Detection of RFLP markers associated with antibody response in meat-type chickens: haplotype/genotype, single-band, and multiband analyses of RFLP in the major histocompatibility complex

Improving disease resistance in poultry by direct selection or by selecting for immune response is hardly feasible due to the quantitative nature of these traits, their low heritability, and the difficulties associated with reliable measurements. In this situation, marker-assisted selection (MAS) is expected to be a more effective breeding approach. The major histocompatibility complex (MHC), known to affect immune response and disease resistance, was examined as a set of candidate genes for association between DNA markers and antibody response. Backcross (BC1) and F2 families were generated from a cross between lines divergently selected for high or low antibody response to Escherichia coli vaccination. Restriction fragment length polymorphism (RFLP) analysis of the highly polymorphic MHC class IV (B-G) region suggested an association with antibody response to several antigens (E. coli, SRBC, NDV). The multiband data generated with the class IV probe were used to compare the efficacies of three alternative analyses: "single-band" (carriers versus noncarriers of each RFLP band separately), "multiband" (multiple regression on all RFLP bands), and "genotype" (determined from family analysis of RFLP patterns/haplotypes). Groups of birds identified by the "multiband" analysis were identical to the haplotype-based genotypes, suggesting that the laborious step of haplotype determination can be omitted without unduly sacrificing power of analysis.     

Designation by restriction fragment length polymorphism of major histocompatibility complex class IV haplotypes in meat-type chickens

Major histocompatiblity complex (MHC) class IV haplotypes were identified in a population of meat-type chickens by restriction fragment length polymorphism (RFLP) analysis. Fourteen different haplotypes were designated on the basis of restriction patterns obtained from Southern blots of PvuII- or BglII-digested DNA, hybridized with the MHC class IV cDNA probe bg32.1. Digestion with each restriction enzyme yielded the same level of polymorphism among individuals. For each haplotype, 4–10 restriction fragments ranging from 0–8 to 8 kb were observed. Such a designation of meat-type chicken MHC class IV haplotypes enables a rapid recognition of previously defined haplotypes, is readily adjustable to additional, newly found restriction patterns and could prove useful in practical breeding programmes.     

三个地方鸡种MHC B-L BII基因遗传变异与免疫性状的关联分析

旨在研究3个地方鸡品种(汶上芦花鸡、莱芜黑鸡、济宁百日鸡)主要组织相容性复合体(Major Histocompatibility Complex,MHC) B-LBII基因遗传变异与绵羊红细胞(Sheep red blood cell,SRBC)、禽流感(Avian influenza,AI)和新城疫(Newcastle disease,ND)抗体滴度等免疫性状的关系,揭示不同品种间基因变异与免疫性状的相关性.以300只地方品种鸡为材料,运用直接测序和聚合酶链式反应-单链构象多态性(PCR-SSCP)等技术检测MHC B-L BⅡ基因的序列变异.在3个地方鸡品种中分别发现了19～22个核苷酸变异位点,可导致其中16～18个氨基酸的变异.3个地方鸡种MHC B-L BⅡ基因中分别有7～8个(Single nucleotide polymorphism,SNP)位点与部分免疫性状存在不同程度的显著相关性.变异位点G97A和T138A在3个品种中均存在,与SRBC、ND、AI抗体滴度均显著相关(P＜0.05).其中,G97A突变在济宁百日鸡中与ND抗体滴度显著相关(P＜0.05),在莱芜黑鸡中与SRBC抗体滴度显著相关(P＜0.05),在汶上芦花鸡中与H9抗体滴度显著相关(P＜0.05);变异位点T138A在汶上芦花鸡和济宁百日鸡中与H9抗体滴度显著相关(P＜0.05).研究表明3个地方鸡种的MHC B-LBII基因遗传变异与免疫性状存在显著关联.     

Restriction fragment length polymorphism analysis of the chicken B-F and B-L genes and their association with serologically defined B haplotypes

Seven serologically defined chicken haplotypes have been analysed by restriction fragment length polymorphism (RFLP) with chicken cDNA probes specific for MHC class I and II. The results demonstrate an excellent correlation between the observed RFLP banding patterns in the investigated haplotypes and the serological B-typing. In future, RFLP analysis in addition to serological B-typing may sharpen the tools in the search for recombinant chromosomes separating B-F and B-L.     

