BioMed Research International
Volume 2021 (2021), Article ID 8851888, 14 pages
https://doi.org/10.1155/2021/8851888

Research Article

Development of Microsatellite Marker System to Determine the Genetic Diversity of Experimental Chicken, Duck, Goose, and Pigeon Populations

Xiulin Zhang,¹ Yang He,¹ Wei Zhang,¹ Yining Wang,¹ Xinmeng Liu,¹ Aique Cui,¹ Yidi Gong,¹ Jing Lu,¹ Xin Liu,¹ Xueyun Huo,¹ Jianyi Lv,¹ Meng Guo,¹ Xiaoyan Du,¹ Lingxia Han,² Hongyan Chen,² Jilan Chen,³ Changlong Li,¹ and Zhenwen Chen¹

¹Capital Medical University, School of Basic Medical Sciences, Beijing 100069, China
²Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin 150069, China
³Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Correspondence should be addressed to Changlong Li; licl@ccmu.edu.cn and Zhenwen Chen; czwen@ccmu.edu.cn

Received 10 September 2020; Revised 9 December 2020; Accepted 5 January 2021; Published 15 January 2021

Academic Editor: Monica Fedele

Copyright © 2021 Xiulin Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

Poultries including chickens, ducks, geese, and pigeons are widely used in the biological and medical research in many aspects. The genetic quality of experimental poultries directly affects the results of the research. In this study, following electrophoresis analysis and short tandem repeat (STR) scanning, we screened out the microsatellite loci for determining the genetic characteristics of Chinese experimental chickens, ducks, geese, and pigeons. The panels of loci selected in our research provide a good choice for genetic monitoring of the population genetic diversity of Chinese native experimental chickens, ducks, geese, and ducks.

1. Introduction

Laboratory animals are important experimental materials for science research. They play key roles in the investigation of pathogenesis, diagnosis of diseases, pharmaceutical research, and other fields [1]. The genetic quality of laboratory animals directly affects the accuracy, repeatability, and scientificity of medical biological research results. Genetic monitoring is one of the effective methods to evaluate population’s genetic diversity. Through genetic monitoring, whether genetic mutations and genetic pollution occurred can be analyzed.

Poultry, including chicken, duck, goose, and pigeon, has become commonly used laboratory animals [2]. They are easy to reproduce and hatch in vitro. Among them, chickens are the most widely used poultry in life science research [3, 4]. Ducks, geese, and pigeons also play important roles in the research of epidemiology, immunology, virology, and pharmacotoxicology [5–9]. There are many genetic analysis and quality control methods applied to chickens [10, 11]. However, at present, we find few reports about the genetic analysis systems and quality control methods of duck, goose, and pigeon populations, especially in the Chinese native groups.

Hence, in this study, we screened out the microsatellite loci with uniform distribution, stable amplification, and rich polymorphism in experimental chickens, ducks, geese, and pigeons with different genetic backgrounds [12]. We developed effective microsatellite marker systems to determine the genetic diversity of experimental chickens, ducks, geese, and pigeons, which will lay the foundation for the genetic quality control of them and promote the application of experimental poultry.

2. Materials and Methods

2.1. Animal Sample

Three outbred groups and three haplotype groups of experimental chicken were used in this research: outbred group BWEL-SPF chickens ((SCXK (black) 2017-005)), 40 samples, 37 weeks old, 6 males and 34 females, which has been closed for 20 generations; outbred group BM chicken (from BWEL chicken lineage (SCXK (black) 2017-005)), 40 samples, 14 weeks old, 6 males and 34 females; outbred group Beijing oil chickens, 46 samples. MHC haplotype chickens were bred from the 13th generation of BWEL chicken, the haplotype was continuously selected based on the MHC core genes, and the half-sibling or sibling mating method was used to breed to the 8th generation [13]. We selected 5 G1 haplotype chickens, 53 weeks old, 1 male and 4 females; 5 G2 haplotype chickens, 93 weeks, 1 male and 4 females; and 5 G7 haplotype chickens, 82 weeks, 1 male and 4 females. The Beijing oil chickens came from the Institute of Animal Science (IAS), Chinese Academy of Agricultural Sciences (CAAS). Other samples were from Harbin Veterinary Research Institute (HVRI), CAAS. All the samples were blood.

