixation index (F ST ), haplotype frequency distribution and analysis of molecular variance revealed varying levels of population structures across populations. No significant signature of population contraction was detected in the grasscutter populations. Evolutionary analyses of CYTB suggests that South African population might have diverged from other populations about 6.1 (2.6-10.18, 95% CI) MYA. Taken together, this study reveals the population status and evolutionary history of grasscutter populations in the region., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LHD declared a shared affiliation with the author CAMSD to the handling editor at the time of review., (Copyright © 2023 Babarinde, Adeola, Djagoun, Nneji, Okeyoyin, Niba, Wanzie, Oladipo, Adebambo, Bello, Ng’ang’a, Olaniyi, Okoro, Adedeji, Olatunde, Ayoola, Matouke, Wang, Sanke, Oseni, Nwani and Murphy.)

Azakheli is relatively smaller riverine breed with a very peculiar characteristics kept under unique traditional husbandry practices in comparison with rest of the Pakistani buffalo breeds; however, milk production is comparable. The present study was conducted to evaluate the genetic diversity of Azakheli breed. A total of sixty-six blood samples were collected for the amplification of mtDNA D-loop region and MC1R gene sequencing analysis. Median-joining network analysis of 191 mtDNA D-loop sequences of Azakheli and eight Indian riverine buffalo breeds clustered into three clades. Ancient Azakheli Region 1 clade was the oldest with the highest mutation steps and was present close to the root of UPGMA phylogenetic tree. There was 5 mutated lines distance between Pakistan buffalo and Indian riverine buffaloes. The populations of neighboring countries did not share any haplotypes with Azakheli buffalo of Pakistan. Possibly, residing for so long in the cold atmosphere and high elevation regions caused the mutation in mtDNA D-loop, though these conditions did not affect the overall performance of Azakheli as milch buffalo breed of Pakistan. MC1R analyses showed high mutations in Azakheli of Albino phenotype and all the black phenotype individuals of Azakheli buffalo share haplotypes with dominant Chinese and Indian black phenotypes buffaloes in MC1R median-joining network, indicating the reason of black coat color is due to MC1R gene. The haplotype diversity and nucleotide diversity was (H. 0.923, Pi: 0.00895) in Azakheli. Current results illustrated Asian ancestry for Azakheli buffalo, and mtDNA and MC1R analyses provided further evidence. Additional genetic analyses and archeological studies may provide further insight into the domestication period and history of Azakheli buffalo breed. The further studies are required on different coat colors with different genes on Azakheli buffalo to understand the phenotype variation., Competing Interests: The authors declare no conflict of interest regarding the publication of this paper., (Copyright © 2022 Sikandar Ali et al.)

African wild suids consist of several endemic species that represent ancient members of the family Suidae and have colonized diverse habitats on the African continent. However, limited genomic resources for African wild suids hinder our understanding of their evolution and genetic diversity. In this study, we assembled high-quality genomes of a common warthog (Phacochoerus africanus), a red river hog (Potamochoerus porcus), as well as an East Asian Diannan small-ear pig (Sus scrofa). Phylogenetic analysis showed that common warthog and red river hog diverged from their common ancestor around the Miocene/Pliocene boundary, putatively predating their entry into Africa. We detected species-specific selective signals associated with sensory perception and interferon signaling pathways in common warthog and red river hog, respectively, which contributed to their local adaptation to savannah and tropical rainforest environments, respectively. The structural variation and evolving signals in genes involved in T-cell immunity, viral infection, and lymphoid development were identified in their ancestral lineage. Our results provide new insights into the evolutionary histories and divergent genetic adaptations of African suids., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Prior studies on transcriptomes of hypothalamus and ovary revealed that AKT3 is one of the candidate genes that might affect egg production in White Muscovy ducks. The role of AKT3 in the uterus during reproductive processes cannot be overemphasized. However, functional role of this gene in the tissues and on egg production traits of Muscovy ducks remains unknown. To identify the relationship between AKT3 and egg production traits in ducks, relative expression profile was first examined prior to identifying the variants within AKT3 that may underscore egg production traits [age at first egg (AFE), number of eggs at 300 d (N300D), and number of eggs at 59 wk (N59W)] in 549 ducks. The mRNA expression of AKT3 gene in high producing (HP) ducks was significantly higher than low producing (LP) ducks in the ovary, oviduct, and hypothalamus (P < 0.05 or 0.001). Three variants in AKT3 (C-3631A, C-3766T, and C-3953T) and high linkage block between C-3766T and C-3953T which are significantly (P < 0.05) associated with N300D and N59W were discovered. This study elucidates novel knowledge on the molecular mechanism of AKT3 that might be regulating egg production traits in Muscovy ducks., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The Muscovy duck (Cairina moschata) is an economically important poultry species, which is susceptible to fatty liver. Thus, the Muscovy duck may serve as an excellent candidate animal model of non-alcoholic fatty liver disease. However, the mechanisms underlying fatty liver development in this species are poorly understood. In this study, we report a chromosome-level genome assembly of the Muscovy duck, with a contig N50 of 11.8 Mb and scaffold N50 of 83.16 Mb. The susceptibility of Muscovy duck to fatty liver was mainly attributed to weak lipid catabolism capabilities (fatty acid β-oxidation and lipolysis). Furthermore, conserved noncoding elements (CNEs) showing accelerated evolution contributed to fatty liver formation by down-regulating the expression of genes involved in hepatic lipid catabolism. We propose that the susceptibility of Muscovy duck to fatty liver is an evolutionary by-product. In conclusion, this study revealed the potential mechanisms underlying the susceptibility of Muscovy duck to fatty liver., (Copyright © 2022. Published by Elsevier Inc.)