Genetic markers linked to quantitative traits in poultry

This study utilized DNA fingerprints and crosses of two genetically distinct lines of layer-type chickens to identify genetic markers linked to quantitative trait loci (QTL). In phase I, back-cross (BC¹) hens were separately ranked for each of eight traits and then blood pools were produced in groups along each phenotypic distribution. The DNA was isolated from the blood pools and used in a gradient analysis to screen for DNA fingerprint bands that exhibited intensity gradients associated with the phenotypic traits. To identify linkage of bands with QTL and to estimate band effects, F₂ progeny were produced in phase II from the phase I BC, population. A single-trait animal model was used for analysis of associations of all individual DNA fingerprint bands of sires and their progeny phenotypic performance. Twenty fingerprint bands, only two of which had shown trait-associated gradients in phase I, were identified by the animal model analysis of the progeny test as QTL linked (P≤005) to specific traits of growth, reproduction and egg quality. These 20 bands warrant further study as potentially valuable molecular markers for QTL.     

Non-association between Rfp-Y major histocompatibility complex-like genes and susceptibility to Marek's disease virus-induced tumours in 63× 72 F2 intercross chickens

Marek's disease (MD) is a lymphoproliferative disease caused by a member of the herpesvirus family, and the best understood genetic resistance to MD involves the chicken major histocompatibility complex (MHC) B -complex. Preliminary observations have suggested that MHC-like Rfp-Y genes might also influence the incidence of MD. This study describes the differentiation and definition of unique Rfp-Y genes in inbred lines 6₃ and 7₂, lines that possess identical B -complex genes, but that are resistant or susceptible to MD, respectively. To assess if Rfp-Y genes affect susceptibility to MD, 265 6₃× 7₂ F₂ chickens were challenged with the JM strain of MD virus at 1 week of age and were evaluated for MD lesions at up to 10 weeks of age. Genotyping of the F₂ chickens for Rfp-Y haplotypes was performed by restriction fragment length polymorphism analysis of genomic DNA using Taq I and a B-FIV probe. Analysis of variance and interval mapping procedures were used to determine association between the Rfp-Y haplotypes and the phenotypic MD values of the F₂ chickens. The cosegregation analysis of 265 F₂ chickens indicated that there was no association between Rfp-Y haplotypes and MD susceptibility. Furthermore, the fact that the Rfp-Y haplotypes fit the 1:2:1 segregation ratio and the Rfp-Y allele frequencies did not differ significantly from 0·5 in the full population or in selected subpopulations (of either 40 MD-resistant or 39 MD-susceptible chickens) also indicated that Rfp-Y haplotypes do not significantly influence MD susceptibility. We conclude that Rfp-Y haplotypes do not play a major role in determining the genetic susceptibility to MD in 6₃× 7₂ F₂ White Leghorn chickens.     

Restriction fragment length polymorphism analysis of major histocompatibility complex class II genes from inbred chicken lines

Summary. High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes ( Pvu II, Hind III, Bg /II, and Bam HI), electrophoresed for 18 or 45h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II β-chain probe (β2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B halotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.     

Restriction fragment length polymorphism analysis of major histocompatibility complex class II genes from inbred chicken lines

High molecular weight DNA was extracted from sperm from chickens of 14 inbred lines. The DNA was digested with each of four restriction enzymes (Pvu II, Hind III, Bgl II, and Bam HI), electrophoresed for 18 or 45 h, blotted onto nitrocellulose, and hybridized to a chicken major histocompatibility complex (MHC, B complex) class II beta-chain probe (beta 2-exon specific). Restriction fragment length polymorphisms (RFLPs) were found with each of the restriction enzymes used. Birds with the same B haplotype always showed the same RFLP pattern; however, some birds of different B haplotypes also shared the same RFLP pattern. To test for the Mendelian inheritance of the RFLP patterns, the F2 progeny of an informative cross were analysed. The RFLP patterns corresponded with the serologically determined B haplotypes of the F2 birds, thereby showing the Mendelian inheritance of the polymorphic bands.     

