Genetic diversity of selected genes that are potentially economically important in feral sheep of New Zealand

  • Grant W McKenzie1,

    Affiliated with

    • Johanna Abbott2,

      Affiliated with

      • Huitong Zhou1,

        Affiliated with

        • Qian Fang1,

          Affiliated with

          • Norma Merrick1,

            Affiliated with

            • Rachel H Forrest3,

              Affiliated with

              • J Richard Sedcole1 and

                Affiliated with

                • Jonathan G Hickford1Email author

                  Affiliated with

                  Genetics Selection Evolution201042:43

                  DOI: 10.1186/1297-9686-42-43

                  Received: 1 March 2010

                  Accepted: 21 December 2010

                  Published: 21 December 2010

                  Abstract

                  Background

                  Feral sheep are considered to be a source of genetic variation that has been lost from their domestic counterparts through selection.

                  Methods

                  This study investigates variation in the genes KRTAP1-1, KRT33, ADRB3 and DQA2 in Merino-like feral sheep populations from New Zealand and its offshore islands. These genes have previously been shown to influence wool, lamb survival and animal health.

                  Results

                  All the genes were polymorphic, but no new allele was identified in the feral populations. In some of these populations, allele frequencies differed from those observed in commercial Merino sheep and other breeds found in New Zealand. Heterozygosity levels were comparable to those observed in other studies on feral sheep. Our results suggest that some of the feral populations may have been either inbred or outbred over the duration of their apparent isolation.

                  Conclusion

                  The variation described here allows us to draw some conclusions about the likely genetic origin of the populations and selective pressures that may have acted upon them, but they do not appear to be a source of new genetic material, at least for these four genes.

                  Background

                  It is thought that livestock genetic variation has decreased through breed substitution and crossing of local and global breeds [1]. Accordingly, interest in feral populations has increased because they are potential sources of genetic variation that may have been lost in commercial sheep flocks [2, 3]. It has been argued that reintroducing genetic variability could enhance production in commercial breeds [4].

                  New Zealand (NZ) has eleven feral sheep populations either on the mainland, or on offshore islands [5]. The mainland populations originated from farmed sheep [6], while those on offshore islands either originated from farms, or were liberated as a food source for mariners [7]. These populations have been described previously [1, 4, 6, 813].

                  In this study, the level of genetic variation of four genes was determined in order to ascertain whether the isolation of these flocks had preserved greater genetic diversity compared to their commercial counterparts in NZ. These four genes are located on three different chromosomes i.e. KRTAP1-1 (chromosome 11; a keratin-associated protein gene that encodes a protein KAP1-1 commonly found in wool), KRT33 (chromosome 11; encoding wool keratin K33), ADRB3 (chromosome 26; encoding the seven-transmembrane domain beta-3 adrenergic receptor ADRB3) and DQA2 (chromosome 20; encoding a class II major histocompatibility complex (MHC) protein DQA2).

                  Previous studies have reported that variations in the keratin and keratin-associated protein genes, including the ones above, influence many wool properties including fibre diameter [14], staple strength [15], mean staple length [16] and the brightness of wool [16]. Accordingly, given the wide phenotypic variation seen in the wool of feral sheep [6, 8], one might expect to see increased variation in these genes.

                  Neonatal lamb mortality, particularly in Merino sheep, represents a large loss to the NZ sheep industry. Allelic variation in ADRB3 has been associated with survival in various sheep breeds [17], thus it might be expected that previously reported or new alleles would be found at a higher frequency in feral populations routinely exposed to harsh environmental conditions.

                  It has been reported that feral sheep may have an increased resistance to a number of diseases. This resistance could imply that variation in key immune function genes such as the highly polymorphic MHC genes is important, as it plays a role in the immune response to pathogens and parasites [1821].

                  Collectively the four genes chosen here cover a variety of different animal traits that could be associated with variation in the ability to survive in remote and potentially more severe environments, and where feed availability was probably reduced relative to farmed sheep.

