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Objective The remarkable adaptability to the environment, high growth rate, meat with good taste and aroma, and ornamental appearance of the Pradu Hang Dam (PDH) and Samae Dam (SD) chickens make them valuable for improvement of poultry production to enhance food security. However, despite their close phenotypic similarity, distinct classification of PDH and SD chickens remains controversial. Thus, this study aimed to clarify genetic origins and variation between PDH and SD chickens, genetic diversity and structures of PDH and SD chickens. Methods This study analyzed 5 populations of PDH and 2 populations of SD chickens using 28 microsatellite markers and compared with those of other indigenous and local chicken breeds using Thailand’s “The Siam Chicken Bioresource Project” database. Results Considerably high genetic variability was observed within PDH (370 total alleles; 4.086±0.312 alleles/locus) and SD chickens (179 total alleles; 3.607±0.349 alleles/locus). A partial overlap of gene pools was observed between SD chickens from the Department of Livestock, Uthai Thani (SD1) and PDH chickens, suggesting a potentially close relationship between the two chicken breeds. A gene pool that partially overlapped with that of the red junglefowl was observed in the SD chicken population from the Sanhawat Farm Uthai Thani population (SD2). Distinct subclusters were observed within SD chickens, indicating the possibility that genetic differentiation occurred early in the process of establishment of SD chickens. Conclusion These findings could offer valuable insights into genetic verification of Thai local chicken breeds and their sustainable conservation and utilization.

IntroductionReproductive isolation and hybrid sterility are mechanisms that maintain the genetic integrity of species and prevent the introgression of heterospecific genes. However, crosses of closely related species can lead to complex evolution, such as the formation of all-female lineages that reproduce clonally. Bighead catfish (Clarias macrocephalus) and North African catfish (C. gariepinus) diverged 40 million years ago. They are cultivated and hybridized in Thailand for human consumption. Male hybrids are sterile due to genome-wide chromosome asynapsis during meiosis. Although female hybrids are sometimes fertile, their chromosome configuration during meiosis has not yet been studied.MethodsWe analyzed meiosis in the hybrid female catfish at pachytene (synaptonemal complexes) and diplotene (lampbrush chromosomes), using immunostaining to detect chromosome pairing and double-stranded break formation, and FISH with species-specific satellite DNAs to distinguish the parental chromosomes.ResultsMore than 95% of oocytes exhibited chromosome asynapsis in female hybrid catfish; however, they were able to progress to the diplotene stage and form mature eggs. The remaining oocytes underwent premeiotic endoreplication, followed by synapsis and crossing over between sister chromosomes, similar to known clonal lineages in fish and reptiles.DiscussionThe occurrence of clonal reproduction in female hybrid catfish suggests a unique model for studying gametogenic alterations caused by hybridization and their potential for asexual reproduction. Our results further support the view that clonal reproduction in certain hybrid animals relies on intrinsic mechanisms of sexually reproducing parental species, given their multiple independent origins with the same mechanism.

ABSTRACT: The thermal stress caused by global climate change adversely affects the welfare, productivity, and reproductive performance of farm animals, including chickens, and causes substantial economic losses. However, the understanding of the genetic basis of the indigenous chicken adaptation to high ambient temperatures is limited. Hence, to reveal the genetic basis of thermal stress adaptation in chickens, this study investigated polymorphisms in the heat shock protein 70 (HSP70) and HSP90 genes, known mechanisms of cellular defense against thermal stress in indigenous and local chicken breeds and red junglefowls in Thailand. The result revealed seven alleles of the HSP70 gene. One allele exhibited a missense mutation, where an amino acid changed from Asn to His in the substrate-binding and peptide-binding domains, which is exclusive to the Lao Pa Koi chicken breed. Twenty new alleles with silent mutations in the HSP90 gene highlighted its greater complexity. Despite this diversity, distinct population structures were not found for either HSP70 or HSP90, which suggests incomplete impact on the domestication process and selection. The low genetic diversity, shown by the sharing of alleles between red junglefowls and Thai indigenous and local chicken breeds, aligns with the hypothesis that these alleles have undergone selection in tropical regions, such as Thailand. Selection signature analysis suggests the purifying selection of HSP70 for thermotolerance. This study provides valuable insights for enhancing the conservation of genetic resources with thermotolerant traits, which are essential for developing breeding programs to increase poultry production in the context of global climate change.

The North African catfish (Clarias gariepinus) is a significant species in aquaculture, which is crucial for ensuring food and nutrition security. Their high adaptability to diverse environments has led to an increase in the number of farms that are available for their production. However, long-term closed breeding adversely affects their reproductive performance, leading to a decrease in production efficiency. This is possibly caused by inbreeding depression. To investigate the root cause of this issue, the genetic diversity of captive North African catfish populations was assessed in this study. Microsatellite genotyping and mitochondrial DNA D-loop sequencing were applied to 136 catfish specimens, collected from three populations captured for breeding in Thailand. Interestingly, extremely low inbreeding coefficients were obtained within each population, and distinct genetic diversity was observed among the three populations, indicating that their genetic origins are markedly different. This suggests that outbreeding depression by genetic admixture among currently captured populations of different origins may account for the low productivity of the North African catfish in Thailand. Genetic improvement of the North African catfish populations is required by introducing new populations whose origins are clearly known. This strategy should be systematically integrated into breeding programs to establish an ideal founder stock for selective breeding.