Two outbred groups and four haplotype groups of experimental duck (bred from Jinding (JD) duck lineage (SCXK (black) 2017-006)) were selected: outbred group 1, 40 samples, 37 weeks old, 6 males and 34 females; outbred group JD duck, 40 samples, 37 weeks old, 6 males and 34 females; 10 A haplotype ducks, 53 weeks old, 1 male and 4 females; 10 B haplotype ducks, 53 weeks old, 1 male and 4 females; 10 C haplotype ducks, 53 weeks old, 1 male and 4 females; 10 D haplotype ducks, 53 weeks old, 1 male and 4 females. All the samples are duck muscle tissue and were from HVRI, CAAS.

We collected two outbred groups of experimental geese: outbred group Guangdong Wuzong goose, 44 samples, 37 weeks old, 6 males and 34 females; outbred group Yangzhou goose, 44 samples, 37 weeks old, 6 males and 34 females. All the samples are goose liver tissue. Guangdong Wuzong geese were from Southern Medical University, and Yangzhou geese were from Yangzhou University.

Forty pigeons were randomly selected from two populations of white king pigeons and silver king pigeons, half male and half female, with no age limit. All the animals were from Liujinlong pigeon farms in Beijing. Their heart tissues were collected.

All breeding is carried out in accordance with Chinese agricultural standards NY/T 1901. What is more, all experiments followed the 3R principle.

2.2. Microsatellite Locus Selection

By searching PubMed and using SSR Hunter software to analyze animal gene information, we obtained microsatellite loci for further screening.

2.3. DNA Extraction

Phenol-chloroform extraction method was used to extract DNA from muscle, liver, and heart tissue. TIANamp Blood DNA Kits (Tiangen, Beijing, China) were used to extract DNA from chicken blood samples. All DNA concentrations were diluted to 50 ng/μL, stored in -20°C.

2.4. PCR Procedure and Agarose Gel Electrophoresis

The PCR was performed in a 20 μL reaction volume containing 10 μL Dream Taq Green PCR Master Mix (Thermo Fisher Scientific, Massachusetts, MA), 2 μL pure water (ddH₂O), 10 pmol each primer, and 50 ng of the extracted DNA template. The PCR protocol was as follows: 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, suitable temperature for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min. Amplified products were stored at -20°C for further analysis.

Amplified products were electrophoresed on a 2% agarose gel at 130 V, 90 min.

2.5. STR Scanning

We performed STR scanning on PCR amplification products of candidate loci. The forward primers of candidate microsatellite loci were fluorescent labelled with FAM, HEX, and TAMRA. The sample genome was amplified with fluorescent primers, and the amplified products were scanned by STR through 3730xl DNA Analyzer (Applied Biosystems, Thermo Fisher Scientific, Massachusetts, USA). All the STR scanning was performed by Beijing Tianyi Huiyuan Biotechnology Co., Ltd.

2.6. Data Analysis

GeneMarker V2.2.0 software was used to analyze the length of amplified fragments from different populations at each microsatellite locus. Popgene 3.2 software was used to analyze the observed number of alleles, effective number of alleles, Shannon’s information index, and effective heterozygosity of microsatellite loci. The polymorphic information content of multiple sites was calculated using PIC calculation software (PIC_CALC.0.6).

3. Results

3.1. Microsatellite Locus Selection

3.1.1. Preliminary Screening of Microsatellite Loci by PCR

Firstly, we obtained the microsatellite locus information of experimental chickens, ducks, geese, and pigeons by searching previous reports on PubMed and using the SSR Hunter software to analyze the genetic information of different populations [14, 15]. We collected 72, 59, 57, and 61 microsatellite loci of experimental chicken, duck, goose, and pigeon, respectively.