                  Materials and methods

                  Sheep and DNA sources

                  Ten feral flocks and two reference flocks (non-feral) were investigated in this study (Table 1). Genomic DNA from these sheep was obtained from whole blood collected on FTA Classic Cards (Whatman BioScience, Middlesex, UK) following the manufacturer's instructions. Reference flock allele frequencies (see Tables 2 and 3) were sourced from published data [17, 2224] and from NZ commercial sheep DNA samples stored at Lincoln University.
                  Table 1

                  Origin of sheep populations and sample numbers (N)

                  Flock type

                  Flock location

                  Origin/Breed/Type

                  N

                  Feral

                  Offshore

                  Arapawa Island I

                  Australia/Merino/unknown

                  17

                    

                  Arapawa Island II

                  Australia/Merino/unknown

                  61

                    

                  Chatham Island

                  Australia/Merinos/Saxon

                  22

                    

                  Pitt Island

                  Australia/Merinos/Saxon

                  519

                    

                  Campbell Island

                  Australia/Merino × longwool

                  105

                   

                  Mainland

                  Woodstock

                  Australia/Merino/unknown

                  31

                    

                  Hokonui I

                  Tasmania/Merino/Saxon

                  12

                    

                  Hokonui II

                  Tasmania/Merino/Saxon

                  73

                    

                  Herbert

                  Tasmania/Merino/unknown

                  24

                    

                  Mohaka

                  Unknown/Merino/unknown

                  14

                      

                  878

                  Domestic reference flocks

                  Mainland

                  Merino1

                  New Zealand/Merino/unknown

                  20 123

                    

                  All breeds2

                  Corriedale, Poll Dorset, Suffolk, Borderdale, Coopworth,

                  Dorset Down × Coopworth, Merino × Coopworth,

                  Merino × Polwarth, Merino, Polwarth, Dorset Down and Hampshire, NZ Romney, Awassi, Finnish Landrace and other NZ crossbred sheep

                  43 737

                  Table 2

                  Within-population sample sizes (N), number of alleles identified (n) and allele frequencies for KRTAP1-1, KRT33 and ADRB3

                  KRTAP1-1

                  Population

                  N

                  n

                  A

                  B

                  C

                       

                  Arapawa Island I

                  14

                  3

                  0.43a

                  0.46a

                  0.11bc

                       

                  Arapawa Island II

                  59

                  2

                  -

                  0.93c

                  0.07bc

                       

                  Chatham Island

                  22

                  2

                  0.18ab

                  0.82ab

                  -

                       

                  Pitt Island

                  477

                  2

                  -

                  0.85b

                  0.15a

                       

                  Campbell Island

                  97

                  3

                  0.02c

                  0.82b

                  0.16a

                       

                  Woodstock

                  28

                  3

                  0.29a

                  0.68ab

                  0.04b

                       

                  Hokonui I

                  11

                  2

                  -

                  0.82ab

                  0.18bc

                       

                  Hokonui II

                  65

                  2

                  -

                  0.88b

                  0.12bc

                       

                  Herbert

                  23

                  2

                  0.15ab

                  0.85b

                  -

                       

                  Mohaka

                  6

                  2

                  -

                  0.83ab

                  0.17bc

                       

                  Merino reference flock

                  795

                  3

                  0.23a

                  0.7a

                  0.07b

                       

                  All Breeds reference flock

                  309

                  3

                  0.06b

                  0.80b

                  0.14c

                       

                  KRT33

                  Population

                  N

                  n

                  A

                  B

                  C

                  D

                  E

                     

                  Arapawa Island I

                  13

                  5

                  0.04a

                  0.19ab

                   

                  0.46ac

                  0.19a

                     

                  Arapawa Island II

                  60

                  3

                  -

                  -

                  0.40a

                  0.23a

                  0.38b

                     

                  Chatham Island

                  22

                  5

                  0.32b

                  0.07a

                  0.16bc

                  0.23c

                  0.23a

                     

                  Pitt Island

                  471

                  4

                  0.04a

                  -

                  0.05c

                  0.43c

                  0.48b

                     