Objective Cambodia is located within the distribution range of the red junglefowl, the common ancestor of domestic chickens. Although a variety of indigenous chickens have been reared in Cambodia since ancient times, their genetic characteristics have yet to be sufficiently defined. Here, we conducted a large-scale population genetic study to investigate the genetic diversity and population genetic structure of Cambodian indigenous chickens and their phylogenetic relationships with other chicken breeds and native chickens worldwide. Methods A Bayesian phylogenetic tree was constructed based on 625 mitochondrial DNA D-loop sequences, and Bayesian clustering analysis was performed for 666 individuals with 23 microsatellite markers, using samples collected from 28 indigenous chicken populations in 24 provinces and three commercial chicken breeds. Results A total of 92 haplotypes of mitochondrial D-loop sequences belonging to haplogroups A to F and J were detected in Cambodian chickens; in the indigenous chickens, haplogroup D (44.4%) was the most common, and haplogroups A (21.0%) and B (13.2%) were also dominant. However, haplogroup J, which is rare in domestic chickens but abundant in Thai red junglefowl, was found at a high frequency (14.5%), whereas the frequency of haplogroup E was considerably lower (4.6%). Population genetic structure analysis based on microsatellite markers revealed the presence of three major genetic clusters in Cambodian indigenous chickens. Their genetic diversity was relatively high, which was similar to findings reported for indigenous chickens from other Southeast Asian countries. Conclusion Cambodian indigenous chickens are characterized by mitochondrial D-loop haplotypes that are common to indigenous chickens throughout Southeast Asia, and may retain many of the haplotypes that originated from wild ancestral populations. These chickens exhibit high population genetic diversity, and the geographical distribution of three major clusters may be attributed to inter-regional trade and poultry transportation routes within Cambodia or international movement between Cambodia and other countries.

Summary: Background: The benefits of artificial nutrition and hydration in patients with head and neck cancer and esophageal cancer in the late stage remain unknown. We performed a secondary analysis of a cohort study to investigate the effects of enteral tube feeding (ETF) and parenteral nutrition and hydration (PNH) on survival in this population. Methods: Patients with head and neck cancer and esophageal cancer admitted to palliative care units were included. Information on primary nutritional administration routes during the first week of admission and data on the averaged calorie sufficiency rate/total calorie intake, e.g., 75%≤/750 kcal/day≤ and

Lao Pa Koi (LPK) chicken is a popular fighting breed in Thailand, prized for (its unique characteristics acquired by selective breeding), and a valuable model for exploring the genetic diversity and admixture of red junglefowls and domestic chickens. In this study, genetic structure and diversity of LPK chicken were assessed using 28 microsatellite markers and mitochondrial DNA (mtDNA) D-loop sequences, and the findings were compared to a gene pool library from "The Siam Chicken Bioresource Project". High genetic variability was observed in LPK chickens using mtDNA D-loop haplotype analysis, and six haplotypes were identified. Microsatellite data revealed 182 alleles, with an average of 6.5 alleles per locus. These results confirmed the occurrence of genetic admixture of red junglefowl and Thai domestic chickens in LPK chicken breed. A maximum entropy modeling approach was used to analyze the spatial suitability and to assess the adaptive evolution of LPK chickens in diverse local environments. The model identified 82.52% of the area studied as unsuitable, and 9.34%, 7.11%, and 2.02% of the area indicated moderate, low, and high suitability, respectively. The highest contribution rate to land suitability for LPK chickens was found at an elevation of 100-250 m, suggesting the importance of elevation for their potential distribution. The results of this study provide valuable insights into the genetic origin of LPK chicken breed and identify resources for future genetic improvement.

Summary: Background & Aims: The prevalence of artificially administered nutrition and hydration (AANH) in different age groups among patients with advanced cancer remains unknown. The present study investigated the current utilization of AANH according to age groups in palliative care units. Methods: This was a secondary analysis of a prospective cohort study. We obtained information on primary nutritional administration routes during the first week of admission and data on the averaged calorie sufficiency rate or total calorie intake on the 7th day of admission. Patients were divided into five age groups (18–39, 40–59, 60–74, 75–89, and 90- years). Among patients receiving AANH, the proportions of higher-calorie AANH were compared between the five age groups. Results: A total of 1453 patients were included. The proportion of patients categorized as receiving nutrition and hydration via the parenteral route was the highest in the 18–39 and 40–59 groups (52.4 and 41.1%, respectively). Among patients receiving AANH (n = 534), the proportions of patients categorized into the

