GENETICS AND GENOMICS

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1 GENETICS AND GENOMICS MHC-B variability within the Finnish Landrace chicken conservation program J. E. Fulton,,1 M. E. Berres, J. Kantanen,, and M. Honkatukia Hy-Line International, Dallas Center, IA; University of Wisconsin, Madison, WI; Green Technology, Natural Resources Institute Finland (LUKE), FI Jokioinen, Finland; and Department of Environmental and Biological Sciences, University of Eastern Finland, PO Box 1627, FI Kuopio, Finland ABSTRACT The major histocompatibility complex maintained by a network of volunteer hobbyist breeders. (MHC) is a cluster of genes involved with immune responses. The chicken MHC has been shown to influence resistance to viruses, bacteria, and infections from both internal and external parasites. The highly variable chicken MHC haplotypes were initially identified by the use of haplotype-specific serological reagents. A novel SNP-based panel encompassing 210,000 bp of the MHC-B locus was developed to allow fine scale genetic analyses including rapid identification of novel haplotypes for which serological reagents are not available. The Finnish Landrace breed of chickens traces its origins to almost 1,000 years ago, with multiple lineages maintained as small populations in isolated villages. The breed is well adapted to the cooler Finnish climate and is considered to be an infrequent egg layer. Conservation efforts to protect this endangered breed were initiated by a hobby breeder in the 1960s. An official conservation program was established in 1998 and now 12 different populations are currently Variation in the MHC-B region in these popu- lations was examined using a panel of 90 selected SNP. A total of 195 samples from 12 distinct populations (average of 15 individuals sampled per population) were genotyped with the 90 SNP panel specific for the MHC-B region, spanning 210,000 bp. There were 36 haplotypes found, 16 of which are a subset of 78 that had been previously identified in either commercially utilized or heritage breeds from North America with the remaining 20 haplotypes being novel. The average number of MHC-B haplotypes found within each Finnish Landrace population was 5.9, and ranged from one to 13. While haplotypes common to multiple populations were found, populationspecific haplotypes were also identified. This study shows that substantial MHC-B region diversity exists in the Finnish Landrace breed and exemplifies the significance tied to conserving multiple populations of rare breeds. Key words: Finnish Landrace breed, MHC-B variability, SNP genotype, haplotype 2017 Poultry Science 96: INTRODUCTION Archaeological evidence suggests that chickens first appeared in Finland almost 1000 years ago, during the Iron Age (Bläuer, 2015). Since then, other chicken breeds were introduced into the ancestral population. Historically, these chickens were kept only by those with elevated socio-economic status. Over time, chickens proliferated throughout the region and eventually were established for family consumption on almost every farm. During this time, they adapted to free-range and modest living conditions in a cold and northern climate with very little nutritional supplementation, even in winter. Despite the ubiquity of chickens C 2017 Poultry Science Association Inc. Received November 28, Accepted April 5, Corresponding author: jfulton@hyline.com throughout Finland, the physical isolation of human villages contributed to the formation of isolated, genetically distinct populations, collectively now known as the Finnish Landrace. Today, only remnants of the Finnish Landrace chicken breed exist. Similar to the situation in the United States and Western Europe, the locally developed and adapted breeds were replaced by highly selected and efficient meat and egg producing breeds during the 1950s. The remaining Finnish Landrace breeds persisted in several isolated populations found only in remote villages. Specific populations are typically named for the town or geographic region where the chickens were found. In general, no artificial selection is applied to the Finnish Landrace breeds, except perhaps for darker color plumage or grey colored legs. Egg production is very modest compared to commercial egg production breeds, producing an egg every second or third day, perhaps an adaptation to limited nutrient 3026