                  Campbell Island

                  92

                  5

                  0.02a

                  0.42b

                  0.01a

                  0.10b

                  0.45b

                     

                  Woodstock

                  30

                  5

                  0.07c

                  0.18c

                  0.30b

                  0.33c

                  0.12a

                     

                  Hokonui I

                  11

                  4

                  0.23bc

                  0.36b

                  -

                  0.23bc

                  0.18a

                     

                  Hokonui II

                  67

                  5

                  0.27b

                  0.28b

                  0.14b

                  0.21a

                  0.10a

                     

                  Herbert

                  24

                  5

                  0.13bc

                  0.35b

                  0.31b

                  0.19b

                  0.02c

                     

                  Mohaka

                  14

                  2

                  -

                  -

                  -

                  0.43c

                  0.57b

                     

                  Merino reference flock

                  739

                  5

                  0.26b

                  0.36b

                  0.19b

                  0.04b

                  0.15a

                     

                  All Breeds reference flock

                  967

                  5

                  0.08c

                  0.04a

                  0.05c

                  0.40c

                  0.43b

                     

                  ADRB3

                  Population

                  N

                  n

                  A 1

                  B 2

                  C 3

                  D 4

                  E 1

                  F 2

                  G 5

                  H 5

                  Arapawa Island I

                  17

                  4

                  0.32bc

                  -

                  0.35bc

                  -

                  0.24bc

                  0.09bc

                  -

                  -

                  Arapawa Island II

                  60

                  4

                  0.17a

                  -

                  0.04a

                  -

                  0.39a

                  0.40a

                  -

                  -

                  Chatham Island

                  22

                  4

                  0.27bc

                  -

                  0.16c

                  -

                  0.55a

                  0.02c

                  -

                  -

                  Pitt Island

                  499

                  6

                  0.20a

                  0.04a

                  0.23c

                  -

                  0.28a

                  0.25a

                  0.002a

                  -

                  Campbell Island

                  102

                  4

                  0.66a

                  0.17a

                  0.1c

                  -

                  -

                  0.01a

                  -

                  -

                  Woodstock

                  30

                  4

                  0.28bc

                  0.05bc

                  0.18c

                  -

                  -

                  0.48a

                  -

                  -

                  Hokonui I

                  11

                  3

                  0.73a

                  0.23bc

                  -

                  -

                  0.05a

                  -

                  -

                  -

                  Hokonui II

                  68

                  4

                  0.53a

                  0.29a

                  -

                  -

                  0.16bc

                  -

                  0.01a

                  -

                  Herbert

                  24

                  4

                  0.77a

                  0.04bc

                  0.17c

                  -

                  -

                  0.02b

                   

                  -

                  Mohaka

                  6

                  3

                  0.25bc

                  0.08bc

                  -

                  -

                  -

                  0.67a

                  -

                  -

                  Merino reference flock

                  4 484

                  6

                  0.35b

                  0.02b

                  0.33b

                  0.06

                  0.20b

                  0.05b

                  -

                  -

                  All Breeds reference flock

                  13 420

                  8

                  0.37c

                  0.09c

                  0.21c

                  0.02

                  0.20c

                  0.10c

                  0.01b

                  0.004

                  1-5 represent the effect of gene on cold survival based on the odd ratios reported in [17]: 1good survival; 2neutral survival; 3below average survival; 4poor survival, 5 data insufficient to determine the effect on survival; a-c allele frequency differences within columns that share no common alphabetic superscripts are significantly different (P < 0.05), while those pair wise comparisons that are not different are represented with the same superscripts; "-" represents alleles or data not available

                  Table 3

                  Within population sample sizes (N), number of alleles identified (n) and allele frequencies for DQA2 1

                  DQA2 alleles

                  Population

                  N

                  n

                  06023

                  0601

                  08011

                  0901

                  0103

                  1101

                  0102-1601

                  0101-1401

                  1201

                   

                  Arapawa Island I

                  17

                  8

                  0.18a

                  -

                  0.15a

                  -

                  0.06a

                  0.06a

                  -

                  -

                  0.03a

                   

