Genetic Variability in Local and Imported Germplasm Chicken Populations as Revealed by Analyzing Runs of Homozygosity

1. Introduction

Assessment of the genome-wide diversity plays a significant role in conserving local and imported genetic resources and maintaining an effective program for breeding commercial populations (e.g., [1,2,3]). Previously, in the framework of a poultry germplasm conservation and utilization research project, we assessed a broad spectrum of the world’s chicken germplasm populations for genetic variation at economically important loci [4,5] and multiple single nucleotide polymorphisms (SNP) loci [6,7,8,9].

A comparative genetic evaluation of livestock populations of different origin, population history, and size is an important source of information for genetic changes in their genome, including degree of homozygosity across genome-wide regions [2,3,10]. This is especially desirable when breeding small conserved groups and evaluating crossbreeding/inbreeding effects [11,12]. Lengths of runs of homozygosity (ROH) in the genome of a particular animal eventually depend on selection, gene drift, and herd size in original population [12], and are useful for assessing information about the degree of inbreeding. Long ROHs are typical for inbred individuals, as haplotypes inherited from a common ancestor do not shorten during recombination. In contrast, short ROHs can inform studies of less pronounced inbreeding intrinsic to heterogeneous animals [13,14,15,16].

Since relevant ROH studies in small closed populations kept in a germplasm collection of local, rare, and endangered chicken breeds are missing, the goal of our study was a genome-wide evaluation of SNP genetic diversity in these populations, and a search for criteria to identify occasional hybridization events in population history in order to control breed purity. Herewith we investigated ROHs and linkage disequilibrium (LD) and hypothesized that, when monitoring intra-breed genetic structure, small purebred populations would have more and longer ROHs compared to F₁ crossbreds, which could be served as a marker to detect potential occasional hybridization events.

2. Materials and Methods

For this study, SNP genotypes of 673 birds were obtained for the following 21 small populations of local and imported breeds that represent a broad sample of the world’s chicken germplasm and are maintained at the RRIFAGB Collective Use Centre ‘Genetic Collection of Rare and Endangered Chicken Breeds’. These included 14 purebred populations: Amroks Cuckoo (A), Brahma Buff (BB), Brahma Light (BL), Bantam Mille Fleur (or Russian Korolyok) (BMF), White Cornish (C; two-way hybrid C1 bred inter se), Frizzle (F), Hamburg Silver Spangled Dwarf (HSSD), Leghorn Light Brown (or Italian Partridge) (LLB), Pushkin (P), Russian White (two populations, RW1 and RW2), Sussex Light (SL), Tsarskoye Selo (or Tsarskoselkaya) (TC), Uzbek Game (UG); and 7 groups of F₁ crossbred progeny: Brahma Light × Sussex Light (BL × SL), Sussex Light × Amroks Cuckoo (SL × A), Tsarskoye Selo × Sussex Light (TC × SL), Uzbek Game × Amroks Cuckoo (UG × A), White Cornish × (Brahma Light × Sussex) (C × BL × SL), White Cornish × (Sussex × Amroks Cuckoo) (C × SL × A), and Tsarskoye Selo × (Sussex Light × Amroks Cuckoo) (TC × SL × A) as shown in

Table 1. Runs of homozygosity (ROH)-based metrics and linkage disequilibrium (LD) values in the 21 purebred and F₁ crossbred chicken populations studied.