MHC variation in birds and reptiles

The major histocompatibility complex (MHC) has been studied in a multitude of mammals by now, but much less is known about its organisation and variation in other vertebrate species. The mammalian MHC is organised as a single gene cluster, but recent studies on birds suggest that this paradigm of MHC organisation has to be supplemented. The domestic chicken thus possesses two separate gene clusters which both contain MHC class I and class II B genes, and we have shown that the ring‐necked pheasant Phasianus colchicus also has two unlinked clusters of class II B genes. We are studying the effect of the MHC on mate choice, survival and reproductive success in natural populations of birds and reptiles. For this reason, we are developing DNA techniques to determine the animals' MHC genotype. The amplification of the hypervariable exon 3 of the class I gene from songbirds and reptiles has provided us with species specific probes that can be used in Southern blot analysis. The first results indicate very extensive variation in all studied species, that is starlings Sturnus vulgaris, great reed warblers Acrocephalus arundinaceus and water pythons Liasis fuscus. The restriction fragment length polymorphism (RFLP) analysis also suggests that the number of MHC genes is significantly larger in these species than in pheasants and domestic chickens. This revised version was published online in July 2006 with corrections to the Cover Date.     

Analysis of the sheep MHC using HLA class I,II, and C4 cDNA probes

Four cDNA probes for the human major histocompatibility complex (MHC) were used to investigate the sheep MHC, in conjunction with serological typing for ovine lymphocyte antigen (OLA). Lymphocytes from a family (two parents and five offspring) of Romanov sheep were subjected to genomic DNA digestion by the restriction endonuclease Eco RI, followed by gel electrophoresis. A single Southern blot representing all seven individuals was then consecutively hybridized with the class I, alpha-DC, beta-DR, and C4 probes, which were originally designed to identify HLA class I, class II (DC and DR), and C4 products, respectively. Using each of the three class I/class II probes, several bands showing DNA polymorphism were detected. The segregation of these bands in the five offspring exactly paralleled the OLA haplotype segregation established by serological typing. A further eight individuals carrying haplotypes which were phenotypically identical to those in the above-mentioned family showed bands in the corresponding positions when tested with the same three probes. Using the C4 probe, no polymorphism was detected in these fifteen individuals.Abbreviations used in this paper MHC major histocompatibility complex - OLA ovine lymphocyte antigen - kbp kilobase pair(s) - MLR mixed lymphocyte reaction - RFLP restriction fragment length polymorphism     

Antibody responses to sheep erythrocytes for White Leghorn chickens differing in haplotypes of the major histocompatibility complex (B)

Summary. Lines of White Leghorn chickens were developed by selection for high (HA) or low (LA) antibody response to sheep red blood cells (SRBC) and then backcrossed to provide individuals segregating for haplotypes B ¹³ and B ²¹ of the major histocompatibility complex (MHC) within each selected line. Although antibody response to SRBC was consistently higher in background genome HA than LA, there was a significant interaction between background genome and MHC haplotypes. The interaction resulted from higher antibody response in B¹³/B²¹ individuals of line HA and in B²¹/ B ²¹ individuals of line LA. Thus, response to SRBC was dependent on particular haplotype combinations present at the MHC as well as the background genome in which they were expressed.     

Correlated responses of chickens to selection for production of antibodies to sheep erythrocytes

A bi directional selection experiment was conducted to measure 5-day antibody titers to sheep erythrocytes in White Leghorn chickens. There was an immediate response to selection with significant differences between lines for the selected trait found in the S₁ and all subsequent generations. Comparisons of S₆, S₇ and S₈ generation females revealed differences between lines in disease resistance and in certain reproductive traits such as age at first egg, percentage hen-day egg production, percentage fertility and duration of fertility. The implications of these correlated responses are important to selection programs for general disease resistance.     

Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana

Recombinant inbred (RI) lines of Arabidopsis thaliana (Arabidopsis) have been generated from a cross between the ecotypes Landsberg erecta and Columbia. Progeny of 300 individual F₂ seedlings were taken by single seed descent to the F₈ generation. Sixty-seven loci, scored using 64 RFLP probes and one phenotypic marker, chosen at approximately 20 cM intervals from the two previously published RFLP maps, were mapped using 100 of these RI lines. More than 500 other new loci are currently being mapped using these RI lines by several other groups. These 100 RI lines thus provide the material to map new probes or phenotypic traits polymorphic between Landsberg erecta and Columbia, relative to an increasing number of molecular markers. Higher resolution mapping of distinct chromosomal regions can be achieved by analysing the segregation of particular markers on the additional 200 RI lines.     