Understanding the genetic diversity of domestic chicken breeds under the impact of socio–cultural and ecological dynamics is vital for the conservation of natural resources. Mae Hong Son chicken is a local breed of North Thai domestic chicken widely distributed in Mae Hong Son Province, Thailand; however, its genetic characterization, origin, and diversity remain poorly understood. Here, we studied the socio–cultural, environmental, and genetic aspects of the Mae Hong Son chicken breed and investigated its diversity and allelic gene pool. We genotyped 28 microsatellite markers and analyzed mitochondrial D-loop sequencing data to evaluate genetic diversity and assessed spatial habitat suitability using maximum entropy modeling. Sequence diversity analysis revealed a total of 188 genotyped alleles, with overall nucleotide diversity of 0.014 ± 0.007, indicating that the Mae Hong Son chicken population is genetically highly diverse, with 35 (M1–M35) haplotypes clustered into haplogroups A, B, E, and F, mostly in the North ecotype. Allelic gene pool patterns showed a unique DNA fingerprint of the Mae Hong Son chicken, as compared to other breeds and red junglefowl. A genetic introgression of some parts of the gene pool of red junglefowl and other indigenous breeds was identified in the Mae Hong Son chicken, supporting the hypothesis of the origin of the Mae Hong Son chicken. During domestication in the past 200–300 years after the crossing of indigenous chickens and red junglefowl, the Mae Hong Son chicken has adapted to the highland environment and played a significant socio–cultural role in the Northern Thai community. The unique genetic fingerprint of the Mae Hong Son chicken, retaining a high level of genetic variability that includes a dynamic demographic and domestication history, as well as a range of ecological factors, might reshape the adaptation of this breed under selective pressure.

Abstract In this study, we aimed to elucidate the origin of domestic chickens and their evolutionary history over the course of their domestication. We conducted a large-scale genetic study using mitochondrial DNA D-loop sequences and 28 microsatellite DNA markers to investigate the diversity of 298 wild progenitor red junglefowl (Gallus gallus) across two subspecies (G. g. gallus and G. g. spadiceus) from 12 populations and 138 chickens from 10 chicken breeds indigenous to Thailand. Twenty-nine D-loop sequence haplotypes were newly identified: 14 and 17 for Thai indigenous chickens and red junglefowl, respectively. Bayesian clustering analysis with microsatellite markers also revealed high genetic diversity in the red junglefowl populations. These results suggest that the ancestral populations of Thai indigenous chickens were large, and that a part of the red junglefowl population gene pool was not involved in the domestication process. In addition, some haplogroups that are distributed in other countries of Southeast Asia were not observed in either the red junglefowls or the indigenous chickens examined in the present study, suggesting that chicken domestication occurred independently across multiple regions in Southeast Asia.

Abstract The trade‐off between reproduction and self‐maintenance is a cornerstone of life history theory, yet its proximate underpinnings are elusive. Here, we used an artificial selection approach to create replicated lines of Japanese quail (Coturnix japonica) that differ genetically in their reproductive investment. Whole transcriptome sequencing revealed that females from lines selected for high reproductive output show a consistent upregulation of genes associated with reproduction but a simultaneous downregulation of immune genes. Concordant phenotypic differences in immune function (i.e., specific antibody response against keyhole limpet hemocyanin) were observed between the selection lines, even in males who do not provide parental care. Our findings demonstrate the key role of obligate transcriptional constraints in the maintenance of life history variation. These constraints set fundamental limits to productivity and health in natural and domestic animal populations.

The precursor of heme, protoporphyrin IX (PPIX), accumulates abundantly in the uteri of birds, such as Japanese quail, Coturnix japonica, which has brown-speckled eggshells; however, the molecular basis of PPIX production in the uterus remains largely unknown. Here, we investigated the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis. We also performed genetic linkage analysis to identify the causative genomic region of the white eggshell phenotype. We found that 11 genes, including 5’-aminolevulinate synthase 1 (ALAS1) and hephaestin-like 1 (HEPHL1), were specifically upregulated in the wild-type uterus and downregulated in the mutant. We mapped the 172 kb candidate genomic region on chromosome 6, which contains several genes, including a part of the paired-like homeodomain 3 (PITX3), which encodes a transcription factor. ALAS1, HEPHL1, and PITX3 were expressed in the apical cells of the luminal epithelium and lamina propria cells of the uterine mucosa of the wild-type quail, while their expression levels were downregulated in the cells of the mutant quail. Biochemical analysis using uterine homogenates indicated that the restricted availability of 5’-aminolevulinic acid is the main cause of low PPIX production. These results suggest that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on these findings, we discussed the molecular basis of PPIX production in the uteri of Japanese quails.