Population	Abbreviation Code	Origin	Type	Sample Size	Number of ROHs per Individual			Length of ROH, Kb			FROH1	LD
					Mean	Minimum	Maximum	Mean	Minimum	Maximum
Pure breeds
Amroks Cuckoo	A	USA	dual purpose	20	18.9 ± 1.5	3	31	4057.2 ± 198.8	2308.9	5998.3	0.105 ± 0.009	0.187 ± 0.001
Brahma Buff	BB	India, USA	fancy, meat	20	36.6 ± 2.1	24	56	4508.4 ± 194.7	3116.2	6347.6	0.167 ± 0.015	0.283 ± 0.001
Brahma Light	BL	“ 2	“	20	37.4 ± 3.0	10	56	5259.3 ± 193.4	3772.7	6916.0	0.203 ± 0.017	0.286 ± 0.001
Bantam Mille Fleur (or Russian Korolyok)	BMF	Russia	fancy	20	61.2 ± 2.1	31	78	5259.0 ± 234.0	4043.3	8082.3	0.291 ± 0.015	0.374 ± 0.001
White Cornish (two-way hybrid C1 bred inter se)	C	England	meat	20	40.4 ± 1.1	33	52	3609.8 ± 80.7	3105.0	4494.0	0.055 ± 0.007	0.155 ± 0.001
Frizzle	F	Asia, Europe	fancy	20	5.9 ± 0.1	1	20	3149.0 ± 234.1	1394.6	6322.4	0.023 ± 0.005	0.171 ± 0.0003
Hamburg Silver Spangled Dwarf	HSSD	Holland	“	20	63.9 ± 1.5	42	76	5735.0 ± 164.2	4672.2	7311.6	0.324 ± 0.011	0.438 ± 0.001
Light Brown Leghorn (or Italian Partridge)	LLB	Italy	egg	19	47.5 ± 2.3	26	62	3903.8 ± 137.7	2949.8	5033.0	0.167 ± 0.011	0.288 ± 0.001
Pushkin	P	Pushkin, USSR/Russia	dual purpose	20	23.8 ± 1.3	15	36	3889.4 ± 209.7	2449.5	5569.3	0.112 ± 0.009	0.232 ± 0.001
Russian White	RW1	Pushkin, Russia	egg	6	68.3 ± 2.0	62	76	5121.9 ± 278.3	4279.8	5850.7	0.307 ± 0.014	0.518 ± 0.001
Russian White	RW2	“	“	170	25.9 ± 1.1	16	36	3749.6 ± 111.3	2431.3	4737.0	0.083 ± 0.006	0.218 ± 0.001
Sussex Light	SL	England	dual purpose	20	23.1 ± 1.5	11	35	4075.4 ± 155.8	3030.5	5773.5	0.127 ± 0.003	0.263 ± 0.001
Tsarskoe Selo (Tsarskoselskaya)	TC	Pushkin, Russia	“	20	18.6 ± 1.2	8	30	4098.5 ± 193.1	2765.8	6303.2	0.102 ± 0.009	0.201 ± 0.001
Uzbek Game	UG	Uzbekistan	game	19	9.0 ± 1.3	0	21	3838.4 ± 349.5	0	6215.3	0.095 ± 0.007	0.178 ± 0.001
F1 crossbreds
Brahma Light × Sussex Light	BL × SL	Pushkin, Russia	meat	12	4.3 ± 0.5	1	7	3602.2 ± 378.5	2347.8	7266.8	0.051 ± 0.008	0.215 ± 0.001
Sussex Light × Amroks Cuckoo	SL × A	“	“	14	2.2 ± 0.3	0	5	2905.1 ± 291.9	0	5126.7	0.033 ± 0.008	0.188 ± 0.001
Tsarskoye Selo × Sussex Light	TC × SL	“	“	14	6.0 ± 0.7	3	10	3032.8 ± 188.2	2050.0	4181.1	0.040 ± 0.009	0.215 ± 0.001
Uzbek Game × Amroks Cuckoo	UG × A	“	“	14	4.9 ± 0.4	3	8	3023.5 ± 170.8	2062.4	4098.3	0.039 ± 0.008	0.206 ± 0.001
White Cornish × (Brahma Light × Sussex Light)	C × BL × SL	“	“	14	0.9 ± 0.3	0	3	1622.8 ± 457.5	0	4427.7	0.024 ± 0.008	0.206 ± 0.001
White Cornish × (Sussex Light × Amroks Cuckoo)	C × SL × A	“	“	14	1.4 ± 0.4	0	4	1840.6 ± 425.2	0	4641.6	0.033 ± 0.008	0.193 ± 0.001
Tsarskoye Selo × (Sussex Light × Amroks Cuckoo)	TC × SL × A	“	“	14	3.8 ± 0.6	0	8	2715.2 ± 383.2	0	5410.2	0.046 ± 0.008	0.195 ± 0.001

¹F_ROH, ROH-based inbreeding coefficient; ² As above.

.

In each population, both hens and cocks were genotyped using Illumina Chicken 60K SNP BeadChip (Illumina, USA) as performed with the assistance of the Geneseek/Neogen Corporation. SNPs were filtered and removed from the further analysis if they met the following criteria: minor allele frequency ≤0.05, Hardy–Weinberg equilibrium probability ≤1 × 10⁻⁴, and call rate ≤95%. After completing the quality control procedures, 44,230 SNP markers from 28 autosomes were available for further analysis.

LD (or r²) coefficients were calculated using the following formula in PLINK 1.9 software [17]:

$$r^2=\frac{{\left(f_{11}{f}_{22}-f_{12}{f}_{21}\right)}^2}{f{\mathrm{A}}_{1}f{\mathrm{A}}_{2}f{\mathrm{B}}_{1}f{\mathrm{B}}_2}$$

(1)

where A and B are two loci each containing two alleles A₁ and A₂, and B₁ and B₂; f₁₁, f₁₂, f₂₁ and f₂₂ are the frequencies of haplotypes A₁B₁, A₁B₂, A₂B₁ and A₂B₂, respectively; and fA₁, fA₂, fB₁ and fB₂ are the frequencies of alleles A₁, A₂, B₁ and B₂, respectively.

Search for homozygous regions was carried out using the PLINK 1.9 program according to the following algorithm: ROHs containing at least 100 SNPs and a total length of ≥1000 Kb were taken into account, each ROH having had at least one SNP per 50 Kb. The scan window contained 50 SNPs and no more than 1 heterozygous genotype.

Values of ROH-based inbreeding coefficients (F_ROH) were found using the following formula [18]:

$$F_{\mathrm{ROH}}=\frac{{{{\displaystyle \sum }}^{}}_k length\left(RO{H}_k\right)}{L}$$

(2)

where k is the number of ROHs, length (ROH_k) is an individual ROH region length, and L is autosomal genome size covered by SNPs.