Objectives: Domestic pigeons ( Columba livia domestica ) have diverse plumage pigmentations. Melanocortin 1 receptor ( MC1R ) gene variation has been correlated with color traits. The association between MC1R and plumage coloration in African domestic pigeons is yet to be investigated., Materials and Methods: Herein, we report the relationships between single nucleotide polymorphisms (SNPs) in MC1R and plumage of 35 domestic pigeons from Nigeria with 4 different plumage phenotypes plus 37 published MC1R sequences from France ( n = 14) and Russia ( n = 11)., Results: We obtained 14 SNP sites among 72 individuals. Missense mutations C206T (Ser69Leu) and G253A (Val85Met) were observed in 16 and 8 Nigerian pigeons, respectively. The chi-squared test ( p < 0.05) for C206T, G253A, and A520G has the advantage of homozygous genotypes CC, GG, and AA, respectively. The association of C206T loci showed the advantage of CC genotype in ash-red, spread, and white pigeons, and TT in blue-bar, spread, and white feather pigeons. For G253A and A520G loci, GG and AA were dominant in all plumages except for genotype AA in G253A, which was prominent in ash-red, spread, and white plumages. The three SNPs were assigned to seven haplotypes. The median-joining network revealed 20 haplotypes, including 5 in Nigeria and 2 shared., Conclusion: This study provides an insight into the association of MC1R variation and plumage diversity in Nigerian domestic pigeons. However, due to the limitation of the current data, we could not make further conclusions; this necessitates the need for more genomics studies on Nigerian pigeons., Competing Interests: The authors declare no conflict of interest., (Copyright: © Journal of Advanced Veterinary and Animal Research.)

Duck is the second-largest poultry species aside from chicken. The rate of egg production is a major determinant of the economic income of poultry farmers. Among the reproductive organs, the ovary is a major part of the female reproductive system which is highly important for egg production. Based on the importance of this organ, several studies have been carried out to identify candidate genes at the transcriptome level, and also the expression level of these genes at different tissues or egg-laying conditions, and single nucleotide polymorphism (SNPs) of genes associated with egg production in duck. In this review, expression profile and association study analyses at SNPs level of different candidate genes with egg production traits of duck were highlighted. Furthermore, different studies on transcriptome analysis, Quantitative Trait Loci (QTL) mapping, and Genome Wide Association Study (GWAS) approach used to identify potential candidate genes for egg production in ducks were reported. This review would widen our knowledge on molecular markers that are associated or have a positive correlation to improving egg production in ducks, for the increasing world populace., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The domestic Muscovy duck ( Cairina moschata ) provide unique genetic resources patterned by both tropical environmental conditions and human activities, the evaluation of their genetic diversity and population structure will shade light on the mechanism of their remarkable adaptive capacities. We therefore analyzed the variation in mtDNA cytochrome b and nuclear DNA CYP2U1 sequences of 378 Nigerian Muscovy ducks (comprising of 287 de novo and 91 downloaded) plus 80 published sequences of Muscovy ducks from India. The results showed high haplotype diversity (0.800 ± 0.023) among Nigerian Muscovy duck populations with 91 distinct haplotypes for the nuclear DNA CYP2U1 gene but low (0.266 ± 0.033) for cytochrome b with 31 haplotypes. The median-joining networks of both markers grouped Nigerian Muscovy ducks into two; the first group consisting of only Nigerian Muscovy duck populations, and the second group Nigerian with Indian populations. Neutrality test results indicated that Nigerian populations experienced recent population expansion and/or genetic hitchhiking. A geographic signal was absent in line with previously studied poultry species in Nigeria. The most prominent haplotype dominated across all regions in Nigeria, which may be due to extensive genetic intermixing except for the Indian population ( F ST = 0.02550, P = 0.01075). This indicated low genetic differentiation between and within Nigerian Muscovy duck as revealed by the suitability of the nuclear DNA CYP2U1 gene., Competing Interests: The authors declare that they have no competing interests., (© 2022 Adeola et al.)