Background: Limited information is available on the relationship between C-reactive protein (CRP) levels and pain in advanced cancer. Objectives: To investigate the relationship between serum levels of CRP and subtypes of pain. Design: A secondary cross-sectional analysis of a prospective cohort study. Setting/Subjects: Patients with advanced cancer admitted to 23 palliative care units in Japan. Measurements: Patients rated the severity of pain on the numerical rating scale (NRS) and physicians evaluated pain on the integrated palliative care outcome scale (IPOS). Physicians assessed neuropathic pain and breakthrough pain based on their presence or absence. Patients were divided into four groups according to CRP levels. Comparisons were performed using the Kruskal?Wallis test or chi-squared test. To evaluate the relationship between CRP and subtypes of pain, adjusted odds ratios (ORs) and 95% confidence intervals (CIs) in logistic models were calculated. Results: We divided 1513 patients into four groups: low CRP (n?=?234), moderate CRP (n?=?513), high CRP (n?=?352), and very high CRP (n?=?414). Spearman's correlation coefficient between CRP and pain NRS and that between CRP and pain IPOS were 0.15 (p?0.001) and 0.16 (p?

Hybrid incompatibility, such as sterility and inviability, prevents gene flow between closely-related populations as a reproductive isolation barrier. F1 hybrids between chickens and Japanese quail (hereafter, referred to as quail), exhibit a high frequency of developmental arrest at the preprimitive streak stage. To investigate the molecular basis of the developmental arrest at the preprimitive streak stage in chicken-quail F1 hybrid embryos, we investigated chromosomal abnormalities in the hybrid embryos using molecular cytogenetic analysis. In addition, we quantified gene expression in parental species and chicken- and quail-derived allele-specific expression in the hybrids at the early blastoderm and preprimitive streak stages by mRNA sequencing. Subsequently, we compared the directions of change in gene expression, including upregulation, downregulation, or no change, from the early blastoderm stage to the preprimitive streak stage between parental species and their hybrids. Chromosome analysis revealed that the cells of the hybrid embryos contained a fifty-fifty mixture of parental chromosomes, and numerical chromosomal abnormalities were hardly observed in the hybrid cells. Gene expression analysis revealed that a part of the genes that were upregulated from the early blastoderm stage to the preprimitive streak stage in both parental species exhibited no upregulation of both chicken- and quail-derived alleles in the hybrids. GO term enrichment analysis revealed that these misregulated genes are involved in various biological processes, including ribosome-mediated protein synthesis and cell proliferation. Furthermore, the misregulated genes included genes involved in early embryonic development, such as primitive streak formation and gastrulation. These results suggest that numerical chromosomal abnormalities due to a segregation failure does not cause the lethality of chicken-quail hybrid embryos, and that the downregulated expression of the genes that are involved in various biological processes, including translation and primitive streak formation, mainly causes the developmental arrest at the preprimitive streak stage in the hybrids.

Abstract Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

The highly repetitive DNA sequence of centromeric heterochromatin is an effective molecular cytogenetic marker for investigating genomic compartmentalization between macrochromosomes and microchromosomes in birds. We isolated four repetitive sequence families of centromeric heterochromatin from three Anseriformes species, viz., domestic duck (Anas platyrhynchos, APL), bean goose (Anser fabalis, AFA), and whooper swan (Cygnus cygnus, CCY), and characterized the sequences by molecular cytogenetic approach. The 190-bp APL-HaeIII and 101-bp AFA-HinfI-S sequences were localized in almost all chromosomes of A. platyrhynchos and A. fabalis, respectively. However, the 192-bp AFA-HinfI-L and 290-bp CCY-ApaI sequences were distributed in almost all microchromosomes of A. fabalis and in approximately 10 microchromosomes of C. cygnus, respectively. APL-HaeIII, AFA-HinfI-L, and CCY-ApaI showed partial sequence homology with the chicken nuclear-membrane-associated (CNM) repeat families, which were localized primarily to the centromeric regions of microchromosomes in Galliformes, suggesting that ancestral sequences of the CNM repeat families are observed in the common ancestors of Anseriformes and Galliformes. These results collectively provide the possibility that homogenization of centromeric heterochromatin occurred between microchromosomes in Anseriformes and Galliformes; however, homogenization between macrochromosomes and microchromosomes also occurred in some centromeric repetitive sequences.

Chicken egg white ovotransferrin (OTF) has been reported to exist in three electrophoretic variants (OTFA, OTFB and OTFC). In this report, we identified a causal polymorphism between the OTFB and OTFC variants in Japanese and Taiwanese native chickens and compared the antibacterial activity between these two variants. The cDNA sequence analyses from Satsumadori oviducts revealed that three non-synonymous SNPs T1809G (Ser52Ala), A2258G (Ile96Val) and G7823A (Asp500Asn) corresponded to the OTF electrophoretic phenotypes. Of the three SNPs, the G7823A mutation perfectly corresponded to the electrophoretic phenotypes OTFB (G/G, Asp500Asp), OTFB/C (G/A, Asp500Asn) and OTFC (A/A, Asn500Asn) in three chicken populations. The variants OTFB and OTFC exhibited similar antibacterial potency against Gram-positive and Gram-negative bacteria. This study provides, for the first time, molecular information on polymorphism of OTFB and OTFC variants of chicken ovotransferrin and its effect on the antimicrobial activity of the respective variants.