Chicken MHC class I and II gene effects on antibody response kinetics in adult chickens

The major histocompatibility complex (MHC) plays an important role in regulation of the immune response. The MHC class I and II genes were selected as candidates to investigate associations with vaccine response to Salmonella enteritidis and kinetics of antibody response to sheep red blood cell (SRBC) and Brucella abortus. Primary antibody response after S. enteritidis vaccination at day 10, and antibody response to SRBC and killed B. abortus after immunization at 19 and 22 weeks were measured in an F2 population. The resource population was derived from males of two highly inbred MHC-congenic Fayoumi chicken lines (M5.1 and M15.2) mated with highly inbred G-B1 Leghorn line hens. Secondary phase parameters of minimum titers ( Y(min)), maximum titers ( Y(max)), and time needed to achieve Y(min) ( t(min)) and Y(max) ( t(max)) were estimated from post-secondary titers by using a non-linear regression model. Associations of single nucleotide polymorphisms (SNPs) in MHC class I and II genes with antibody response parameters were determined by a general linear model. Significant associations were found primarily in the M15.2 grandsire haplotype. There were significant associations between MHC class I alpha(1) and alpha(2) SNPs and antibody response to S. enteritidis, primary antibody response to B. abortus, Y(min) to SRBC, and Y(max) to both SRBC and B. abortus. There were significant effects of the MHC class II beta(1) domain SNP on S. enteritidis antibody and Y(max) to SRBC. The results suggest that the characterized SNPs might be used in future applications by marker-assisted selection to improve vaccine response and immunocompetence in chickens.     

Isolation and characterization of three class II MHC genomic clones from the chicken

A genomic library was constructed from sperm DNA from an individual of the inbred chicken line G-B2, MHC haplotype B6. The library was screened with a chicken class II probe (beta 2 exon specific) and three MHC class II beta chain genomic clones were isolated. The restriction maps of the three clones showed that each of the three clones was unique. The position of the beta chain sequence was located in each of the three genomic clones by Southern blot hybridization. Subclones containing the beta chain gene were produced from each of the genomic clones and the orientation of the leader peptide, beta 1, beta 2, transmembrane, and cytoplasmic exons was determined by Southern blot hybridization and nucleotide sequencing. The complete nucleotide sequence of two of the three subclones was determined. Comparison of the nucleotide and predicted amino acid sequences of the two subclones with other class II beta chain sequences showed that the B6 chicken beta chain genes are evolutionarily related to the class II beta chain genes from chickens of other MHC haplotypes, and to class II beta chain genes from other species. Analysis of Southern blots of B6 chicken DNA, as well as the isolation of the three beta chain genes, suggests that chickens of the B6 haplotype possess at least three MHC class II beta chain genes.     

Association of MHC class I and class II gene polymorphisms with vaccine or challenge response to Salmonella enteritidis in young chicks

Salmonella enteritidis (SE) is a worldwide source of salmonellosis in humans, caused mainly by consumption of contaminated poultry products. The major histocompatibility complex (MHC) class I and class II molecules, which function as antigen-presentation structures, are involved in both macrophage and dendritic cell immune response to Salmonella and may, therefore, play an important role in host resistance to infections. The chicken MHC class I and class II were investigated as candidate genes for immune response to SE. We characterized the complete MHC class I cDNA sequences for an outbred broiler line and four diverse highly inbred lines: two MHC-congenic Leghorn (G-B2 and G-B1), one Egyptian Fayoumi (M15.2), and one Spanish (Sp21.1), to define the allelic sequences within these lines, so that we might study the association between particular MHC polymorphisms and response to SE. The F1 offspring of outbred broiler sires crossed with three inbred lines (G-B1, G-B2, and Fayoumi) were evaluated as young chicks for either bacterial load in spleen and cecum after pathogenic SE inoculation or antibody level after SE vaccination. Alleles defined by a Lys(148)-->Met(148) polymorphism in the MHC class I alpha(2) domain were associated with spleen bacterial load after SE challenge. These results suggest that particular MHC haplotypes may contribute to control of responses to SE, and that particular polymorphisms may serve as markers for genetic resistance to SE in the chicken.     

