Results
Genome sequencing and identification of variants. To
detect genome-wide variation in three Chinese chicken breeds that have
been conserved in situ , we genotyped 91 individuals using
high-throughput sequencing (Figure 1). Alignment of 79.95 Gb of sequence
data against the Gallus gallus 5.0 reference genome yielded
> 8× average read depth (Table S1). In combination with
genomic data obtained from the same three breeds conserved in ex
situ programs (Zhang et al., 2018), a total of 5,070,414 variants were
identified, including 4,709,112 SNPs and 361,302 short insertions and
deletions (indels). Variants were evenly distributed across the genome
(Figure S1a and Figure S2). 31.58% of the total SNPs were novel and had
not been included in the dbSNP database at NCBI (Table S2 and Figure
S1b). After removal of variations that did not meet quality criteria
(MAF 0.01, HWE 10e-6), 1,518,758 SNPs remained for further analysis.
Population structure analysis. To investigate
phylogenetic relationships and population structure amongst the 361
chickens, we constructed a neighbor-joining tree using a pairwise
genetic distance matrix (Figure 2a) and performed principal component
analysis (PCA) based on the variance-standardized genotype relationship
matrix (Figure 2b). The neighbor-joining tree suggests that the samples
form six major clusters that correspond to the three Chinese domestic
chicken breeds, with further subdivision of each breed intoin-situ and ex-situ populations. This pattern was further
confirmed by PCA. The first principal component (PC1, variance explained
= 11.65%) successfully separated the Langshan chicken breed from the
other groups. The second principal component (variance explained =
10.7%) separated all populations in the three chicken breeds (Figure
S3). Notably, the PCA separated the in situ and ex situpopulations, especially for Beijing You chicken (Figure S3). To better
understand p population ancestry, we used ADMIXTURE to estimate the
number of ancestral populations (Alexander et al., 2009) and allowed
population number (K) to vary from 2 to 9. The minimum estimated
cross-validation error occurred at K=6 (Figure S4). These results
suggest that the three Chinese domestic chicken breeds have distinct
backgrounds and also differ between in-situ and ex-situpopulations, consistent with the results from the NJ tree and principal
components analyses. The likelihood model based on K=6 resolves the
three Chinese domestic chicken populations into six genetic clusters
(Figure 2c). One individual from the in-situ conserved population
of Beijing You chickens (YBYC) had a genetic background that was
distinct from other individuals in the YBYC population, based on the NJ
tree, PCA, and ADMIXTURE results. We therefore removed this individual
from subsequent analyses.
Genomic diversity assessment. Analyses of genomic
genetic variability parameters for the six sub-populations are presented
in Figure 3 and Table 2. The parameters include observed (Ho) and
expected (He) heterozygosity, allelic richness (AR),
proportion of polymorphic SNPs (PN ), and
inbreeding coefficient (F ). The genomic diversity in populations
conserved in situ was higher than in those conserved ex
situ . Observed heterozygosity (Ho) and expected heterozygosity (He)
were similar for all three breeds in both in situ and ex
situ conserved populations. For example, changes in genetic diversity
between in situ conserved populations of the Beijing You chicken
(YBYC, Ho = 0.2646, He = 0.2714) vs. ex situ (BYC15, Ho = 0.2729,
He = 0.2658) were less than 5%. In contrast, allelic richness
(AR) and proportion of polymorphic markers
(PN ) for in situ conserved populations
(AR = 1.209, PN = 0.7891) were
higher than for ex situ (AR = 1.198,
PN = 0.7258).
Estimation of inbreeding coefficients. To estimate the
degree of inbreeding in in situ and ex situ conserved
populations, we calculated FES andFROH across subpopulations. As expected,FES values increased while conservation
procedures were maintained. This trend is also evident in the comparison
of FES in in situ vs. ex situconserved chicken populations. Conservation practices have been applied
for a longer period (conservation time; CT) for the in situpopulation than the ex situ population, and theFES values for the in situ population are
correspondingly higher.
Since FROH is better at detecting both rare and
common variants, we focused on this measurement in subsequent analyses.
The inbreeding coefficient based on runs of homozygosity
(FROH ) was relatively low, ranging from 0.0463 to
0.0958. Except for Langshan chickens, FROH inin situ conserved populations was lower thanFROH inex
situ populations. The difference may be caused by the small size of the
Langshan chicken in situ conserved population and the long
conservation time (CT = 60 years). Inbreeding coefficients are compared
for the current generation of all three chicken breeds in Figure 3 and
Table 2.