In order to clarify the amplification conditions of the microsatellite loci and exclude the loci with poor specificity, we performed temperature gradient PCR and agarose gel electrophoresis of microsatellite loci. Then, we performed PCR amplification on the most suitable conditions and subjected the PCR products to agarose gel electrophoresis to screen out loci with suitable length, good polymorphism in outbred groups, good monomorphism in haplotypes, and high specificity. Taking the chicken GGNCAMZO locus and duck AY264 locus as example, the results are shown in Figure 1. GGNCAMZO locus is monomorphic in the haplotype chicken population, and AY264 locus is polymorphic in the outbred duck group.

(a)

(b)

Figure 1: Results of agarose gel electrophoresis of microsatellite DNA locus GGNCAMZO in experimental chickens and locus AY264 in experimental ducks. (a) GGNCAMZO in haplotype chicken line G1. (b) AY264 in the outbred group of experimental ducks.

In summary, we selected 37 and 32 microsatellite loci with good polymorphism in the outbred groups and haplotypes of chicken, respectively [12, 16, 17]. In addition, 15 and 23 loci were screened in the outbred groups and haplotypes of duck, respectively [14, 18, 19]. In the outbred groups of goose and pigeon, 14 and 20 microsatellite loci were chosen [18, 20–23]. Loci in these panels would be candidate for the final microsatellite marker evaluation systems.

3.1.2. STR Scanning Analysis

In order to further complete the microsatellite marker system, we performed STR scanning on the candidate microsatellite DNA loci matched microsatellite criteria and analyzed the length of the amplified product at the peak with GeneMarker software (V1.75). Taking the UU-CliμT47 locus as an example, it showed polymorphism in the outbred group of pigeon (Figure 2).

(a)

(b)

(c)

Figure 2: Results of UU-CliμT47 scan of the experimental pigeons. (a) The STR graph corresponding to the sample of haplotype under primer UU-CliμT47 shows homozygote with a wave peak of 201 bp. (b) The STR diagram corresponding to the sample of outbred groups under primer UU-CliμT47 shows heterozygote with two wave peaks of 201 bp and 205 bp, respectively. (c) The STR diagram corresponding to the sample of outbred groups under primer UU-CliμT47 shows heterozygote with two wave peaks of 201 bp and 209 bp, respectively.

We finally determined that in experimental chickens, 28 loci were selected for genetic monitoring in the outbred groups and 14 loci for haplotypes. All microsatellite DNA loci are shown in Table 1. There are 13 common loci.

Table 1: Number of alleles, optimal amplification conditions, and fragment length of 29 alleles for the laboratory chickens.

