Microsatellite variation in the equine MHC

Genes within the major histocompatibility complex (MHC) encode proteins involved in innate and adaptive immune responses. Genetic variation in this region can influence the immune response of an individual animal to challenges from a variety of pathogens; however, a complete documentation of genetic variation in the MHC is lacking for most domestic animals, including horses. To provide additional genetic markers for study of the horse MHC, or ELA (equine lymphocyte antigen), we identified 37 polymorphic microsatellite repeats in ELA and used these variations separately and together with published SNPs to investigate linkage disequilibrium (LD) and haplotype structure in a sample of Thoroughbred horses. ELA SNPs alone detected little LD, but microsatellites, either separately or combined with SNPs, revealed substantially more LD. A subset of markers in very high LD across the breadth of ELA may be predictive of structural polymorphisms or linked epistases that are important drivers of haplotype structure in Thoroughbreds.     

Rapid loss of MHC class II variation in a bottlenecked population is explained by drift and loss of copy number variation

Population bottlenecks may reduce genetic variation and potentially increase the risk of extinction. Here, we present the first study to use historic samples to analyse loss of variation at the major histocompatibility complex (MHC), which plays a central role in vertebrate disease resistance. Balancing selection acts on the MHC and could moderate the loss of variation expected from drift; however, in a Wisconsin population of greater prairie-chickens (Tympanuchus cupido), the number of MHC class II B alleles per individual declined by 44% following a population bottleneck, compared to a loss of only 8% at microsatellites. Simulations indicate that drift likely reduced MHC variation at the population level, as well as within individuals by reducing the number of gene copies per individual or by fixing the same alleles across multiple loci. These multiple effects of genetic drift on MHC variation could have important implications for immunity and fitness.     

Reduced MHC variation in a threatened tuatara species

The relationship between neutral and adaptive genetic diversity is important to understand in assessing the implications of a population bottleneck. Fitness-related genes, such as those of the major histocompatibility complex (MHC), may be influenced by selection, and so retain diversity even when it is lost at neutral markers. We measured MHC class I variation in an archaic reptile species Sphenodon guntheri [North Brother Island (NBI) tuatara], which naturally occurs on one 4 ha island in Cook Strait, New Zealand, and has low levels of microsatellite diversity. MHC variation in S. guntheri was compared with microsatellite DNA variation, and with MHC variation in a large population of Sphenodon punctatus (Cook Strait tuatara) on Stephens Island. The NBI population shows significantly decreased levels of genetic diversity compared with the Stephens Island population. Only three different MHC sequences and three genotypes were found on NBI, compared with 15 sequences and 21 genotypes in a similar sample size from Stephens Island. Two sequences appear to be unique to the NBI population. The assortment of sequence variants into genotypes suggests strong gametic disequilibrium between two MHC class I loci in S. guntheri , and only two haplotypes that were present in Hardy–Weinberg proportions were identified. MHC diversity in NBI tuatara appears to be largely influenced by genetic drift, consistent with a recent population bottleneck. This may compromise the ability of this population to respond to novel disease threats.     

Balancing selection on MHC class I in wild brown trout Salmo trutta

Evidence is reported for balancing selection acting on variation at major histocompatibility complex (MHC) in wild populations of brown trout Salmo trutta. First, variation at an MHC class I (satr-uba)-linked microsatellite locus (mhc1) is retained in small S. trutta populations isolated above waterfalls although variation is lost at neutral microsatellite markers. Second, populations across several catchments are less differentiated at mhc1 than at neutral markers, as predicted by theory. The population structure of these fish was also elucidated.     