Calculation of nucleotide diversity The results of a
nucleotide diversity (Pi) survey are shown for the three breeds in
Figure 4a. The YLSC (Pi = 0.000112582) had the highest average
nucleotide diversity amongst the 12 subpopulations, followed in
descending order by YBEC, LSC15, LSC12, YBYC, BEC10, BEC07, LSC10,
BYC07, BYC10, BEC15, and BYC15. Pi was markedly higher in in situconserved populations than in ex situ in all three chicken
breeds, and highly significant differences (P<0.001) were observed
between populations within breeds.
Linkage disequilibrium decay Differences in LD decay
between in situ and ex situ conserved populations are
shown in Figure 4b. The highest maximum average LD
(r2 = 0.2235) was observed in Beijing You
chickens (BYC15), and the lowest (r2 = 0.1806)
occurred in Baier Yellow chickens (YBEC). Compared to the current
generation of ex situ conserved populations (BYC15, BEC15, and
LSC15), maximum average LD values were lower in the in situconserved Beijing You chicken and Baier Yellow chicken populations,
while higher values were observed in Langshan chickens.
This
may indicate that YBYC and YLSC have greater genetic diversity than
BYC15 and LSC15. As expected, LD declined as the physical distance
increased between pairwise SNPs. As shown in Figure 4b, LD decay inin situ conserved populations declined markedly compared withex situ populations for Beijing You chicken and Baier Yellow
chicken. In contrast, LD decay. is similar in in situ andex situ conserved populations for Langshan chickens. Using
Beijing You chickens as an example, r2decreased by half (from 0.1982 to 0.0991) over a span of 11.84 kb in thein situ group, while LD decayed by half over a span of 14.68 kb
in the ex situ conserved population (BYC15).
Estimation of population differentiation using Fst To
estimate population differentiation, we calculated pairwiseFST values across the sub-populations (Table S3).
Values ranged from 0.004826 to 0.1508. FST values
for all pair-wise comparisons are shown in Figure 5. For all three
breeds, FST values amongst three successive
generations were lower than 0.05. This result indicates that no or
little genetic differentiation has occurred in the conserved populations
from one generation to the next. Significant or moderate genetic
differentiation is observed between breeds, and the maximumFST value was calculated between LSC15 and BYC15
(FST = 0.1508). Notably,FST values between in situ and ex
situ conserved populations for all three breeds were greater than 0.05.
In the case of the Beijing You chicken, FSTvalues have increased with time of conservation, and the maximumFST value was 0.1379 between BYC15 and YBYC.
Overall, moderate genetic differentiation has occurred in in situand ex situ conserved populations among the three chicken breeds.
Effective population size (Ne) Ne is an important
measure in conservation genetics, and conservation efforts strive to
increase Ne. In order to estimate
current
Ne for conserved Chinese domestic chicken breeds, we used NeEstimator v2
(Do et al., 2014b), which applies a method based on linkage
disequilibrium (LD) to calculate Ne using whole-genome SNPs markers.
Effective population size (Ne) was estimated for autosomal chromosomes
gga1 through gga28 (Table S4). Ne ranged from 2.7 to 167.4, with a mean
of 43.81. Amongst macro-chromosomes (gga1-gga5), BEC15 exhibited the
smallest estimated Ne (50.96), suggesting that BEC15 is a limited pool,
whereas YBEC had the largest value (130.28), suggesting much higher
genetic diversity. Importantly, Ne in in situ conserved
populations was higher than in current generations of ex situconserved populations (Figure 6).
Runs of homozygosity The abundance and genomic
distribution of ROH provide information about the demographic history of
a livestock species. ROH were identified in the genomes of all in
situ and ex situ conserved populations (Table S5). A genome-wide
survey for autozygosity was conducted to identify regions with
signatures that reflect ancient or recent inbreeding effects. Using
estimates of FROH , maximum values were found in
Beijing You chickens subjected to in ex situ conservation. In
contrast, the minimum values occurred in Baier Yellow chicken breeds
enrolled in in situ conservation programs (Table 3). BYC15, the
current generation in an ex situ conserved population, had the
highest level of inbreeding (0.1018). As expected, YBYC in the in
situ conservation population had a lower level of inbreeding (0.0777)
than BYC15. YBEC had the lowest level of inbreeding (0.0463) amongst all
populations. However, within the Langshan chicken breed, YLSC
(FROH = 0.0745) had a higher level of inbreeding
than LSC15 (FROH = 0.0604).
All ROH were then assessed to determine whether any populations exhibit
evidence of recent inbreeding. For BYC and BEC, the ex situconserved populations had longer ROH and lower genomic diversity than
the in situ conserved populations in these breeds (Figure 7a). In
contrast, the in situ conserved LSC population had a higher level
of inbreeding than the ex situ conserved population. We also
mapped ROH to the genome, and found that the homozygosity segments in
populations subjected to in situ vs. ex situ conservation
were distributed differently (Figure 7b).