Loci
Primer sequence (5-3)
Temperature(°C)
Allele range
Applicable groups

MCW0029
GTGGACACCCATTTGTACCCTATG
63.8
139-188
Outbred group

CATGCAATTCAGGACCGTGCA

ADL0293
GTAATCTAGAAACCCCATCT
53.9
106-120
Outbred group

ACATACCGCAGTCTTTGTTC

ADL0317
AGTTGGTTTCAGCCATCCAT
58.5
177-219
Outbred group

CCCAGAGCACACTGTCACTG

GCT0016
TCCAAGGTTCTCCAGTTC
52.2
111-148
Outbred group

GGCATAAGGATAGCAACAG

ADL0304
GGGGAGGAACTCTGGAAATG
53.9
138-161
Outbred group

CCTCATGCTTCGTGCTTTTT

LEI0074
GACCTGGTCCTGACATGGGTG
58.5
221-243
Outbred group

GTTTGCTGATTAGCCATCGCG

ADL328
CACCCATAGCTGTGACTTTG
53.9
107-120
Outbred group

AAAACCGGAATGTGTAACTG

GGANTECl
GCGGGGCCGTTATCAGAGCA
65.0
139-194
Outbred group

AGTGCAGGGCGCTCCTGGT

LEI094
CAGGATGGCTGTTATGCTTCCA
56.0
176-211
Outbred group

CACAGTGCAGAGTGGTGCGA

MCW0330
TGGACCTCATCAGTCTGACAG
58.5
217-287
Outbred group

AATGTTCTCATAGAGTTCCTGC

LEI0141
CGCATTTGATGCATAACACATG
52.2
221-245
Outbred group

AAGGCAAACTCAGCTGGAACG

MCW0087
ATTTCTGCAGCCAACTTGGAG
58.5
268-289
Outbred group

CTCAGGCAGTTCTCAAGAACA

MCW0347
GCTTCCAGATGAGCTCCATGG
52.0
121-149
Outbred group

CACAGCGCTGCAGCAACTG

ADL176
TTGTGGATTCTGGTGGTAGC
58.5
183-200
Outbred group

TTCTCCCGTAACACTCGTCA

ADL0201
GCTGAGGATTCAGATAAGAC
58.5
111-151
Outbred group

AATGGCYGACGTTTCACAGC

GGNCAMZO
GTCACTAGGTTAGCAGCATG
56.0
234
Outbred group

GCTGGATACAGACCTCGATT
Haplotype

GGAVIR
AGAGATGGTGCACGCAACCT
60.7
86-89
Outbred group

CGAGCACTTTCTGGCAGAGA
Haplotype

MCW0063
GGCTCCAAAAGCTTGTTCTTAGCT
53.9
116-146
Outbred group

GAAAACCAGTAAAGCTTCTTAC
Haplotype

ADL185
CATGGCAGCTGACTCCAGAT
58.5
116-142
Outbred group

AGCGTTACCTGTTCGTTTGC
Haplotype

GGMYC
CGAGGCGCTCTGCGAGTTTA
62.4
139-151
Outbred group

TGGGGACCTCTGGCTCTGAC
Haplotype

LEI0094
GATCTCACCAGTATGAGCTGC
53.9
250-283
Outbred group

TCTCACACTGTAACACAGTGC
Haplotype

GGVITC
AGCCATCATTCAGGGCATCT
58.5
86
Outbred group

GATGTCCTGAGTGATGCTCA
Haplotype

ADL0292
CCAAATCAGGCAAAACTTCT
58.5
110-136
Outbred group

AAATGGCCTAAGGATGAGGA
Haplotype

GGVITIIG
GGCAGGTTTCTAATGCCTGA
56.0
186-189
Outbred group

CCCATCGTTTCAACTGTATG
Haplotype

ADL166
TGCCAGCCCGTAATCATAGG
58.5
131-154
Outbred group

AAGCACCACGACCCAATCTA
Haplotype

MCW0014
AAAATATTGGCTCTAGGAACTGTC
58.5
172-195
Outbred group

ACCGGAAATGAAGGTAAGACTAGC
Haplotype

GGCYMA
AGCGAGGCGCTCTGCGAGTT
64.6
140-153
Outbred group

GGGCACCTCTGGCTCTGACC
Haplotype

MCW0402
ACTGTGCCTAGGACTAGCTG
56.0
141-229
Outbred group

CCTAAGTCTGGGCTCTTCTG
Haplotype

STMSGGHU2-1A
CTTAATATGTGTGAGGTGGC
53.9
235-238
Haplotype

GTTCTCACAATTGCATTAGC

In experimental duck populations, we chose 25 loci and 15 loci for genetic monitoring in the outbred duck groups and haplotype groups. There are 12 common loci. Microsatellite loci are shown in Table 2.

Table 2: Number of alleles, optimal amplification conditions, and fragment length of 28 alleles for the laboratory ducks.