Family‐assisted inference of the genetic architecture of major histocompatibility complex variation

With their direct link to individual fitness, genes of the major histocompatibility complex (MHC) are a popular system to study the evolution of adaptive genetic diversity. However, owing to the highly dynamic evolution of the MHC region, the isolation, characterization and genotyping of MHC genes remain a major challenge. While high‐throughput sequencing technologies now provide unprecedented resolution of the high allelic diversity observed at the MHC, in many species, it remains unclear (i) how alleles are distributed among MHC loci, (ii) whether MHC loci are linked or segregate independently and (iii) how much copy number variation (CNV) can be observed for MHC genes in natural populations. Here, we show that the study of allele segregation patterns within families can provide significant insights in this context. We sequenced two MHC class I (MHC‐I) loci in 1267 European barn owls (Tyto alba), including 590 offspring from 130 families using Illumina MiSeq technology. Coupled with a high per‐individual sequencing coverage (~3000×), the study of allele segregation patterns within families provided information on three aspects of the architecture of MHC‐I variation in barn owls: (i) extensive sharing of alleles among loci, (ii) strong linkage of MHC‐I loci indicating tandem architecture and (iii) the presence of CNV in the barn owl MHC‐I. We conclude that the additional information that can be gained from high‐coverage amplicon sequencing by investigating allele segregation patterns in families not only helps improving the accuracy of MHC genotyping, but also contributes towards enhanced analyses in the context of MHC evolutionary ecology.     

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.     

MHC class I variation associates with parasite resistance and longevity in tropical pythons

Using restriction fragment length polymorphism (RFLP) we identified 26 unique major histocompatibility complex (MHC) genotypes in 104 water pythons. We observed a significant independent association between reduced blood parasite load (Hepatozoon sp.) and python body length/age, presence of a specific RFLP fragment (C-fragment) and the overall number of fragments. The parasite has a negative impact on several python life-history traits such as growth, nutritional status and longevity. Thus, the C-fragment could be considered a 'good gene' (a fitness-enhancing genetic element). However, while the number of fragments affected parasite load, the association between level of parasitaemia and fragment number was not linear, and, hence, minimum parasite infection level was achieved at an intermediate number of fragments. Intermediate MHC fragment numbers were also observed among the largest/oldest pythons, suggesting that both a specific fragment and intermediate levels of MHC polymorphism enhanced python longevity. Thus, our results suggest python MHC is subject to both frequency-dependent and balancing selection.     

PCR‐based isolation of multigene families: lessons from the avian MHC class IIB

The amount of sequence data available today highly facilitates the access to genes from many gene families. Primers amplifying the desired genes over a range of species are readily obtained by aligning conserved gene regions, and laborious gene isolation procedures can often be replaced by quicker PCR‐based approaches. However, in the case of multigene families, PCR‐based approaches bear the often ignored risk of incomplete isolation of family members. This problem is most prominent in gene families with highly variable and thus unpredictable number of gene copies among species, such as in the major histocompatibility complex (MHC). In this study, we (i) report new primers for the isolation of the MHC class IIB (MHCIIB) gene family in birds and (ii) share our experience with isolating MHCIIB genes from an unprecedented number of avian species from all over the avian phylogeny. We report important and usually underappreciated problems encountered during PCR‐based multigene family isolation and provide a collection of measures to help significantly improving the chance of successfully isolating complete multigene families using PCR‐based approaches.     

MHC diversity, malaria and lifetime reproductive success in collared flycatchers

Major histocompatibility complex (MHC) genes encode proteins involved in the recognition of parasite-derived antigens. Their extreme polymorphism is presumed to be driven by co-evolution with parasites. Host-parasite co-evolution was also hypothesized to optimize within-individual MHC diversity at the intermediate level. Here, we use unique data on lifetime reproductive success (LRS) of female collared flycatchers to test whether LRS is associated with within-individual MHC class II diversity. We also examined the association between MHC and infection with avian malaria. Using 454 sequencing, we found that individual flycatchers carry between 3 and 23 functional MHC class II B alleles. Predictions of the optimality hypothesis were not confirmed by our data as the prevalence of blood parasites decreased with functional MHC diversity. Furthermore, we did not find evidence for an association between MHC diversity and LRS.     