Background: Cattle are considered to be the most desirable livestock by small scale farmers. In Africa, although comprehensive genomic studies have been carried out on cattle, the genetic variations in indigenous cattle from Nigeria have not been fully explored. In this study, genome-wide analysis based on genotyping-by-sequencing (GBS) of 193 Nigerian cattle was used to reveal new insights on the history of West African cattle and their adaptation to the tropical African environment, particularly in sub-Saharan region.  RESULTS: The GBS data were evaluated against whole-genome sequencing (WGS) data and high rate of variant concordance between the two platforms was evident with high correlated genetic distance matrices genotyped by both methods suggestive of the reliability of GBS applicability in population genetics. The genetic structure of Nigerian cattle was observed to be homogenous and unique from other African cattle populations. Selection analysis for the genomic regions harboring imprints of adaptation revealed genes associated with immune responses, growth and reproduction, efficiency of feeds utilization, and heat tolerance. Our findings depict potential convergent adaptation between African cattle, dogs and humans with adaptive genes SPRY2 and ITGB1BP1 possibly involved in common physiological activities., Conclusion: The study presents unique genetic patterns of Nigerian cattle which provide new insights on the history of cattle in West Africa based on their population structure and the possibility of parallel adaptation between African cattle, dogs and humans in Africa which require further investigations., (© 2022. The Author(s).)

Speciation is a process whereby the evolution of reproductive barriers leads to isolated species. Although many studies have addressed large-effect genetic footprints in the advanced stages of speciation, the genetics of reproductive isolation in nascent stage of speciation remains unclear. Here, we show that pig domestication offers an interesting model for studying the early stages of speciation in great details. Pig breeds have not evolved the large X-effect of hybrid incompatibility commonly observed between "good species." Instead, deleterious epistatic interactions among multiple autosomal loci are common. These weak Dobzhansky-Muller incompatibilities confer partial hybrid inviability with sex biases in crosses between European and East Asian domestic pigs. The genomic incompatibility is enriched in pathways for angiogenesis, androgen receptor signaling and immunity, with an observation of many highly differentiated cis-regulatory variants. Our study suggests that partial hybrid inviability caused by pervasive but weak interactions among autosomal loci may be a hallmark of nascent speciation in mammals., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Over the last several hundred years, donkeys have adapted to high-altitude conditions on the Tibetan Plateau. Interestingly, the kiang, a closely related equid species, also inhabits this region. Previous reports have demonstrated the importance of specific genes and adaptive introgression in divergent lineages for adaptation to hypoxic conditions on the Tibetan Plateau. Here, we assessed whether donkeys and kiangs adapted to the Tibetan Plateau via the same or different biological pathways and whether adaptive introgression has occurred. We assembled a de novo genome from a kiang individual and analyzed the genomes of five kiangs and 93 donkeys (including 24 from the Tibetan Plateau). Our analyses suggested the existence of a strong hard selective sweep at the EPAS1 locus in kiangs. In Tibetan donkeys, however, another gene, i.e., EGLN1 , was likely involved in their adaptation to high altitude. In addition, admixture analysis found no evidence for interspecific gene flow between kiangs and Tibetan donkeys. Our findings indicate that despite the short evolutionary time scale since the arrival of donkeys on the Tibetan Plateau, as well as the existence of a closely related species already adapted to hypoxia, Tibetan donkeys did not acquire adaptation via admixture but instead evolved adaptations via a different biological pathway.