                  Arapawa Island II

                  61

                  7

                  0.02a

                  0.02a

                  -

                  -

                  -

                  -

                  0.03a

                  0.17a

                  -

                   

                  Chatham Island

                  22

                  6

                  0.23a

                  -

                  -

                  0.05a

                  -

                  0.09a

                  -

                  -

                  -

                   

                  Pitt Island

                  519

                  13

                  0.07b

                  0.02b

                  -

                  0.11b

                  0.03b

                  0.02a

                  0.04a

                  0.14a

                  0.11b

                   

                  Campbell Island

                  105

                  9

                  -

                  0.01b

                  -

                  -

                  0.07a

                  0.17b

                  -

                  0.40b

                  -

                   

                  Woodstock

                  31

                  10

                  0.03b

                  -

                  -

                  -

                  0.05a

                  0.31b

                  -

                  -

                  0.24b

                   

                  Hokonui I

                  12

                  6

                  0.05a

                  -

                  0.13a

                  -

                  0.08a

                  -

                  -

                  0.63b

                  0.04b

                   

                  Hokonui II

                  73

                  9

                  0.07a

                  0.08a

                  0.21b

                  -

                  0.12a

                  0.13b

                  -

                  0.23a

                  0.11b

                   

                  Herbert

                  24

                  7

                  0.06a

                  -

                  0.35b

                  -

                  0.02b

                  0.08a

                  -

                  0.23a

                  -

                   

                  Mohaka

                  8

                  2

                  -

                  -

                  -

                  -

                  -

                  0.94b

                  -

                  0.06a

                  -

                   

                  Merino reference flock

                  20123

                  -

                  -

                  -

                  -

                  -

                  -

                  -

                  0.04a

                  -

                  -

                   

                  All breeds reference flock

                  43737

                  -

                  0.11a

                  0.04a

                  0.03a

                  0.08a

                  0.10a

                  0.01a

                  0.05a

                  0.15a

                  0.13b

                   

                  DQA2 alleles

                  Population

                    

                  08012-0201

                  0701-1401

                  0701-1301

                  0401-1501

                  0702-1401

                  0301

                  0501

                  0402-1701

                  0401-1601

                   

                  Arapawa Island I

                    

                  0.32a

                  -

                  -

                  -

                  0.09bc

                  -

                  0.12b

                  -

                  -

                   

                  Arapawa Island II

                    

                  0.11a

                  -

                  -

                  -

                  0.07bc

                  -

                  0.58a

                  -

                  -

                   

                  Chatham Island

                    

                  0.27a

                  -

                  -

                  -

                  0.23a

                  -

                  0.14b

                  -

                  -

                   

                  Pitt Island

                    

                  0.002c

                  -

                  -

                  -

                  0.33a

                  0.001a

                  0.01a

                  0.13a

                  -

                   

                  Campbell Island

                    

                  -

                  -

                  -

                  -

                  0.16a

                  0.17a

                  0.005a

                  0.02bc

                  0.005a

                   

                  Woodstock

                    

                  0.05bc

                  0.02a

                  0.02a

                  0.23a

                  0.03bc

                  -

                  -

                  0.03bc

                  -

                   

                  Hokonui I

                    

                  -

                  -

                  -

                  -

                  0.08bc

                  -

                  -

                  -

                  -

                   

                  Hokonui II

                    

                  -

                  -

                  -

                  0.01c

                  0.05bc

                  -

                  -

                  -

                  -

                   

                  Herbert

                    

                  0.19a

                  -

                  -

                  -

                  -

                  -

                  0.06b

                  -

                  -

                   

                  Mohaka

                    

                  -

                  -

                  -

                  -

                  -

                  -

                  -

                  -

                  -

                   

                  Merino Reference flock

                    

                  0.03b

                  -

                  0.001a

                  0.04b

                  0.03b

                  -

                  -

                  0.02b

                  0.05b

                   

                  All breeds reference flock

                    