Mapping of QTL on chromosome X for fat deposition, muscling and growth traits in a wild boar x Meishan F2 family using a high-density gene map

Quantitative trait loci (QTL) for fat deposition, growth and muscling traits have been previously mapped on the basis of low-density linkage maps in a wild boar × Meishan F₂ family to the chromosome X region flanked by SW2456 and SW1943 . Improved QTL resolution was possible using data for F₂ animals with a marker density of 2.7 cM distance in the SW2456 to SW1943 region, including AR , SERPINA7 and ACSL4 as candidate genes. The resolution of the QTL scan was increased substantially, as evidenced by the higher F -ratio values for all QTL. Maxima of F -ratio values for fat deposition, muscling and growth traits were 28.6, 18.2 and 16.5 respectively, and those QTL positions accounted for 7.9%, 5.0% and 4.5% of the F₂ phenotypic variance (VF₂) respectively. QTL for fatness and growth and for most muscling traits mapped near ACSL4 , with the exception of the QTL for ham traits that mapped proximally, in the vicinity of AR . An analysis performed separately for F₂ male animals showed the predominant QTL affecting fat deposition traits (up to 13.6% VF₂) near AR and two QTL for muscling traits (up to 9.9% VF₂) mapped close to ACSL4 . In the F₂ female animals, QTL affecting muscling (up to 12.1% VF₂) mapped at ACSL4 and SW2456 , and QTL for fat deposition (10% VF₂) and growth (up to 10.5% VF₂) mapped at ACSL4 .     

Genome-wide association study reveals the genetic determinism of growth traits in a Gushi-Anka F2 chicken population

Chicken growth traits are economically important, but the relevant genetic mechanisms have not yet been elucidated. Herein, we performed a genome-wide association study to identify the variants associated with growth traits. In total, 860 chickens from a Gushi-Anka F₂ resource population were phenotyped for 68 growth and carcass traits, and 768 samples were genotyped based on the genotyping-by-sequencing (GBS) method. Finally, 734 chickens and 321,314 SNPs remained after quality control and removal of the sex chromosomes, and these data were used to carry out a GWAS analysis. A total of 470 significant single-nucleotide polymorphisms (SNPs) for 43 of the 68 traits were detected and mapped on chromosomes (Chr) 1–6, -9, -10, -16, -18, -23, and -27. Of these, the significant SNPs in Chr1, -4, and -27 were found to be associated with more than 10 traits. Multiple traits shared significant SNPs, indicating that the same mutation in the region might have a large effect on multiple growth or carcass traits. Haplotype analysis revealed that SNPs within the candidate region of Chr1 presented a mosaic pattern. The significant SNPs and pathway enrichment analysis revealed that the MLNR, MED4, CAB39L, LDB2, and IGF2BP1 genes could be putative candidate genes for growth and carcass traits. The findings of this study improve our understanding of the genetic mechanisms regulating chicken growth and carcass traits and provide a theoretical basis for chicken breeding programs.Subject terms: Genome-wide association studies, Genetic linkage study, Development, DNA sequencing, Animal breeding     

DNA fingerprint bands applied to linkage analysis with quantitative trait loci in chickens

Summary
An efficient approach to detect association between quantitative traits and bands of DNA fingerprint patterns uses intra-family tail analysis, which compares fingerprints of DNA mixes from individuals at the two tails of a phenotypic distribution. In analysis of 67 paternal half-sibs of a meat-type chicken family, of 57 sire bands generated by two probes, one sire-specific band (S6–6) was associated with abdominal fat deposition. The band effect was estimated by a linear model analysis to be 0–88 standard deviations, or about 30% of the family mean. The association between band S6–6 and abdominal fat was further examined by testing progeny of paternal half-sibs of the chickens which were used in the tail analysis, establishing genetic linkage between the DNA marker and a genetic locus affecting abdominal fat deposition.