Domestication of the helmeted guinea fowl (HGF; Numida meleagris) in Africa remains elusive. Here we report a high-quality de novo genome assembly for domestic HGF generated by long- and short-reads sequencing together with optical and chromatin interaction mapping. Using this assembly as the reference, we performed population genomic analyses for newly sequenced whole-genomes for 129 birds from Africa, Asia, and Europe, including domestic animals (n = 89), wild progenitors (n = 34), and their closely related wild species (n = 6). Our results reveal domestication of HGF in West Africa around 1,300-5,500 years ago. Scanning for selective signals characterized the functional genes in behavior and locomotion changes involved in domestication of HGF. The pleiotropy and linkage in genes affecting plumage color and fertility were revealed in the recent breeding of Italian domestic HGF. In addition to presenting a missing piece to the jigsaw puzzle of domestication in poultry, our study provides valuable genetic resources for researchers and breeders to improve production in this species., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Development of thoracolumbar vertebra (TLV) and rib primordium (RP) is a common evolutionary feature across vertebrates, although whole-organism analysis of the expression dynamics of TLV- and RP-related genes has been lacking. Here, we investigated the single-cell transcriptome landscape of thoracic vertebra (TV), lumbar vertebra (LV), and RP cells from a pig embryo at 27 days post-fertilization (dpf) and identified six cell types with distinct gene expression signatures. In-depth dissection of the gene expression dynamics and RNA velocity revealed a coupled process of osteogenesis and angiogenesis during TLV and RP development. Further analysis of cell type-specific and strand-specific expression uncovered the extremely high level of HOXA10 3'-UTR sequence specific to osteoblasts of LV cells, which may function as anti-HOXA10-antisense by counteracting the HOXA10-antisense effect to determine TLV transition. Thus, this work provides a valuable resource for understanding embryonic osteogenesis and angiogenesis underlying vertebrate TLV and RP development at the cell type-specific resolution, which serves as a comprehensive view on the transcriptional profile of animal embryo development., (Copyright © 2021 The Author. Published by Elsevier B.V. All rights reserved.)

Guenons (tribe Cercopithecini) are the most widely distributed nonhuman primate in the tropical forest belt of Africa and show considerable phenotypic, taxonomic, and ecological diversity. However, genomic information for most species within this group is still lacking. Here, we present a high-quality de novo genome (total 2.90 Gb, contig N50 equal to 22.7 Mb) of the mona monkey (Cercopithecus mona), together with genome resequencing data of 13 individuals sampled across Nigeria. Our results showed differentiation between populations from East and West of the Niger River ∼84 ka and potential ancient introgression in the East population from other mona group species. The PTPRK, FRAS1, BNC2, and EDN3 genes related to pigmentation displayed signals of introgression in the East population. Genomic scans suggest that immunity genes such as AKT3 and IL13 (possibly involved in simian immunodeficiency virus defense), and G6PD, a gene involved in malaria resistance, are under positive natural selection. Our study gives insights into differentiation, natural selection, and introgression in guenons., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

The African cattle provide unique genetic resources shaped up by both diverse tropical environmental conditions and human activities, the assessment of their genetic diversity will shade light on the mechanism of their remarkable adaptive capacities. We therefore analyzed the genetic diversity of cattle samples from Nigeria using both maternal and paternal DNA markers. Nigerian cattle can be assigned to 80 haplotypes based on the mitochondrial DNA (mtDNA) D-loop sequences and haplotype diversity was 0.985 + 0.005. The network showed two major matrilineal clustering: the dominant cluster constituting the Nigerian cattle together with other African cattle while the other clustered Eurasian cattle. Paternal analysis indicates only zebu haplogroup in Nigerian cattle with high genetic diversity 1.000 ± 0.016 compared to other cattle. There was no signal of maternal genetic structure in Nigerian cattle population, which may suggest an extensive genetic intermixing within the country. The absence of Bos indicus maternal signal in Nigerian cattle is attributable to vulnerability bottleneck of mtDNA lineages and concordance with the view of male zebu genetic introgression in African cattle. Our study shades light on the current genetic diversity in Nigerian cattle and population history in West Africa., Competing Interests: The authors declare that they have no competing interests., (© 2021 Mauki et al.)

The ichthyofauna diversity of the Jebba Hydroelectric Power (HEP) Dam, Jebba, North-central Nigeria was studied. Fishes were sampled for 24 months using gill net, hook and line, and cast net. Individuals were identified using morphological and molecular (mitochondrial Cytochrome c Oxidase subunit I) data. A total of 9605 freshwater fishes were recorded during the sampling period. The use of an integrative taxonomic approach enabled the identification of 83 species belonging to 42 genera. Additionally, the study recorded three unidentified species - Ctenopoma sp, Malapterurus sp., and Protopterus sp. Analyses showed that individuals belonging to families Cichlidae and Mochokidae dominated the dam. The diversity analyses revealed relatively high fish diversity during the rainy season at the downstream section of Jebba HEP dam compared to the upstream section. The study, therefore, showed the presence of a diverse fish community comprising high species richness and diversity across the Jebba HEP dam. Finally, we recommend proper biodiversity monitoring and assessment of freshwater fish diversity across Nigeria. In addition, the use of an integrated taxonomic approach is recommended for appropriate species' identification and studies of freshwater fishes from Nigeria.