The mangrove killifish Kryptolebias marmoratus, and its close relative Kryptolebias hermaphroditus, are the only vertebrate species known to reproduce by self-fertilization due to functional ovotestis development. To improve our understanding of their genomes, we constructed a genetic map. First, a single F1 fish was made by artificial fertilization between K. marmoratus and K. hermaphroditus strains. F2 progeny were then obtained by self-fertilization of the F1 fish. We used RAD-seq to query genomic DNAs from the two parental strains, the F1 individual and 49 F2 progeny. Results identified 9904 polymorphic RAD-tags (DNA markers) that mapped to 24 linkage groups, corresponding to the haploid chromosome number of these species. The total length of the map was 1248 cM, indicating that about one recombination occurred for each of the 24 homologous chromosome pairs in each meiosis. Markers were not evenly distributed along the chromosomes: in all chromosomes, many markers (> 8% of the total markers for each chromosome) mapped to chromosome tips. Centromeres suppress recombination, and this uneven distribution is probably due to the species’ acrocentric chromosomes. Mapped marker sequences were compared to genomic sequences of medaka and platyfish, the next most closely related species with sequenced genomes that are anchored to genetic maps. Results showed that each mangrove killifish chromosome corresponds to a single chromosome of both platyfish and medaka, suggesting strong conservation of chromosomes over 100 million years of evolution. Our genetic map provides a framework for the K. marmoratus/K. hermaphroditus genome sequence and an important resource for understanding the biology of hermaphroditism.

To assess the genetic diversity of domestic Japanese quail (Coturnix japonica) populations, and their genetic relationships, we examined mitochondrial DNA (mtDNA) D-loop sequences and microsatellite markers for 19 Japanese quail populations. The populations included nine laboratory lines established in Japan (LWC, Quv, RWN, WE, AWE, AMRP, rb-TKP, NIES-L, and W), six meat-type quail lines reimported from Western countries (JD, JW, Estonia, NIES-Br, NIES-Fr, and NIES-Hn), one commercial population in Japan, and three wild quail populations collected from three Asian areas. The phylogenetic tree of mtDNA D-loop sequences revealed two distinct haplotype groups, Dloop-Group1 and Dloop-Group2. Dloop-Group1 included a dominant haplotype representing most of the quail populations, including wild quail. Dloop-Group2 was composed of minor haplotypes found in several laboratory lines, two meat-type lines, and a few individuals in commercial and wild quail populations. Taking the breeding histories of domestic populations into consideration, these results suggest that domestic quail populations may have derived from two sources, i.e., domestic populations established before and after World War II in Japan. A discriminant analysis of principal components and a Bayesian clustering analysis with microsatellite markers indicated that the domestic populations are clustered into four genetic groups. The two major groups were Microsat-Group1, which contained WE, and four WE-derived laboratory lines (LWC, Quv, RWN, and AWE), and Microsat-Group2 consisting of NIES-L, JD, JW, Estonia, NIES-Br, NIES-Fr, NIES-Hn, W, and commercial and wild populations. The remaining two lines (AMRP and rb-TKP) were each clustered into a separate clade. This hierarchical genetic difference between domestic quail populations is attributed to the genetic background derived from two different genetic sources-the pre-war and post-war populations-which is well supported by their breeding histories.

To better understand the process of karyotype evolution in Galloanserae (Galliformes and Anseriformes), we performed comparative chromosome painting with chicken chromosome-specific DNA probes and FISH mapping of the 18S-28S ribosomal RNA (rRNA) genes, telomeric (TTAGGG)n repeats, and cDNA clones of 37 genes for three anserid species, the domestic duck (Anas platyrhynchos), Muscovy duck (Cairina moschata), and Chinese goose (Anser cygnoides). Each chicken probe of chromosomes 1-9 and the Z chromosome painted a single pair of chromosomes in the three species except for the chromosome 4 probe, which painted acrocentric chromosome 4 and a pair of microchromosomes. The 18S-28S rRNA genes were localized to four pairs of microchromosomes in the domestic duck and Muscovy duck, and eight pairs of microchromosomes in the Chinese goose. The (TTAGGG)n repeats were localized to both telomeric ends of all chromosomes in the three species, and were additionally located in the interstitial region of the short arm of chromosome 1 in the domestic duck and in the centromeric region of chromosome 1 in the Muscovy duck. Comparative gene mapping of 37 chicken chromosome 1-4-linked and Z-linked genes revealed high chromosome homologies among three anserid species and also between the chicken and the three anserid species, although there were several chromosome rearrangements: pericentric inversion in chromosome 2, pericentric and paracentric inversions or centromere repositioning in the Z chromosome between the chicken and the anserid species, and pericentric inversions in chromosome 4 and the Z chromosome between the Chinese goose and the two duck species. These results collectively suggest that karyotypes have been highly conserved in Anseriformes and that chromosome rearrangements also occurred less frequently between Galliformes and Anseriformes after they diverged around 100 million years ago.