Population variability of SNP markers in purebred and F₁ crossbred populations was estimated in the admixture 1.3.0 program [19]. To compute the maximum likelihood estimates, the most probable number of ancestral populations (K) was selected using genotype data from 44,230 SNPs. For this purpose, a cross-validation procedure was performed. The lowest cross-validation error was found at K = 10, and charts were plotted using the function barplot() in R [20].

Multidimensional scaling (MDS) was performed in Plink 1.9 software [21] using a matrix of identity by state (IBS) distances between samples. To eliminate the effect of individual’s sex on IBS distances, SNP markers located on the sex chromosomes were excluded from the MDS analysis and, therefore, genotype data from 44,230 autosomal SNPs were employed.

Additionally, to assess between population diversity, Wright’s [22] fixation index (F_ST) values were computed using eigensoft 6.1.4 software [23]. Phylogeny tree was plotted using Neighbor Joining algorithm in PHYLIP (PHYLogeny Inference Package) [24] and Interactive Tree Of Life (iTOL v4) [25].

3. Results and Discussion

Using whole-genome SNP genotyping, we found that the examined populations of local and imported chicken breeds significantly differed from each other in genetic architecture and total length of ROHs (Figure 1). Most conserved purebred populations had greater numbers of ROHs and longer ROHs as well as higher ROH-based inbreeding coefficients (F_ROH) than F₁ inter-breed crossbreds produced by crossing two or three breeds (Table 1). For example, HSSD, an old Dutch breed that underwent a strict intra-breed type of breeding, was characterized by a greater number of ROHs (63.9 ± 1.5 vs. 32.0 ± 5.2 averaged across other 13 breed populations) and a greater length of ROH (5735.0 ± 164.2 Kb vs. an average of 4193.8 ± 182.7 Kb), with the inbreeding coefficient F_ROH being the highest one (0.324 ± 0.011 vs. an average of 0.141 ± 0.023; Table 1). All this suggests that this breed and its particular small population were affected by inbreeding both in earlier times and more recently. The presence of SNPs with higher rates of LD (0.438 ± 0.001; Table 1) also testifies to inbreeding effects in this population.

There were other small closed purebred populations with a higher content of ROHs in their genome, e.g., BB (F_ROH = 0.167 ± 0.015), BMF (F_ROH = 0.291 ± 0.015), etc. (Table 1). Among these, we found an exception with F_ROH = 0.023 ± 0.005 in the decorative F breed known in Europe since 1676 and characterized by a specific structure of feathers due to the F (frizzling) gene. A lower content of ROHs in this breed population can be associated with a targeted introgression of genes of the other breeds to reduce the effect of feather fragility as reflected by the admixture analysis (Figure 2).

The original Russian White population (RW1) existed from 1953–2003, had one founder and underwent intensive selection for chick tolerance to cold [7,26]. In 2005, in order to maintain genetic diversity in a small population of this breed, a single introductory crossing with White Leghorns resulted in producing the present population RW2. Accordingly, we observed a higher F_ROH in RW1 (0.307 ± 0.014) and a lower one in RW2 (0.083 ± 0.006; p < 0.0001; Table 1). Also, these two populations differed in LD, with a lower value being in RW2 as compared to RW1 (p < 0.0001; Table 1). Despite a relatively small size (~200 animals), RW2 is not expected to raise homozygosity because of the previous crossing with White Leghorns that was likely to significantly increase its genetic diversity.

In two-breed F₁ crossbreds, we revealed a decrease in ROH metrics, while three-breed F₁ crossbreds appeared to show a further lowering in their genomic content of ROH (Table 1).

Number of ROHs could reflect the type of breeding program. Small local and imported conserved populations bred inter se had a greater (>20) number of ROHs (e.g., BMF, BB, BL, RW1 and C), meaning a reduced genetic diversity similar to what was shown in small livestock populations kept by individual breeders or in local breeds [2,27,28,29,30,31]. In contrast, the genomes of two- and three-breed F₁ crossbred progenies contained a lesser (<6) number of ROHs (Table 1; Figure 1), which was consistent with lower ROH metrics observed in other studies on crossbred animals (e.g., [3,32]).

Characterization of genetic differentiation in conserved populations of local and imported breeds and monitoring of their purity/variability are an important component of breeding/conservation programs [1,2]. In the presence of ancestral purebred populations, use of the admixture program effectively detects F₁ crossbred progenies (Figure 2). However, in the absence of ancestral populations, it is difficult to determine whether it is an F₁ crossbred progeny or a purebred population (data not shown). If data on the origin are not taken into account, F₁ crossbred progenies are difficult to identify. In contrast, when conventional MDS and F_ST-based analyses (Figure 3a,b) are performed, one cannot always describe properly the population structure, differentiation, and gene flow due to crossbreeding, especially when comparing related breeds.

4. Conclusions

Exploration of the length and number of ROHs can be helpful in discriminating between purebred and F₁ crossbred animals, and this approach can be used as a tool in selecting purebred individuals for conserved propagation of local and imported breeds. ROH-based characterization in individuals and in the whole population can be used in adjusting germplasm breeding/conservation programs and identifying events of inter-breed gene transfer.