Accurate identification of species is a prerequisite for successful biodiversity management and further genetic studies. Species identification techniques often require both morphological diagnostics and molecular tools, such as DNA barcoding, for correct identification. In particular, the use of the subunit I of the mitochondrial cytochrome c oxidase (COI) gene for DNA barcoding has proven useful in species identification for insects. However, to date, no studies have been carried out on the DNA barcoding of Nigerian butterflies. We evaluated the utility of DNA barcoding applied for the first time to 735 butterfly specimens from southern Nigeria. In total, 699 DNA barcodes, resulting in a record of 116 species belonging to 57 genera, were generated. Our study sample comprised 807 DNA barcodes based on sequences generated from our current study and 108 others retrieved from BOLD. Different molecular analyses, including genetic distance-based evaluation (Neighbor-Joining, Maximum Likelihood and Bayesian trees) and species delimitation tests (TaxonDNA, Automated Barcode Gap Discovery, General Mixed Yule-Coalescent, and Bayesian Poisson Tree Processes) were performed to accurately identify and delineate species. The genetic distance-based analyses resulted in 163 well-separated clusters consisting of 147 described and 16 unidentified species. Our findings indicate that about 90.20% of the butterfly species were explicitly discriminated using DNA barcodes. Also, our field collections reported the first country records of ten butterfly species-Acraea serena, Amauris cf. dannfelti, Aterica galena extensa, Axione tjoane rubescens, Charaxes galleyanus, Papilio lormieri lormeri, Pentila alba, Precis actia, Precis tugela, and Tagiades flesus. Further, DNA barcodes revealed a high mitochondrial intraspecific divergence of more than 3% in Bicyclus vulgaris vulgaris and Colotis evagore. Furthermore, our result revealed an overall high haplotype (gene) diversity (0.9764), suggesting that DNA barcoding can provide information at a population level for Nigerian butterflies. The present study confirms the efficiency of DNA barcoding for identifying butterflies from Nigeria. To gain a better understanding of regional variation in DNA barcodes of this biogeographically complex area, future work should expand the DNA barcode reference library to include all butterfly species from Nigeria as well as surrounding countries. Also, further studies, involving relevant genetic and eco-morphological datasets, are required to understand processes governing mitochondrial intraspecific divergences reported in some species complexes.

The continuous variation of the seasonal influenza viruses, particularly A(H1N1)pdm09, persistently threatens human life and health around the world. In local areas of southwest china, the large time-scale genomic research on A(H1N1)pdm09 is still insufficient. Here, we sequenced 45 whole-genome sequences of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018, by next-generation sequencing technology to characterize molecular mechanisms of their origin and evolution. Our phylogenetic analyses suggest that the A(H1N1)pdm09 strains circulating in Lincang belong to clade 6B and the subclade 6B.1A predominates in 2018. Further, the strains in 2018 possess elevated evolutionary rate as compared to strains in other years. Several newly emerged mutations for HA (hemagglutinin) in 2018 are revealed (i.e., S183P and R221K). Intriguingly, the substitution R221K falls into the RBS (receptor binding site) of HA protein, which could affect antigenic properties of influenza A(H1N1)pdm09 viruses, and another substitution S183P near to RBS with a high covering frequency (11/14 strains) in 2018 is exactly located at the epitope B. Notably, the NA (neuraminidase) protein harbors a new mutation I23T, potentially involved in N-glycosylation. Based on the background with a higher evolutionary rate in 2018 strains, we deeply evaluate the potential vaccine efficacy against Lincang strains and discover a substantive decline of the vaccine efficacy in 2018. Our analyses reaffirm that the real-time molecular surveillance and timely updated vaccine strains for prevention and control of influenza A(H1N1)pdm09 are crucial in the future., Competing Interests: The authors have declared that no competing interests exist.

Background: The remarkable abilities of the human brain are distinctive features that set us apart from other animals. However, our understanding of how the brain has changed in the human lineage remains incomplete, but is essential for understanding cognition, behavior, and brain disorders in humans. Here, we compared the expression trajectory in brain development between humans and rhesus macaques (Macaca mulatta) to explore their divergent transcriptome profiles., Results: Results showed that brain development could be divided into two stages, with a demarcation date in a range between 25 and 26 postconception weeks (PCW) for humans and 17-23PCWfor rhesus macaques, rather than birth time that have been widely used as a uniform demarcation time of neurodevelopment across species. Dynamic network biomarker (DNB) analysis revealed that the two demarcation dates were transition phases during brain development, after which the brain transcriptome profiles underwent critical transitions characterized by highly fluctuating DNB molecules. We also found that changes between early and later brain developmental stages (as defined by the demarcation points) were substantially greater in the human brain than in the macaque brain. To explore the molecular mechanism underlying prolonged timing during early human brain development, we carried out expression heterochrony tests. Results demonstrated that compared to macaques, more heterochronic genes exhibited neoteny during early human brain development, consistent with the delayed demarcation time in the human lineage, and proving that neoteny in human brain development could be traced to the prenatal period. We further constructed transcriptional networks to explore the profile of early human brain development and identified the hub gene RBFOX1 as playing an important role in regulating early brain development. We also found RBFOX1 evolved rapidly in its non-coding regions, indicating that this gene played an important role in human brain evolution. Our findings provide evidence that RBFOX1 is a likely key hub gene in early human brain development and evolution., Conclusions: By comparing gene expression profiles between humans and macaques, we found divergent expression trajectories between the two species, which deepens our understanding of the evolution of the human brain.