2 MHC-B VARIABILITY WITHIN FINNISH LANDRACE BREED 3027 availability. A high percentage of hens exhibit broody behaviors. Historical physiological and environmental stressors have shaped the few remaining Finnish Landrace breeds into very active, robust breeds with limited husbandry requirements. These same conditions may have contributed to enhanced disease resistance. The contemporary conservation program, established in 1998, aims to insure breed purity by maintaining genetic and phenotypic diversity of the Finnish Landrace chicken. Instead of having a centralized conservation program (e.g., zoological park, or university), the Finnish conservation program relies entirely on contributions by public participants. Coordinated by Natural Resources Institute, Finland, a voluntary network of almost 400 non-professional breeders maintains localized populations of the Finnish Landrace breeds. The ongoing program also actively encourages new participants. Upon discovery, each flock is evaluated based on known history and phenotypic characteristics. The Finnish Landrace breed has previously been included within two chicken diversity studies using neutral genetic markers (microsatellites) (Vanhala et al., 1998; Hillel et al., 2003), but not with studies involving variation with known influence on population survival and balancing selection such as the major histocompatibility complex (MHC). The chicken MHC encompasses two clusters (MHC-B and MHC-Y) of highly polymorphic immune response genes located on chromosome. MHC-B haplotypes have been well studied and specific haplotypes have been shown to provide enhanced resistance to multiple viral and bacterial pathogens including Marek s Disease Virus (Hansen et al., 1967; Briles et al., 1977), Rous Sarcoma Virus (Taylor et al., 1992), Salmonella enteritis (Cotter et al., 1998), and Pasteurella multicida (Lamont et al., 1987). MHC-B has also been shown to influence the severity of parasitic infections such as Eimeria acervulina (Lillehoj et al., 1988), Ascaridia galli (Schou et al., 2010), and Northern Fowl Mite (Ornithonyssus sylviarum) (Owens et al., 2008). While specific haplotypes have been shown to exhibit significant differential responses to particular diseases, the overall MHC heterogeneity of a flock is also important. Thus variability within the MHC can have a significant influence on overall health and robustness of a population of chicken populations. Historically, MHC-B haplotypes were identified by the use of alloantisera (Briles and Briles, 1982). DNAbased technologies including Southern blots (Miller et al., 1988; Iglesias et al., 2004), single-stranded conformation polymorphisms (Goto et al., 2002; Iglesias et al., 2004) and microsatellites (Lima-Rosa et al., 2005; Fulton et al., 2006) have also been used to identify MHC- B variability. Recently a 90 SNP panel encompassing 210,000 nucleotides of the chicken MHC-B has been described (Fulton et al., 2016a). This rapid and relatively inexpensive methodology allows for identification of MHC-B polymorphism in large numbers of samples and has been used successfully to examine MHC-B variability in commercially utilized egg layer breeds and university held heritage breeds (Fulton et al., 2016a, 2016b). These studies have identified 78 diverse MHC- B haplotypes, many of them not previously identified. This same MHC-B SNP panel was utilized with samples from wild Red Jungle Fowl, revealing considerable MHC-B diversity within Vietnamese wild populations not subjected to artificial selection (Nguyen-Phuc et al., 2016). The goal of our current research is to characterize the diversity of the MHC-B region within twelve known Finnish Landrace populations utilizing the 90 SNP panel. Achievement of this goal will serve to provide a genetically-based criterion useful for conservation management. MATERIALS AND METHODS Twelve distinct populations of Finnish Landrace chickens were sampled in 2012, with one population (Jussila) having two subpopulations. The number of samples per population averaged 15, with a range from 7 to 27 and a total of 195 samples. Blood samples (approximately 2 ml) were obtained from the wing vein with a 21 ga, 1.5 in needle. Blood was stored at 20 C in sodium heparin tubes. One drop of thawed blood was transferred to FTA Elute Micro Card (GE Healthcare, Piscataway, NJ) and allowed to dry. DNA was extracted from the dried blood spot on the FTA card following the manufacturer s instructions. The MHC-B SNP panel used was previously described by Fulton et al., 2016b. The genotyping done herein utilized a subset of 90 SNPs (from MHCJ6 through MHC178, which encompasses genes LOC through CD1A1 as defined by Shiina et al., 2007, GenBank AB268588), omitting those SNP that may involve a gene deletion/duplication event. Genotypes at each SNP site were determined using allele specific PCR assays with the fluorescence based Kompetitive Allele-Specific PCR (KASP) chemistry (Semagn et al., 2013), and results were analyzed with Kraken software (LGC, Hoddesdon, UK) as described by Fulton et al 2016b. Haplotypes were determined within each population using a two-step process with visualization of genotypes. First, individuals that were homozygous for all 90 SNP were used to define the initial set of haplotypes. Then, individuals that were heterozygous for multiple SNP were compared to the homozygous haplotype set and the alternate haplotype within each heterozygote was identified. Haplotypes found in all 12 populations were compared and common haplotypes identified. All haplotypes found within the Finnish Landrace populations were then compared with those previously reported in heritage breeds and commercially utilized egg layer elite lines (Fulton et al., 2016a, 2016b). RESULTS AND DISCUSSION Table 1 lists the populations tested, the number of samples per population, and the number of haplo-