Loci
Primer sequence(5-3)
Temperature (°C)
Allele range
Applicable groups

CAUD007
ACTTCTCTTGTAGGCATGTCA
60.8
100-190
Outbred group

CACCTGTTGCTCCTGCTGT

CAUD004
TCCACTTGGTAGACCTTGAG
60.8
234-385
Outbred group

TGGGATTCAGTGAGAAGCCT

CAUD023
CACATTAACTACATTTCGGTCT
51.4
163-234
Outbred group

CAGCCAAAGAGTTCAACAGG

CAUD027
AGAAGGCAGGCAAATCAGAG
66.0
70-180
Outbred group

TCCACTCATAAAAACACCCACA

CAUD001
ACAGCTTCAGCAGACTTAGA
55.5
150-247
Outbred group

GCAGAAAGTGTATTAAGGAAG

CAUD031
AGCATCTGGACTTTTTCTGGA
51.4
107-187
Outbred group

CACCCCAGGCTCTGAGATAA

CAUD032
GAAACCAACTGAAAACGGGC
58.1
96-206
Outbred group

CCTCCTGCGTCCCAATAAG

AY314
CTCATTCCAATTCCTCTGTA
50.3
112-329
Outbred group

CAGCATTATTATTTCAGAAGG

CMO211
GGATGTTGCCCCACATATTT
55.0
112-205
Outbred group

TTGCCTTGTTTATGAGCCATT

APH09
GGATGTTGCCCCACATATTT
58.0
134-190
Outbred group

TTGCCTTGTTTATGAGCCATTA

APH11
GGACCTCAGGAAAATCAGTGTA
58.5
183-185
Outbred group

GCAGGCAGAGCAGGAAATA

APL2
GATTCAACCTTAGCTATCAGTCTCC
58.5
115-125
Outbred group

CGCTCTTGGCAAATGTCC

CAUD011
TGCTATCCACCCAATAAGTG
50.3
145-223
Outbred group

CAAAGTTAGCTGGTATCTGC

CAUD006
ATGGTTCTCTGTAGGCAATC
63.5
183-290
Outbred group

TTCTGCTTGGGCTCTTGGA
Haplotype

CAUD018
TTAGACAAATGAGGAAATAGTA
50.3
100-180
Outbred group

GTCCAAACTAAATGCAGGC
Haplotype

CAUD010
GGATGTGTTTTTCATTATTGAT
50.3
138-200
Outbred group

AGAGGCATAAATACTCAGTG
Haplotype

CAUD012
ATTGCCTTTCAGTGGAGTTTC
63.5
182-286
Outbred group

CGGCTCTAAACACATGAATG
Haplotype

CAUD014
CACAACTGACGGCACAAAGT
58.1
136-200
Outbred group

CTGAGTTTTTCCCGCCTCTA
Haplotype

CAUD034
TACTGCATATCACTAGAGGA
55.5
160-296
Outbred group

TAGGCATACTCGGGTTTAG
Haplotype

CAUD035
GTGCCTAACCCTGATGGATG
63.5
174-282
Outbred group

CTTATCAGATGGGGCTCGGA
Haplotype

APL579
ATTAGAGCAGGAGTTAGGAGAC
55.0
116-227
Outbred group

GCAAGAAGTGGCTTTTTTC
Haplotype

AY258
ATGTCTGAGTCCTCGGAGC
58.1
89-162
Outbred group

ACAATAGATTCCAGATGCTGAA
Haplotype

CMO212
CTCCACTAGAACACAGACATT
58.0
186-272
Outbred group

CATCTTTGGCATTTTGAAG
Haplotype

CAUD028
TACACCCAAGTTTATTCTGAG
55.5
152-226
Outbred group

ACTCTCCAGGGCACTAGG
Haplotype

CAUD026
ACGTCACATCACCCCACAG
60.8
134-196
Outbred group

CTTTGCCTCTGGTGAGGTTC
Haplotype

APH18
TTCTGGCCTGATAGGTATGAG
58.0
178-325
Haplotype

GAATTGGGTGGTTCATACTGT

CAUD002
CTTCGGTGCCTGTCTTAGC
60.8
200-231
Haplotype

AGCTGCCTGGAGAAGGTCT

CAUD005
CTGGGTTTGGTGGAGCATAA
60.8
184-290
Haplotype

TACTGGCTGCTTCATTGCTG

14 microsatellite loci with good polymorphism were considered as microsatellite markers in the outbred group of goose. Table 3 demonstrates the number of alleles, optimal amplification conditions, and fragment length of 14 alleles for the outbred experiment geese.

Table 3: Number of alleles, optimal amplification conditions, and fragment length of 14 alleles for the outbred colony laboratory geese.