The silver butter catfish (Schilbe intermedius) is widely distributed across African river systems. To date, information on its mitochondrial genetic diversity, population structure, and historical demography are not well-established. Herein, we combined newly generated mitochondrial cytochrome c oxidase (COI) subunit I gene sequences with previously published COI sequences in the global databases to reconstruct its phylogeography, population genetic structure, and historical demography. Results from the mtDNA phylogeography and species delimitation tests (Cluster algorithm - Species Identifier, Automatic Barcode Gap Discovery and Poison Tree Process model) revealed that S. intermedius comprises at least seven geographically defined matrilines. Although the overall haplotype diversity of S. intermedius was high (h = 0.90), results showed that East (Kenya) and West (Nigeria) African populations had low levels of haplotype diversity (h = ~0.40). In addition, population genetic polymorphism and historical demographics showed that S. intermedius populations in both East and West Africa underwent severe contractions as a result of biogeographic influences. The patterns of genetic diversity and population structure were consistent with adaptive responses to historical biogeographic factors and contemporary environmental variations across African river systems. This is suggestive of the influence of historical biogeographic factors and climatic conditions on population divergence of S. intermedius across African river systems. Given our discovery of previously underappreciated diversity within S. intermedius, we recommend that this species be considered for increased conservation and management.

Background: Polydactyly is one of the most common congenital limb dysplasia in many animal species. Although preaxial polydactyly (PPD) has been comprehensively studied in humans as a common abnormality, the genetic variations in other animal species have not been fully understood. Herein, we focused on the pig, as an even-toed ungulate mammal model with its unique advantages in medical and genetic researches, two PPD families consisting of four affected and 20 normal individuals were sequenced., Results: Our results showed that the PPD in the sampled pigs were not related to previously reported variants. A strong association was identified at ABCC4 and it encodes a transmembrane protein involved in ciliogenesis. We found that the affected and normal individuals were highly differentiated at ABCC4, and all the PPD individuals shared long haplotype stretches as compared with the unaffected individuals. A highly differentiated missense mutation (I85T) in ABCC4 was observed at a residue from a transmembrane domain highly conserved among a variety of organisms., Conclusions: This study reports ABCC4 as a new candidate gene and identifies a missense mutation for PPD in pigs. Our results illustrate a putative role of ciliogenesis process in PPD, coinciding with an earlier observation of ciliogenesis abnormality resulting in pseudo-thumb development in pandas. These results expand our knowledge on the genetic variations underlying PPD in animals.

Molecular studies on donkey mitochondrial sequences have clearly defined two distinct maternal lineages involved in domestication. However, domestication histories of these two lineages remain enigmatic. We therefore compared several population characteristics between these two lineages based on global sampling, which included 171 sequences obtained in this study (including Middle Asian, East Asian, and African samples) plus 536 published sequences (including European, Asian, and African samples). The two lineages were clearly separated from each other based on whole mitochondrial genomes and partial non-coding displacement loop (D-loop) sequences, respectively. The Clade I lineage experienced an increase in population size more than 8 000 years ago and shows a complex haplotype network. In contrast, the population size of the Clade II lineage has remained relatively constant, with a simpler haplotype network. Although the distribution of the two lineages was almost equal across the Eurasian mainland, they still presented discernible but complex geographic bias in most parts of Africa, which are known as their domestication sites. Donkeys from sub-Saharan Africa tended to descend from the Clade I lineage, whereas the Clade II lineage was dominant along the East and North coasts of Africa. Furthermore, the migration routes inferred from diversity decay suggested different expansion across China between the two lineages. Altogether, these differences indicated non-simultaneous domestication of the two lineages, which was possibly influenced by the response of pastoralists to the desertification of the Sahara and by the social expansion and trade of ancient humans in Northeast Africa, respectively.