                  0.003c

                  0.02a

                  0.002a

                  0.03c

                  0.02c

                  0.02b

                  0.06b

                  0.02c

                  0.16c

                   

                  1 DQA2 nomenclature [24]; a-c allele frequency differences within columns that share no common alphabetic superscripts are significantly different (P < 0.05), while the pair-wise comparisons that are not different are represented with different superscripts; "-" represents alleles or data not available

                  PCR amplification and genotyping

                  PCR amplifications and genotyping approaches were carried out using previously described methods [17, 2426]

                  Data analysis

                  Allele frequencies, number of alleles, observed heterozygosity (H O ), expected heterozygosity (H E with a Levene's correction) and coefficient of inbreeding (F IS) estimates based on the method of Weir and Cockerham [27] were determined using GENEPOP version 4.0.7 [28]. This software was also used to determine deviations from Hardy-Weinberg equilibrium (HWE) using the Exact Test with a Markov Chain Method [29] (10 batches, 5 000 iterations per batch and a dememorization number of 10 000). Corrections for multiple significance tests were performed using Fisher's method and by applying a sequential Bonferroni type correction [29]. F IS estimates were calculated across all the populations and genes (global F IS) and for individual populations and genes. Allelic richness, a measure of genetic diversity at a single locus, was determined using FSTAT version 2.9.3 [30] and included rarefaction to correct for sample size variation [31].

                  Allele frequencies for each feral population were compared to those of Merino sheep sourced from NZ commercial farms [17] and to the combined allele frequencies in breeds commonly found in NZ [17, 2224]. This was undertaken to determine which groups were more closely related to each other based on "distance" measured by the Pearson χ2-statistic for each possible pair of breeds and their respective estimated gene frequencies.

                  Results

                  All genes investigated in this study were polymorphic and allele frequencies for each gene varied among the studied flocks (Table 2 and 3). No new allele was identified for any of the genes in any of the sheep typed in this study. All the KRTAP1-1 alleles previously described were present in the feral sheep except allele A absent in four breeds, including one population from Arapawa Island (Hokonui sheep) and one from Pitt Island (Mohaka sheep), and allele C absent in the populations from Herbert Forest and Chatham Island.

                  Previous studies have reported five KRT33 alleles [25], all of which occurred in the feral populations. Alleles D and E were found in all the populations whereas alleles A and B were absent in the sheep from Arapawa Island and the Mohaka populations, allele C was absent in those from Mohaka and alleles B and C in those from the Pitt Island and Hokonui, respectively.

                  Six different ADRB3 alleles were detected in the feral sheep. The lowest diversity was observed in the Mohaka population with only three alleles while it was greatest in the sheep from Pitt Island with six alleles. It is interesting to note that alleles D and H, which occur at relatively low frequencies in other commercial breeds in NZ [17], were absent in all the feral populations. The frequency of allele G is low in NZ commercial sheep and was only found at a low frequency in the sheep from Pitt Island and Hokonui.

                  The distribution of DQA2 alleles varied considerably among populations with some alleles completely absent in some populations. The lowest diversity was observed in the sheep from Mohaka with only two DQA2 alleles. Conversely, thirteen DQA2 alleles were present in the sheep from Pitt Island.

                  For all four genes, in most cases allele frequencies in the feral populations differed significantly (p ≤ 0.025) from the frequencies in the reference flocks, most of the differences being highly significant (p < 0.001). The following exceptions were found: (1) frequencies of KRTAP1-1 alleles of sheep from Chatham Island (p = 0.113), Woodstock (p = 0.434), Herbert (p = 0.055) and Mohaka (p = 0.098) were not significantly different from those of the Merino reference flock, and those of sheep from Mohaka (p = 0.673), Campbell Island (p = 0.084) and Hokonui I (p = 0.454) were not different from those of all breeds and (2) frequencies of ADRB3 alleles of sheep from the Arapawa Island I did not differ from those of either reference flock (Merino p = 0.332; All breeds p = 0.771).