3 3028 FULTON ET AL. Table 1. Finnish Landrace populations tested, the sample size, and number of haplotypes found. Population No. samples No. haplotypes Previous Novel Alho Häme Hornio Iitti Ilmajoki Jussila/Jämijärvi Jussila/Lepola Kiuruvesi Lindell Luumäki Piikkiö Savitaipale Tyrnävä types found within each population. Haplotype number averaged 5.9 per population, and ranged from a single haplotype found in the Jussila/Lepola subpopulation to 13 found in the Kiuruvesi population. Many of the haplotypes were found in multiple populations and overall, these Finnish Landrace populations contained a total of 36 different haplotypes. Comparison of the SNP haplotypes found within the Finnish Landrace populations with those previously reported from multiple commercial and heritage breeds in North America (Fulton et al., 2016a, 2016b) showed that while 16 of these haplotypes had been previously identified in commercial egg layer populations and heritage breeds there were 20 haplotypes that were novel. The 16 previously identified haplotypes found in the Finnish Landrace populations are summarized in Table 2. The most frequently found haplotypes were BSNP-K03 (8 occurrences) which had been previously found in broilers and White Leghorns, BSNP-B03 (6 occurrences) which had been found previously in broilers, New Hampshire, Rhode Island Red, and White Plymouth Rock and BNSP-L01 (5 occurrences) previously reported in broilers and White Leghorns (Fulton et al., 2016b). These previously defined haplotypes had been identified in a variety of breeds and populations within North America, suggesting some historic commonality of the Finnish Landrace breed with white shell egg layers, dual purpose, and heavier meat breeds (i.e., the original breeds used to develop the modern high yield commercial broiler). Table 3 defines the 20 novel haplotypes found in the Finnish Landrace populations and indicates within which populations these haplotypes were found. Provisional haplotype names were provided to distinguish among each haplotype. The haplotype BSNP-Fin11, found only in the Iitti population was identical to a haplotype recently identified within the Sebright breed (Taylor et al., 2016). The haplotype BSNP-Fin15 found only in the Kiuruvesi population is identical to a haplotype found in a Red Jungle fowl zoo population (Fulton, unpublished). Only one population (Jussila/Lepola) did not contain any previously identified haplotypes and this population was homozygous for a novel haplotype (BSNP-Fin04) that was found also in Jussila/Jämijärvi and Häme. Each population was examined in detail to determine the presence of potential recombinant haplotypes. Fulton et al. (2016b) had applied a very stringent requirement that both parental haplotypes must be found within the population to definitively define a novel haplotype as recombinant. Using this same criterion, there were no unequivocal recombinants identified within any of the populations. However, there were several examples where a novel haplotype had large segments of SNP identity with another haplotype. These may have been derived from a recombination event, and the other parental allele was either lost since the initial recombination event or may simply have not been sampled. Specific examples are present in the Häme population. Haplotype BSNP-Fin04 is identical to BSNP-K03 from SNPMHC-91 to SNPMHC-178 (encompassing 115,000 bp of the MHC-B), and haplotype BSNP-Fin07 is identical to BSNP-V03 from SNPMHC- Table 2. Haplotypes originally found in other breeds and their presence in the Finnish Landrace populations. BSNP Type Source & Alho Häme Hornio Iitti Ilmajoki Jussila/Jämijärvi Kiuruvesi Lindell Luumäki Piikkiö Savitaipale Tyrnävä BSNP-A04 WL + BSNP-A08 RIR, WPR + BSNP-B02 BPR, BRL, RIR + BSNP-B03 BRL, NH, RIR, WPR BSNP-D04 BRL, WL BSNP-I01 NH + + BSNP-J06 BPR, BRL, NH, WPR + BSNP-K03 BRL, WL BSNP-K05 LS + BSNP-L01 BPR, WL BSNP-M01 RIR, WPR + + BSNP-O01 DBL + BSNP-O02 NH, RIR BSNP-O03 BRL, NH, RIR, WL BSNP-T04 BPR, BRL, NH, WPR + + BSNP-V03 BRL & Breed source identified in Fulton et al., 2016b. BPR=Barred Plymouth Rock; BRL=Broiler; DBL=Dark Brown Leghorn; LS=Light Sussex; NH=New Hampshire. RIR=Rhode Island Red; WL=White Leghorn; WPR=White Plymouth Rock.