Nigeria is an Afrotropical region with considerable ecological heterogeneity and levels of biotic endemism. Among its vertebrate fauna, reptiles have broad distributions, thus, they constitute a compelling system for assessing the impact of ecological variation and geographic isolation on species diversification. The red-headed rock agama, Agama agama , lives in a wide range of habitats and, thus, it may show genetic structuring and diversification. Herein, we tested the hypothesis that ecology affects its genetic structure and population divergence . Bayesian inference phylogenetic analysis of a mitochondrial DNA (mtDNA) gene recovered four well-supported matrilines with strong evidence of genetic structuring consistent with eco-geographic regions. Genetic differences among populations based on the mtDNA also correlated with geographic distance. The ecological niche model for the matrilines had a good fit and robust performance. Population divergence along the environmental axes was associated with climatic conditions, and temperature ranked highest among all environmental variables for forest specialists, while precipitation ranked highest for the forest/derived savanna, and savanna specialists. Our results cannot reject the hypothesis that niche conservatism promotes geographic isolation of the western populations of Nigerian A. agama . Thus, ecological gradients and geographic isolation impact the genetic structure and population divergence of the lizards . This species might be facing threats due to recent habitat fragmentation, especially in western Nigeria. Conservation actions appear necessary., (© The Author(s) (2019). Published by Oxford University Press on behalf of Editorial Office, Current Zoology.)

Tibetan pig is native to the Qinghai-Tibet Plateau and has adapted to the high-altitude environmental condition such as hypoxia. However, its origin and genetic mechanisms underlying high-altitude adaptation still remain controversial and enigmatic. Herein, we analyze 229 genomes of wild and domestic pigs from Eurasia, including 63 Tibetan pigs, and detect 49.6 million high-quality variants. Phylogenomic and structure analyses show that Tibetan pigs have a close relationship with low-land domestic pigs in China, implying a common domestication origin. Positively selected genes in Tibetan pigs involved in high-altitude physiology, such as hypoxia, cardiovascular systems, UV damage, DNA repair. Three of loci with strong signals of selection are associated with EPAS1, CYP4F2, and THSD7A genes, related to hypoxia and circulation. We validated four non-coding mutations nearby EPAS1 and CYP4F2 showing reduced transcriptional activity in Tibetan pigs. A high-frequency missense mutation is found in THSD7A (Lys561Arg) in Tibetan pigs. The selective sweeps in Tibetan pigs was found in association with selection against non-coding variants, indicating an important role of regulatory mutations in Tibetan pig evolution. This study is important in understanding the evolution of Tibetan pigs and advancing our knowledge on animal adaptation to high-altitude environments.

The Bama Xiang pig (BMX) is a famous early-maturing Chinese indigenous breed with a two-end black coat. To uncover the genetic basis of the BMX phenotype, we conducted comparative genomic analyses between BMX and East Asian wild boars and Laiwu pigs, respectively. Genes under positive selection were enriched in pathways associated with gonadal hormone and melanin synthesis, consistent with the phenotypic changes observed during development in BMX pigs. We also performed differentially expressed gene analysis based on RNA-seq data from pituitary tissues of BMX and Large White pigs. The CTTNBP2NL , FRS2 , KANK4 , and KATNAL1 genes were under selection and exhibited expressional changes in the pituitary tissue, which may affect BMX pig puberty. Our study demonstrated the positive selection of early maturity in the development of BMX pigs and advances our knowledge on the role of regulatory elements in puberty evolution in pigs.

This study examines the utility of morphology and DNA barcoding in species identification of freshwater fishes from north-central Nigeria. We compared molecular data (mitochondrial cytochrome c oxidase subunit I ( COI ) sequences) of 136 de novo samples from 53 morphologically identified species alongside others in GenBank and BOLD databases. Using DNA sequence similarity-based (≥97% cutoff) identification technique, 50 (94.30%) and 24 (45.30%) species were identified to species level using GenBank and BOLD databases, respectively. Furthermore, we identified cases of taxonomic problems in 26 (49.00%) morphologically identified species. There were also four (7.10%) cases of mismatch in DNA barcoding in which our query sequence in GenBank and BOLD showed a sequence match with different species names. Using DNA barcode reference data, we also identified four unknown fish samples collected from fishermen to species level. Our Neighbor-joining (NJ) tree analysis recovers several intraspecific species clusters with strong bootstrap support (≥95%). Analysis uncovers two well-supported lineages within Schilbe intermedius . The Bayesian phylogenetic analyses of Nigerian S. intermedius with others from GenBank recover four lineages. Evidence of genetic structuring is consistent with geographic regions of sub-Saharan Africa. Thus, cryptic lineage diversity may illustrate species' adaptive responses to local environmental conditions. Finally, our study underscores the importance of incorporating morphology and DNA barcoding in species identification. Although developing a complete DNA barcode reference library for Nigerian ichthyofauna will facilitate species identification and diversity studies, taxonomic revisions of DNA sequences submitted in databases alongside voucher specimens are necessary for a reliable taxonomic and diversity inventory.