                  Allelic richness, observed (H O ) and expected (H E ) levels of heterozygosity and coefficient of inbreeding (F IS) are shown in Table 4. On average between 2.03 and 4.86 alleles were detected per polymorphic gene across all the populations. The lowest number of alleles was observed for the ADRB3 gene (1.59) in the sheep from Arapawa Island II while the greatest number of alleles was found for KRT33 (6.12) in the Hokonui II sheep. Allelic richness was highest for KRT33 and lowest for ADRB3 in all feral populations except for the Mohaka sheep.
                  Table 4

                  Allelic richness (r), expected (H E ) and observed (H O ) heterozygosity, F IS 1 values for feral sheep populations of New Zealand

                  Population

                  Arapawa Island I

                  Arapawa Island II

                  Chatham Island

                  Pitt Island

                  locus

                  r

                  H O

                  H E

                  F IS

                  r

                  H O

                  H E

                  F IS

                  r

                  H O

                  H E

                  F IS

                  r

                  H O

                  H E

                  F IS

                  KRTAP1-1

                  3.72

                  0.71

                  0.61

                  -0.18

                  3.30

                  0.14

                  0.13

                  -0.06

                  3.17

                  0.36

                  0.30

                  -0.20

                  4.24

                  0.23

                  0.26

                  0.11*

                  KRT33

                  5.96

                  0.69

                  0.73

                  0.05

                  4.00

                  0.58

                  0.65

                  0.11**

                  5.02

                  0.81

                  0.78

                  -0.05

                  5.95

                  0.59

                  0.58

                  -0.02

                  ADRB3

                  2.83

                  0.71

                  0.73

                  0.03

                  1.59

                  0.75

                  0.66

                  -0.13

                  1.94

                  0.59

                  0.62

                  0.04

                  1.87

                  0.77

                  0.77

                  -0.01

                  DQA2

                  4.26

                  0.82

                  0.84

                  0.02*

                  2.96

                  0.66

                  0.62

                  -0.06

                  4.48

                  0.95

                  0.81

                  -0.18

                  2.83

                  0.82

                  0.83

                  0.00

                  Mean

                  4.19

                  0.73

                  0.73

                  -

                  2.96

                  0.53

                  0.52

                  -

                  3.65

                  0.68

                  0.63

                  -

                  3.72

                  0.60

                  0.61

                  -

                  Population

                  Campbell Island

                  Woodstock

                  Hokonui I

                  Hokonui II

                  locus

                  r

                  H O

                  H E

                  F IS

                  r

                  H O

                  H E

                  F IS

                  r

                  H O

                  H E

                  F IS

                  r

                  H O

                  H E

                  F IS

                  KRTAP1-1

                  2.90

                  0.06

                  0.30

                  0.79**

                  3.41

                  0.36

                  0.47

                  0.24

                  2.54

                  0.36

                  0.31

                  -0.18

                  3.05

                  0.20

                  0.21

                  0.03

                  KRT33

                  4.71

                  0.59

                  0.61

                  0.04

                  5.49

                  0.63

                  0.76

                  0.17

                  4.41

                  1.00

                  0.77

                  -0.33

                  6.12

                  0.85

                  0.78

                  -0.09

                  ADRB3

                  2.06

                  0.53

                  0.51

                  -0.37

                  2.38

                  0.80

                  0.66

                  -0.21

                  1.97

                  0.55

                  0.44

                  -0.26

                  1.79

                  0.69

                  0.61

                  -0.13

                  DQA2

                  3.03

                  0.74

                  0.76

                  0.02

                  4.33

                  0.94

                  0.81

                  -0.16**

                  3.95

                  0.67

                  0.60

                  -0.11

                  4.52

                  0.89

                  0.86

                  -0.04

                  Mean

                  3.18

                  0.48

                  0.55

                  -

                  3.90

                  0.68

                  0.68

                  -

                  3.22

                  0.65

                  0.53

                  -

                  3.87

                  0.66

                  0.62

                  -

                  Population

                  Herbert

                  Mohaka

                  Allele richness averages

                      

                  locus

                  r

                  H O

                  H E

                  F IS

                  r

                  H O

                  H E

                  F IS

                          