Hybrid sterility is a postzygotic reproductive isolation mechanism that prevents successful interbreeding between different species. The mule duck, an intergeneric F1 hybrid between the domestic duck (Anas platyrhynchos) and Muscovy duck (Cairina moschata), displays sterility with gametogenesis failure in both sexes. Although the F1 hybrid male is known to exhibit large-sized testes that produce no sperm, the spermatogenic phenotype has not been well described. In this study, we revealed the abnormal meiotic phenotype of the F1 hybrid spermatocytes and dissimilarity in the karyotypes between the two parental species. Histological examination of the F1 hybrid testis showed the accumulation of primary spermatocytes with irregular highly condensed chromosomes in the seminiferous epithelium, whereas secondary spermatocytes and postmeiotic cells were absent and many testicular cells undergoing apoptosis were present. Cytogenetic analyses of spermatogenic cells from the F1 hybrid male revealed that meiosis succeeded in entering pachytene, but failed to progress beyond diakinesis-metaphase I in primary spermatocytes, and that a number of degenerated spermatocytes were present at pachytene. Karyological observations showed morphological differences in chromosome 1 and the Z chromosome between the parental species. These results collectively suggest that the main cause of abnormal spermatogenesis in the F1 hybrid is pachytene and/or metaphase I arrest, which possibly resulted from the failure of homologous chromosome pairing, recombination, and subsequent chromosome segregation due to chromosomal incompatibility between the parental species.

The Injustice Experience Questionnaire (IEQ) assesses injury-related perceived injustice. This study aimed to (1) develop a Japanese version (IEQ-J), (2) examine its factor structure, validity, and reliability, and (3) discover which demographic variable(s) positively contributed to prediction of IEQ-J scores.Data from 71 patients (33 male, 38 female; age = 20+) with injury pain were employed to investigate factor structure by exploratory and confirmatory factor analyses. Concurrent validity was examined by Pearson correlation coefficients among the IEQ-J, Brief Pain Inventory (BPI), and Pain Catastrophizing Scale (PCS). Internal consistency was investigated by Cronbach's alpha, and test-retest reliability was indicated with intra-class correlations (ICCs) in 42 of 71 patients within four weeks. Relations between demographic variables and IEQ-J scores were examined by covariance analysis and linear regression models.IEQ-J factor structure differed from the original two-factor model. A three-factor model with Severity/irreparability, Blame/unfairness, and Perceived lack of empathy was extracted. The three-factor model showed goodness-of-fit with the data and sufficient reliability (Cronbach's alpha of 0.90 for total IEQ-J; ICCs = 0.96). Pearson correlation coefficients among IEQ-J, BPI, and PCS ranged from 0.38 to 0.73. Pain duration over a year (regression coefficient, 11.92, 95%CI; 5.95-17.89) and liability for injury on another (regression coefficient, 12.17, 95%CI; 6.38-17.96) predicted IEQ-J total scores.This study evidenced the IEQ-J's sound psychometric properties. The three-factor model was the latter distinctive in the Japanese version. Pain duration over a year and injury liability by another statistically significantly increased IEQ-J scores.

Ovoglobulin G2 (G2) has long been known as a major protein constituent of chicken egg white. However, little is known about the biochemical properties and biological functions of G2 because the gene encoding G2 has not been identified. Therefore, the identification of the gene encoding G2 and an analysis of its genetic variability is an important step toward the goal of understanding the biological functions of the G2 protein and its utility in poultry production. To identify and characterize the gene encoding G2, we separated G2 from egg white using electrophoresis on a non-denaturing polyacrylamide gel. Two polymorphic forms of G2 protein (G2A and G2B), with different mobilities (fast and slow respectively), were detected by staining. The protein band corresponding to G2B was electro-eluted from the native gel, re-electrophoresed under denaturing conditions and its N-terminal sequence was determined by Edman degradation following transfer onto a membrane. Sequencing of the 47 kDa G2B band revealed it to be identical to TENP (transiently expressed in neural precursors), also known as BPI fold-containing family B, member 2 (BPIFB2), a protein with strong homology to a bacterial permeability-increasing protein family (BPI) in mammals. Full-length chicken TENP cDNA sequences were determined for 78 individuals across 29 chicken breeds, lines, and populations, and consequently eleven non-synonymous substitutions were detected in the coding region. Of the eleven non-synonymous substitutions, A329G leading to Arg110Gln was completely associated with the noted differential electrophoretic mobility of G2. Specifically G2B, with a slower mobility is encoded by A329 (Arg110), whereas G2A, with a faster mobility, is encoded by G329 (Gln110). The sequence data, derived from the coding region, also revealed that the gene encoding G2 demonstrates significant genetic variability across different chicken breeds/lines/populations. These variants, and how they correlate with egg white properties, may allow us to understand further G2's functions.