The dog mtDNA diversity picture from wide geographical sampling but from a small number of individuals per region or breed, displayed little geographical correlation and high degree of haplotype sharing between very distant breeds. For a clear picture, we extensively surveyed Iranian native dogs (n = 305) in comparison with published European (n = 443) and Southwest Asian (n = 195) dogs. Twelve haplotypes related to haplogroups A, B and C were shared by Iranian, European, Southwest Asian and East Asian dogs. In Iran, haplotype and nucleotide diversities were highest in east, southeast and northwest populations while western population had the least. Sarabi and Saluki dog populations can be assigned into haplogroups A, B, C and D; Qahderijani and Kurdi to haplogroups A, B and C, Torkaman to haplogroups A, B and D while Sangsari and Fendo into haplogroups A and B, respectively. Evaluation of population differentiation using pairwise F ST generally revealed no clear population structure in most Iranian dog populations. The genetic signal of a recent demographic expansion was detected in East and Southeast populations. Further, in accordance with previous studies on dog-wolf hybridization for haplogroup d2 origin, the highest number of d2 haplotypes in Iranian dog as compared to other areas of Mediterranean basin suggests Iran as the probable center of its origin. Historical evidence showed that Silk Road linked Iran to countries in South East Asia and other parts of the world, which might have probably influenced effective gene flow within Iran and these regions. The medium nucleotide diversity observed in Iranian dog calls for utilization of appropriate management techniques in increasing effective population size.

Natural selection in domestic dogs is of great interest in evolutionary biology since dogs have migrated to every inhabited continent of the world alongside humans, and adapted to diverse environments. Here, we explored their demographic history and genetic basis of adaptation to the tropical African environment using whole genome analyses of 19 African indigenous dogs from Nigeria. Demographic analysis suggests that the ancestors of these dogs migrated into Africa from Eurasia 14,000 years ago and underwent a severe founder effect before population expansion. Admixture analysis further reveals that African dog genomes contain about 1.88-3.50% introgression from African golden wolves (Canis anthus). Population genetic analysis identifies 50 positively selected genes linked with immunity, angiogenesis, ultraviolet protection, as well as insulin secretion and sensitivity that may contribute to adaptation to tropical conditions. One of the positively selected genes, adhesion G protein-coupled receptor E1 (ADGRE1), has also been found to be association with severe malaria resistance in African human populations. Functional assessments showed that ADGRE1 provides protective host defense against Plasmodium infections. This result, together with the fact that the inflammatory response to canine babesiosis is similar to complicated falciparum malaria in humans, support the dogs as a model for the study of malaria control and treatment., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The complete mitochondrial genomes of two Sri Lankan junglefowl ( Gallus lafayetti : CJF) individuals were sequenced by using next-generation sequencing technique. Samples were collected from Rathnapura and Pelmadulla areas in Sri Lanka. The complete mitochondrial DNA is 16,839 bp in length, with a typical mitogenome structure composed of a non-coding control region, 22 tRNA, two rRNA, and 13 protein-coding genes. Overall base composition is 30% A, 23.9% T, 32.3% C, and 13.6% G indicating high content of 54.0% A + T for both individuals. Phylogenetic analysis reveals that CJF samples cluster with the clade of the green junglefowl ( Gallus varius ) and red junglefowl ( Gallus gallus ) than to grey junglefowl ( Gallus sonerattii : GyJF). This result can be subsequently used to provide essential information for junglefowl evolution., Competing Interests: The authors have declared that no competing interests exist and are solely responsible for this paper., (© 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

Capture hybridization coupled with high-throughput sequencing (HTS) has become one of the most popular approaches to address some scientific problems not only for fundamental evolution but also for ecology and human disease in recent years. However, the technical problem of limited probe capture ability affects its widespread application. Here, we propose to capture hybridize long-range DNA fragments for HTS (termed LR-LCH). We provide a case of three amphibian samples to examine LR-LCH with 2 kb libraries and comparison of standard capture hybridization with 480 bp libraries. Capture sensitivity increased from an average 13.57% of standard capture hybridization to an average 19.80% of LR-LCH; capture efficiency also increased from an average 72.56% of standard capture hybridization to an average 97.71% of LR-LCH. These indicate that longer fragments in the library generally contain both relatively variable regions and relatively conservative regions. The divergent parts of target DNA are enriched along with conservative parts of DNA sequence that effectively captured during hybridization. We present a protocol that allows users to overcome the low capture sensitivity problem for high divergent regions.