                  KRTAP1-1

                  2.61

                  0.30

                  0.26

                  -0.16

                  3.00

                  0.33

                  0.30

                  -0.11

                  3.19

                    

                  KRT33

                  5.04

                  0.79

                  0.74

                  -0.07

                  1.86

                  0.57

                  0.53

                  -0.09

                  4.86

                      

                  ADRB3

                  1.90

                  0.46

                  0.38

                  -0.20

                  2.00

                  0.67

                  0.53

                  -0.29

                  2.03

                      

                  DQA2

                  4.03

                  0.79

                  0.79

                  -0.01

                  2.00

                  0.14

                  0.14

                  -

                  3.64

                      

                  Mean

                  3.40

                  0.59

                  0.54

                  -

                  2.22

                  0.43

                  0.38

                  -

                          

                  1Significance of F IS is indicated * P< 0.05, ** P< 0.01, figure in bold character shows a tendency towards significance (P < 0.10); negative values indicate outbreeding while positive values indicate inbreeding; "-" represents data that could not be obtained

                  Observed and expected heterozygosity values ranged from a low of 0.06 observed for KRTAP1-1 to a high of 1.0 for KRT33, and a low of 0.13 for KRTAP1-1 and a high of 0.86 for DQA2, respectively. Arapawa I sheep had the highest mean estimate for H O and H E over all of the genes (0.73 and 0.73, respectively), while Mohaka sheep had the lowest mean estimate for Ho and He (0.43 and 0.38, respectively). Allele sharing was high between animals originating from Campbell Island and Pitt Island for KRTAP1-1 and among the Arapawa II flock of feral sheep for KRT33 and lower among the Arapawa I flock for DQA2. Finally, allele sharing among sheep from Woodstock was very low for DQA2.

                  Discussion

                  This is the first report describing DNA variation in feral sheep from NZ. The genes investigated in this study were chosen because they had previously been shown to influence wool traits [16], cold survival [17] and footrot resistance [20, 21]. No new allele was identified for any of the genes in the feral sheep, suggesting that they will not be a source of alternative genetic variability, at least for these genes. The allelic richness and heterozygosity results (observed and expected) are comparable with those presented in previous studies of non-NZ wild sheep populations [3234].

                  Although the feral sheep sampled were chosen so that they were representative of their populations, there is no guarantee that the farmers who maintain these populations on the NZ mainland have been able to maintain genetic diversity, especially because the flocks sizes are small compared to the original populations. Allele sharing among four offshore island flocks (Arapawa I and II, Pitt Island and Campbell Island) was significant for one gene but not necessarily for the same gene. Sheep populations from both Pitt and Campbell Islands, have undergone extensive size reduction before being relocated to the mainland and it is surprising that the level of inbreeding is not higher. In contrast, among the mainland Woodstock sheep, many different alleles are detected for DQA2 suggesting this flock is outbred, although other loci would need to be typed to confirm this. This is most likely due to the ongoing introduction of new genetic material from other Merino sheep which are typically farmed in areas adjacent to this population.

                  Sources of genetic variation in the feral sheep populations include founder effects, random drift, balancing selection, genetic bottlenecks, or combinations of these. Each will be discussed below.

                  Genetic drift may have affected these feral populations [35]. However, in some feral populations allele frequencies were similar to those in commercially farmed Merino sheep. This may not be surprising since both the feral and commercial merino sheep share the same Australian origin, and the two groups have been separated at most by 50 generations.

                  In some cases, allele frequencies in the feral populations were not "Merino-like" and tended to show greater similarity to allele frequencies in other common farmed sheep in NZ. This provides support for the anecdotal contention that these feral sheep have at times interbred with farmed non-Merino sheep.