Chromosomal mapping of the butterfly lizards Leiolepis belliana belliana and L. boehmei was done using the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences. The karyotype of L. b. belliana was 2n = 36, whereas that of L. boehmei was 2n = 34. The 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, while the 5S rRNA genes were found in the pericentromeric region of chromosome 6 in both species. Hybridization signals for the (TTAGGG)n sequence were observed at the telomeric ends of all chromosomes, as well as interstitially at the same position as the 18S-28S rRNA genes in L. boehmei. This finding suggests that in L. boehmei telomere-to-telomere fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located or, alternatively, at the secondary constriction of chromosome 1. The absence of telomeric sequence signals in chromosome 1 of L. b. belliana suggested that its chromosomes may have only a few copies of the (TTAGGG)n sequence or that there may have been a gradual loss of the repeat sequences during chromosomal evolution.

The Hokou gecko (Gekko hokouensis: Gekkonidae, Gekkota, Squamata) has the chromosome number 2n = 38, with no microchromosomes. For molecular cytogenetic characterization of the gekkotan karyotype, we constructed a cytogenetic map for G. hokouensis, which retains the ancestral karyotype of Gekkota, with 86 functional genes, and compared it with cytogenetic maps for four Toxicofera species that have many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) and that for a lacertid species (Lacerta agilis) with only one pair of autosomal microchromosomes. Ten pairs of G. hokouensis chromosomes [GHO1, 2, 3, Z(4), 6, 7, 8, 13, 14, and 15] showed highly conserved linkage homology with macrochromosomes and/or macrochromosome arms of the four Toxicofera species and corresponded to eight L. agilis macrochromosomes (LAG). However, GHO5, GHO9, GHO10, GHO11, and LAG6 were composed of chromosome segments that have a homology with Toxicofera microchromosomes, and no homology was found in the chromosomes between G. hokouensis and L. agilis. These results suggest that repeated fusions of microchromosomes may have occurred independently in each lineage of Gekkota and Lacertidae, leading to the disappearance of microchromosomes and appearance of small-sized macrochromosomes.

A mutation that confers white plumage with black eyes was identified in the Minohiki breed of Japanese native chicken (Gallus gallus domesticus). The white plumage, with a few partially pigmented feathers, was not associated with the tyrosinase gene, and displayed an autosomal recessive mode of inheritance against the pigmented phenotype. All F1 offspring derived from crosses with mottled chickens (mo/mo), which show characteristic pigmented feathers with white tips, had plumage with a mottled-like pattern. This result indicates that the white plumage mutation is a novel allele at the mo locus; we propose the gene symbol mo(w) for this mutant allele. Furthermore, the F1 hybrid between the mo(w) /mo(w) chicken and the panda (s/s) mutant of Japanese quail (Coturnix japonica), whose causative gene is the endothelin receptor B2 (EDNRB2) gene, showed a mo(w)/mo(w) chicken-like plumage, suggesting the possibility that the mutations in parental species are alleles of the same gene, EDNRB2. Nucleotide sequencing of the entire coding region of EDNRB2 revealed a non-synonymous G1008T substitution, which causes Cys244Phe amino acid substitution in exon 5 (which is part of the extracellular loop between the putative fourth and fifth transmembrane domains of EDNRB2) in the mutant chicken. This Cys244Phe mutation was also present in individuals of four Japanese breeds with white plumage. We also identified a non-synonymous substitution leading to Arg332His substitution that was responsible for the mottled (mo/mo) plumage phenotype. These results suggest that the EDN3 (endothelin 3)-EDNRB2 signaling is essential for normal pigmentation in birds, and that the mutations of EDNRB2 may cause defective binding of the protein with endothelins, which interferes with melanocyte differentiation, proliferation, and migration.

Reptiles have a wide diversity of sex-determining mechanisms and types of sex chromosomes. Turtles exhibit temperature-dependent sex determination and genotypic sex determination, with male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) sex chromosomes. Identification of sex chromosomes in many turtle species and their comparative genomic analysis are of great significance to understand the evolutionary processes of sex determination and sex chromosome differentiation in Testudines. The Mexican giant musk turtle (Staurotypus triporcatus, Kinosternidae, Testudines) and the giant musk turtle (Staurotypus salvinii) have heteromorphic XY sex chromosomes with a low degree of morphological differentiation; however, their origin and linkage group are still unknown. Cross-species chromosome painting with chromosome-specific DNA from Chinese soft-shelled turtle (Pelodiscus sinensis) revealed that the X and Y chromosomes of S. triporcatus have homology with P. sinensis chromosome 6, which corresponds to the chicken Z chromosome. We cloned cDNA fragments of S. triporcatus homologs of 16 chicken Z-linked genes and mapped them to S. triporcatus and S. salvinii chromosomes using fluorescence in situ hybridization. Sixteen genes were localized to the X and Y long arms in the same order in both species. The orders were also almost the same as those of the ostrich (Struthio camelus) Z chromosome, which retains the primitive state of the avian ancestral Z chromosome. These results strongly suggest that the X and Y chromosomes of Staurotypus turtles are at a very early stage of sex chromosome differentiation, and that these chromosomes and the avian ZW chromosomes share the same origin. Nonetheless, the turtles and birds acquired different systems of heterogametic sex determination during their evolution.