4 MHC-B VARIABILITY WITHIN FINNISH LANDRACE BREED 3029 Table 3. Novel haplotypes found in Finnish Landrace chicken populations. 25 to SNPMHC-178 (encompassing 173,000 bp of the MHC-B). This sampling of the MHC-B variation contained within multiple subpopulations of Finnish Landrace chickens has been informative. Commonality of MHC-B haplotypes was seen across the 12 populations but also there were numerous unique haplotypes at the population level. This indicates that there is considerable diversity within the MHC-B region of the Finnish Landrace breed and that the conservation efforts have been successful in maintaining the diversity. Genetic diversity analyses utilizing neutral allele information from the rest of the genome is in progress to determine the overall diversity of this unique landrace. From a species conservation perspective, endemic breeds like the Finnish Landrace deserve special consideration given their irreplaceability as a breed resulting from a unique, predominantly natural selection process. Low genetic diversity has been repeatedly implicated in increasing extinction risk and lowering evolutionary potential in wild populations of animals. Considering the potential effects of a changing climate and the predicted increase in catastrophic weather events and spread of novel pathogens, species with low genetic diversity may have increased difficulty in withstanding these perturbations. No evidence exists to exclude domestic lines from these phenomena, which in fact may be at even greater risk (Fulton and Delany, 2003). Efforts by the Natural Resources Institute Finland are incorporating important long-term populationlevel genetic conservation techniques into their program. Maintenance of small captive populations distributed across multiple locations also provides more immediate and valuable biosecurity protection. Continued preservation of this landrace should be encouraged as current populations could provide the foundation for a genetic rescue, should one be required. Establishing genetically informed conservation programs for other rare chicken breeds should also be promoted in advance of potential devastating population collapses. Future poultry genetic management depends on additional initiatives to further identify ancestral lineages, and resiliency to current environmental changes The isolated population structure and natural selection of a founding ancestral population has created a unique assemblage of Finnish Landrace populations and promoted diversification of the MHC-B locus. Conservation efforts made by the Natural Resources Institute Finland, which involves direct participation of small farm-holders, are broadly applicable and do not require significant up-front economic expenditures. This type of management plan provides a readily implemented means to sustain allelic diversity among the myriad of heritage breeds, not only in Finland but throughout the world. Not only will this insure the preservation of the Finnish Landrace and other unique breeds, but also offers access to a reservoir of unique genetic information currently unutilized by commercial poultry enterprises. We encourage and support efforts to further conserve and characterize evolutionary distinct chicken populations. ACKNOWLEDGMENTS The authors wish to acknowledge Miika Tapio from Natural Resources Institute Finland, Jokioinen, Finland for assistance in collection of samples. All samples were obtained following approval by the Animal Experiment Board of LUKE. REFERENCES Bläuer, A Voita, villaa ja vetoeläimiä [English translation; Butter, Wool and Draft Animals] ISBN Briles, W. E., and R. W. Briles Identification of haplotypes of the chicken Major Histocompatibility Complex (B). Immunogenetics 15: Briles, W. E., H. A. Stone, and R. K. Cole Marek s disease: effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science 165: Cotter, P. F., R. L. Taylor, Jr., and H. Abplanalp B-complex associated immunity to Salmonella enteritidis challenge in congenic chickens. Poult. Sci. 77: Fulton, J. E., and M. E. Delany Poultry Genetic Resourcesoperation rescue needed. Science 300: Fulton, J. E., H. R. Juul-Madsen, C. M. Ashwell, A. M. McCarron, J. A. Arthur, N. P. O Sullivan, and R. L. Taylor, Jr., Molecular genotype identification of the Gallus gallus major histocompatibility complex. Immunogenet. 58:

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