MHC standing genetic variation and pathogen resistance in wild Atlantic salmon

Pathogens are increasingly emerging in human-altered environments as a serious threat to biodiversity. In this context of rapid environmental changes, improving our knowledge on the interaction between ecology and evolution is critical. The objective of this study was to evaluate the influence of an immunocompetence gene, the major histocompatibility complex (MHC) class IIβ, on the pathogen infection levels in wild Atlantic salmon populations, Salmo salar, and identify selective agents involved in contemporary coevolution. MHC variability and bacterial infection rate were determined throughout the summer in juvenile salmon from six rivers belonging to different genetic and ecological regions in Québec, Canada. A total of 13 different pathogens were identified in kidney by DNA sequence analysis, including a predominant myxozoa, most probably recently introduced in North America. Infection rates were the highest in southern rivers at the beginning of the summer (average 47.6±6.3% infected fish). One MHC allele conferred a 2.9 times greater chance of being resistant to myxozoa, while another allele increased susceptibility by 3.4 times. The decrease in frequency of the susceptibility allele but not other MHC or microsatellite alleles during summer was suggestive of a mortality event from myxozoa infection. These results supported the hypothesis of pathogen-driven selection in the wild by means of frequency-dependent selection or change in selection through time and space rather than heterozygous advantage, and underline the importance of MHC standing genetic variation for facing pathogens in a changing environment.     

Microsatellite and major histocompatibility complex variation in an endangered rattlesnake,the Eastern Massasauga (Sistrurus catenatus)

Genetic diversity is fundamental to maintaining the long‐term viability of populations, yet reduced genetic variation is often associated with small, isolated populations. To examine the relationship between demography and genetic variation, variation at hypervariable loci (e.g., microsatellite DNA loci) is often measured. However, these loci are selectively neutral (or near neutral) and may not accurately reflect genomewide variation. Variation at functional trait loci, such as the major histocompatibility complex (MHC), can provide a better assessment of adaptive genetic variation in fragmented populations. We compared patterns of microsatellite and MHC variation across three Eastern Massasauga (Sistrurus catenatus) populations representing a gradient of demographic histories to assess the relative roles of natural selection and genetic drift. Using 454 deep amplicon sequencing, we identified 24 putatively functional MHC IIB exon 2 alleles belonging to a minimum of six loci. Analysis of synonymous and nonsynonymous substitution rates provided evidence of historical positive selection at the nucleotide level, and Tajima's D provided support for balancing selection in each population. As predicted, estimates of microsatellite allelic richness, observed, heterozygosity, and expected heterozygosity varied among populations in a pattern qualitatively consistent with demographic history and abundance. While MHC allelic richness at the population and individual levels revealed similar trends, MHC nucleotide diversity was unexpectedly high in the smallest population. Overall, these results suggest that genetic variation in the Eastern Massasauga populations in Illinois has been shaped by multiple evolutionary mechanisms. Thus, conservation efforts should consider both neutral and functional genetic variation when managing captive and wild Eastern Massasauga populations.     

Coevolutionary relationship between helminth diversity and MHC class II polymorphism in rodents

Parasite-mediated selection on major histocompatibility complex (MHC) genes has mainly been explored at the intraspecific level, although many molecular studies have revealed trans-species polymorphism. Interspecific patterns of MHC diversity might reveal factors responsible for the long-term evolution of MHC polymorphism. We hypothesize that host taxa harbouring high parasite diversity should exhibit high levels of MHC genetic diversity. We test this assumption using data on rodent species and their helminth parasites compiled from the literature. Controlling for similarity due to common descent, we present evidence indicating that high helminth species richness in rodent species is associated with increased MHC class II polymorphism. Our results are consistent with the idea that parasites sharing a long-term coevolutionary history with their hosts are the agents of selection explaining MHC polymorphism.     