                  There is evidence of genetic differences between groups of sheep on remote yet neighbouring islands. Chatham and Pitt Island sheep are thought to be descendents of the same founding Merino sheep, yet they show quite different allele frequencies for many of the genes studied here. Pitt and Chatham Island feral sheep have distinct wool colours but whether this is a result of the differences in the genes studied cannot be ascertained here.

                  Founder effects may influence the genetic diversity of feral populations [36]. It is apparent from early farming records that many of these flocks were initiated with 50 or less animals and hence the likelihood of finding rare alleles in the founding individuals might be small. Both ADRB3 variants D and H are rare in farmed NZ sheep [17] and they are absent from the feral populations studied here.

                  An alternative explanation to the founder effect is that particular ADRB3 alleles have been lost in the feral populations because they provide no selective advantage. This is called balancing selection and it reflects the situation where alleles are retained in a population by forms of selection such as heterozygote advantage, frequency-dependent selection [37] or selection varying in space and time that favours some alleles in certain environments [38]. ADRB3 alleles A and E are associated with cold survival, alleles C and F are linked to cold-related mortality, and allele D has a strong association with cold-related mortality and total mortality [17]. The complete absence of ADRB3 allele D in the feral populations could be due to the fact that these flocks were exposed to cold climatic conditions during lambing and death of lambs carrying the allele.

                  A number of studies have suggested that feral sheep show few signs of susceptibility to infection by ectoparasites [9, 12] and fly strike [39] when compared to other domesticated sheep breeds. The reason why these animals may be more resistant to parasites remains unknown, but may involve genetic variation or reduced/non-exposure to the pathogens.

                  Charbonnel and Pemberton [40] have suggested that infection with Teladorsagia circumcincta imposes a selection pressure in the Soay sheep of the island of Hirta in Scotland, and that this is reflected in the temporal divergence of the MHC genes over a relatively short period between 1988 and 2000. In the context of the results reported here, while the MHC allelic richness is at times low, in the absence of any data or evidence of on-going disease challenge it would be speculative to attempt to draw any conclusions. It should be noted that for DQA2, allele sharing was high within one island population but low within the mainland feral population, suggesting that the island population may have undergone some selection pressure.

                  Allele sharing at KRT33 and KRTAP1-1 was typically low suggesting the flocks may be outbred. Allele richness was highest for KRT33 indicating that the level of genetic diversity has remained quite high in these feral sheep populations. Feral sheep populations have some unique wool characteristics including at times a hairy birth-coat type, which has been shown to offer some advantage in improving lamb survival [4143], the ability to shed their wool [7], tightly curled wool [12] and various coat colours and markings [8]. The genes responsible for these traits have yet to be identified, but may include some of the genes for keratins and KAPs that constitute wool fibre.

                  Genetic bottlenecks can cause loss of genetic diversity [44]. Like founder effects, they are largely responsible for the loss of low-frequency alleles and tend to increase the abundance of intermediate- and high-frequency alleles [45]. It is generally admitted that sheep populations from Pitt and Campbell Islands originated from a small number of founding animals that multiplied subsequently. After reaching a size of approximately 4 000 sheep on both islands, genetic bottlenecks most likely occurred, when the majority of the sheep were slaughtered, and small numbers of sheep were transferred to NZ to create the flocks studied here. Thus these island populations may have been subject to both founder and bottleneck effects, but the data presented here does not show any strong evidence in favour of the historically documented bottlenecks and there are no obvious differences in allelic richness between the Pitt and Campbell island populations compared to the other feral sheep populations.

                  Declarations

                  Acknowledgements

                  This research was supported by the Brian Mason Scientific and Technical Trust. We appreciate the time and effort of the farmers who maintain these sheep and for their generosity in supplying the DNA samples. We also thank members of the Gene-Marker Laboratory for completing the genotyping of samples.

                  Authors’ Affiliations

                  (1)
                  Department of Agricultural Science, Faculty of Agriculture and Life Sciences, Lincoln University
                  (2)
                  Environment Canterbury
                  (3)
                  Faculty of Sport and Health Sciences, Eastern Institute of Technology

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                  Copyright

                  © McKenzie et al. 2010

                  This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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