Transitions between sex determination systems have occurred in many lineages of squamates and it follows that novel sex chromosomes will also have arisen multiple times. The formation of sex chromosomes may be reinforced by inhibition of recombination and the accumulation of repetitive DNA sequences. The karyotypes of monitor lizards are known to be highly conserved yet the sex chromosomes in this family have not been fully investigated. Here, we compare male and female karyotypes of three Australian monitor lizards, Varanus acanthurus, V. gouldii and V. rosenbergi, from two different clades. V. acanthurus belongs to the acanthurus clade and the other two belong to the gouldii clade. We applied C-banding and comparative genomic hybridization to reveal that these species have ZZ/ZW sex micro-chromosomes in which the W chromosome is highly differentiated from the Z chromosome. In combination with previous reports, all six Varanus species in which sex chromosomes have been identified have ZZ/ZW sex chromosomes, spanning several clades on the varanid phylogeny, making it likely that the ZZ/ZW sex chromosome is ancestral for this family. However, repetitive sequences of these ZW chromosome pairs differed among species. In particular, an (AAT)n microsatellite repeat motif mapped by fluorescence in situ hybridization on part of W chromosome in V. acanthurus only, whereas a (CGG)n motif mapped onto the W chromosomes of V. gouldii and V. rosenbergi. Furthermore, the W chromosome probe for V. acanthurus produced hybridization signals only on the centromeric regions of W chromosomes of the other two species. These results suggest that the W chromosome sequences were not conserved between gouldii and acanthurus clades and that these repetitive sequences have been amplified rapidly and independently on the W chromosome of the two clades after their divergence.

Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal fusions probably occurred independently in the amphibian, squamate, crocodilian, and mammalian lineages.

The endemic cichlid fishes in Lake Victoria are a model system for speciation through adaptive radiation. Although the evolution of the sex-determination system may also play a role in speciation, little is known about the sex-determination system of Lake Victoria cichlids. To understand the evolution of the sex-determination system in these fish, we performed cytogenetic analysis in 11 cichlid species from Lake Victoria. B chromosomes, which are present in addition to standard chromosomes, were found at a high prevalence rate (85%) in these cichlids. In one species, B chromosomes were female-specific. Cross-breeding using females with and without the B chromosomes demonstrated that the presence of the B chromosomes leads to a female-biased sex ratio in this species. Although B chromosomes were believed to be selfish genetic elements with little effect on phenotype and to lack protein-coding genes, the present study provides evidence that B chromosomes have a functional effect on female sex determination. FISH analysis using a BAC clone containing B chromosome DNA suggested that the B chromosomes are derived from sex chromosomes. Determination of the nucleotide sequences of this clone (104.5 kb) revealed the presence of several protein-coding genes in the B chromosome, suggesting that B chromosomes have the potential to contain functional genes. Because some sex chromosomes in amphibians and arthropods are thought to be derived from B chromosomes, the B chromosomes in Lake Victoria cichlids may represent an evolutionary transition toward the generation of sex chromosomes.

Introduction: Reproductive isolation and hybrid sterility are mechanisms that maintain the genetic integrity of species and prevent the introgression of heterospecific genes. However, crosses of closely related species can lead to complex evolution, such as the formation of all-female lineages that reproduce clonally. Bighead catfish (Clarias macrocephalus) and North African catfish (C. gariepinus) diverged 40 million years ago. They are cultivated and hybridized in Thailand for human consumption. Male hybrids are sterile due to genome-wide chromosome asynapsis during meiosis. Although female hybrids are sometimes fertile, their chromosome configuration during meiosis has not yet been studied. Methods: We analyzed meiosis in the hybrid female catfish at pachytene (synaptonemal complexes) and diplotene (lampbrush chromosomes), using immunostaining to detect chromosome pairing and double-stranded break formation, and FISH with species-specific satellite DNAs to distinguish the parental chromosomes. Results: More than 95% of oocytes exhibited chromosome asynapsis in female hybrid catfish; however, they were able to progress to the diplotene stage and form mature eggs. The remaining oocytes underwent premeiotic endoreplication, followed by synapsis and crossing over between sister chromosomes, similar to known clonal lineages in fish and reptiles. Discussion: The occurrence of clonal reproduction in female hybrid catfish suggests a unique model for studying gametogenic alterations caused by hybridization and their potential for asexual reproduction. Our results further support the view that clonal reproduction in certain hybrid animals relies on intrinsic mechanisms of sexually reproducing parental species, given their multiple independent origins with the same mechanism. [ABSTRACT FROM AUTHOR]