Methods for MHC genotyping in non-model vertebrates

Genes of the major histocompatibility complex (MHC) are considered a paradigm of adaptive evolution at the molecular level and as such are frequently investigated by evolutionary biologists and ecologists. Accurate genotyping is essential for understanding of the role that MHC variation plays in natural populations, but may be extremely challenging. Here, I discuss the DNA-based methods currently used for genotyping MHC in non-model vertebrates, as well as techniques likely to find widespread use in the future. I also highlight the aspects of MHC structure that are relevant for genotyping, and detail the challenges posed by the complex genomic organization and high sequence variation of MHC loci. Special emphasis is placed on designing appropriate PCR primers, accounting for artefacts and the problem of genotyping alleles from multiple, co-amplifying loci, a strategy which is frequently necessary due to the structure of the MHC. The suitability of typing techniques is compared in various research situations, strategies for efficient genotyping are discussed and areas of likely progress in future are identified. This review addresses the well established typing methods such as the Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Reference Strand Conformational Analysis (RSCA) and cloning of PCR products. In addition, it includes the intriguing possibility of direct amplicon sequencing followed by the computational inference of alleles and also next generation sequencing (NGS) technologies; the latter technique may, in the future, find widespread use in typing complex multilocus MHC systems.     

MHC及其在种群遗传学和保护遗传学中的应用

主要组织相容性复合体（major histocompatibility complex,MHC）是脊椎动物体内与免疫应答调节密切相关的一个基因家族,是基因组中多态性最丰富的区域。通过MHC的遗传变异分析可以提供物种的遗传多样性水平、进化历史和种群动态,以及种群遗传结构等信息,并在濒危物种饲养繁殖种群的遗传管理中有重要应用。 MHC and Its Application in the Population and Conservation Genetics YANG Guang,CHEN Xu-yan,REN Wen-hua,YAN Jie Institute of Genetic Resources,Nanjing Normal University,Nanjing 210097,China Abstract:The major histocompatibility complex (MHC),with the highest genetic polymorphism,is a cluster of genes involved in immune response regulation in the vertebrates.MHC can provide information such as population genetic diversity,evolutionary history and population dynamics,and population genetic structure etc.It can also be applied in the captive breeding programme for endangered vertebrate species. Key words：major histocompatibility complex (MHC);genetic diversity,population viability;population genetic structure;captive breeding     

Disentangling the roles of natural selection and genetic drift in shaping variation at MHC immunity genes

The major histocompatibility complex (MHC) forms an integral component of the vertebrate immune response and, due to strong selection pressures, is one of the most polymorphic regions of the entire genome. Despite over 15 years of research, empirical studies offer highly contradictory explanations of the relative roles of different evolutionary forces, selection and genetic drift, acting on MHC genes during population bottlenecks. Here, we take a meta-analytical approach to quantify the results of studies into the effects of bottlenecks on MHC polymorphism. We show that the consequences of selection acting on MHC loci prior to a bottleneck event, combined with drift during the bottleneck, will result in overall loss of MHC polymorphism that is ～15% greater than loss of neutral genetic diversity. These results are counter to general expectations that selection should maintain MHC polymorphism, but do agree with the results of recent simulation models and at least two empirical studies. Notably, our results suggest that negative frequency-dependent selection could be more important than overdominance for maintaining high MHC polymorphism in pre-bottlenecked populations.     

Perspective: detecting adaptive molecular polymorphism: lessons from the MHC

Abstract. In the 1960s, when population geneticists first began to collect data on the amount of genetic variation in natural populations, balancing selection was invoked as a possible explanation for how such high levels of molecular variation are maintained. However, the predictions of the neutral theory of molecular evolution have since become the standard by which cases of balancing selection may be inferred. Here we review the evidence for balancing selection acting on the major histocompatibility complex (MHC) of vertebrates, a genetic system that defies many of the predictions of neutrality. We apply many widely used tests of neutrality to MHC data as a benchmark for assessing the power of these tests. These tests can be categorized as detecting selection in the current generation, over the history of populations, or over the histories of species. We find that selection is not detectable in MHC datasets in every generation, population, or every evolutionary lineage. This suggests either that selection on the MHC is heterogeneous or that many of the current neutrality tests lack sufficient power to detect the selection consistently. Additionally, we identify a potential inference problem associated with several tests of neutrality. We demonstrate that the signals of selection may be generated in a relatively short period of microevolutionary time, yet these signals may take exceptionally long periods of time to be erased in the absence of selection. This is especially true for the neutrality test based on the ratio of nonsynonymous to synonymous substitutions. Inference of the nature of the selection events that create such signals should be approached with caution. However, a combination of tests on different time scales may overcome such problems.     

MHC heterozygosity and survival in red junglefowl

Genes of the major histocompatibility complex (MHC) form a vital part of the vertebrate immune system and play a major role in pathogen resistance. The extremely high levels of polymorphism observed at the MHC are hypothesised to be driven by pathogen‐mediated selection. Although the exact nature of selection remains unclear, three main hypotheses have been put forward; heterozygote advantage, negative frequency‐dependence and fluctuating selection. Here, we report the effects of MHC genotype on survival in a cohort of semi‐natural red junglefowl (Gallus gallus) that suffered severe mortality as a result of an outbreak of the disease coccidiosis. The cohort was followed from hatching until 250 days of age, approximately the age of sexual maturity in this species, during which time over 80% of the birds died. We show that on average birds with MHC heterozygote genotypes survived infection longer than homozygotes and that this effect was independent of genome‐wide heterozygosity, estimated across microsatellite loci. This MHC effect appeared to be caused by a single susceptible haplotype (CD_c) the effect of which was masked in all heterozygote genotypes by other dominant haplotypes. The CD_c homozygous genotype had lower survival than all other genotypes, but CD_c heterozygous genotypes had survival probabilities equal to the most resistant homozygote genotype. Importantly, no heterozygotes conferred greater resistance than the most resistant homozygote genotype, indicating that the observed survival advantage of MHC heterozygotes was the product of dominant, rather than overdominant processes. This pattern and effect of MHC diversity in our population could reflect the processes ongoing in similarly small, fragmented natural populations.     

Organization and polymorphism of bovine major histocompatibility complex class II genes as revealed by genomic hybridizations with bovine probes

Previous studies on restriction fragment length polymorphism of bovine major histocompatibility complex class II genes have primarily been based on the use of human probes. In the present study bovine probes for DQA, DQB, DRB and DYA were used for RFLP analysis of cattle genomic DNA digested with PvuII and TaqI. There was an excellent agreement between the RFLP results obtained with homologous and heterologous probes. Although a few 'new' restriction fragments were revealed with the bovine probes there was no discrepancy with regard to the classification of allelic types with the two types of probes. The major advantages of using bovine probes were a better hybridization signal and reduced cross-hybridization between loci. Hybridization experiments with DQA probes for the first domain exon from two different genomic clones revealed the presence of two distinct types of bovine DQA genes. Surprisingly, these probes did not cross-hybridize at high stringency, indicating that the two genes are quite divergent. Hybridization with a recently described genomic clone for a novel bovine alpha-chain gene confirmed that it corresponds to the DYA gene which had previously been identified by cross-hybridization to a human DQA probe.     

Restriction fragment length polymorphism of DQ and DR class II genes of the bovine major histocompatibility complex

DQ alpha, DQ beta, DR alpha and DR beta class II genes of the bovine major histocompatibility complex (MHC) were investigated by Southern blot hybridizations using human probes. Hybridizations of these probes to genomic DNA, digested with PvuII or TaqI, revealed extensive restriction fragment length polymorphisms (RFLPs). The polymorphisms were interpreted genetically by analysing a family material, comprising five sires, 48 dams and 50 offspring, and a population sample comprising 197 breeding bulls. The analysis resolved 20 DQ alpha, 17 DQ beta, 5 DR alpha and 25 DR beta RFLP types. The segregation data were consistent with simple Mendelian inheritance of the RFLPs. The analysis of the bull sample showed that it is possible to apply the RFLP method for routine typing of class II polymorphism in population samples. The linkage disequilibrium in the DQ-DR region was found to be extremely strong as only about 20 DQ and about 30 DQ-DR haplotypes were observed despite the large number of possible haplotypes. Close linkage to the blood group locus M was also found; the M' allele occurred in strong linkage disequilibrium with the class II haplotype DQ1BDR alpha 4DR beta 1B. A population genetic analysis of the DQ data in the sample of breeding bulls revealed that the frequency of homozygotes was significantly lower than Hardy-Weinberg expectation and that the allele frequency distribution deviated significantly from the one expected for selectively neutral alleles.