Loci
Primer sequence(5-3)
Temperature (°C)
Allele range

G-Ans17
ACAAATAACTGGTTCTAAGCAC
51.0
111–123

AGAGGACTTCTATTCATAAATA

G-TTUCG1
CCCTGCTGGTATACCTGA
53.0
113-115

GTGTCTACACAACAGC

G-APH13
CAACGAGTGACAATGATAAAA
53.0
163-165

CAATGATCTCACTCCCAATAG

G-Ans02
TTCTGTGCAGGGGCGAGTT
58.0
202–230

AGGGAACCGATCACGACATG

G-Ans07
GACTGAGGAACTACAATTGACT
62.0
236–246

ACAAAGACTACTACTGCCAAG

G-Ans18
GTGTTCTCTGTTTATGATATTAC
56.0
229–237

AACAGAATTTGCTTGAAACTGC

G-Ans25
CACTTATTAATGGCACTTGAAA
62.0
261–277

GTTCTCTTGTCACAACTGGA

G-Hhiμ1b
ATCAAAGGCACAATGTGAAAT
60.0
212–216

AGTAAGGGGGCTTCCACC

G-CKW47
AACTTCTGCACCTAAAAACTGTCA
56.0
213-215

TGCTGAGGTAACAGGAATTAAAA

G-Bcaμ5
AGTGTTTCTTTCATCTCCACAAGC
56.0
197-201

AGACCACAATCGGACCACATATTC

G-Bcaμ7
TAGTTTCTATTTGCACCCAATGGAG
60.0
171-175

CGGTCCTGTCCTTGTGCTGTAA

G-Bcaμ8
CCCAAGACTCACAAAACCAGAAAT
58.0
155-159

ATGAAAGAAGAGTTAAACGTGTGCAA

G-CAUD006
ATGGTTCTCTGTAGGCAATC
56.0
170-210

TTCTGCTTGGGCTCTTGGA

G-APH20
ACCAGCCTAGCAAGCACTGT
53.0
140-150

GAGGCTTTAGGAGAGATTGAAAAA

In the outbred group of pigeon, we finally screened out 16 microsatellite loci with good polymorphism, several alleles, and typical stutter peaks. All microsatellite locus information is shown in Table 4.

Table 4: Number of alleles, optimal amplification conditions, and fragment length of 16 alleles for the outbred colony laboratory pigeons.

Loci
Primer sequence(5-3)
Temperature (°C)
Allele range

UU-Cli02
TGGGCAAGGTACACTTTTAGGT
61.0
158-170

CTTTATGCTCCCCCTTGAGAT

UU-Cli06
TTTGAAAAACATGGATTGTGC
56.0
140-145

AATTTGCAGAGGGTGAGTGG

PG5
GTTCTTGGTGTTGCATGGATGC
59.0
262-266

AGTTACGAAATGATTGCCAGAAG

C26L9(1265223)
CAAAGCTGCTGACGTGAATCAA
59.0
467-472

AGAGACGCTCCATGCAAAAG

UU-Cli14
CAGAACGTTTTGTTCTGTTTGG
58.0
265-292

TCTTGCTGCAGTCTTCATCC

C12L1(532572)
GTTGTTTGGCTGAGTGGACG
62.0
126-136

TCAACCAGGGGAATTGGCAG

C12L4(906353)
GCTGCTGTCTTCTTCATTGGG
60.0
210-250

TTAAAACCTCCCGTCTCCCTG

CliμD11
CCAATCCCAAAGAGGATTAT
58.0
78-98

ACTGTCCTATGGCTGAAGTG

C26L10(1404758)
GCTGTCAGGTATCAGCCACAA
59.0
211-226

TCAGACCCACGAAAGCTGTAA

C26L4(568923)
CAACCCCATGTGGGTGAGAC
63.0
357-432

CACCACCACGTGGGACATC

PG4
CCCATCTCCTGCCTGATGC
64.0
136-170

CACAGCAGGATGCTGCCTGC

UU-Cli12
CGCCAGACTGTATTGTGAGC
61.0
231-265

AGCATGGCTGTTCTTTGAGG

CliμT47
ATGTGTGTTTGTGCATGAAG
56.0
183-214

ATGAAAGCCTGTTAGTGGAA

CliμD32
GAGCCATTTCAGTGAGTGACA
60.0
136-158

GTTTGCAGGAGCGTGTAGAGAAGT

UU-Cli07
GCTGCCTGTTACTACCTGAGC
61.0
277-310

CTGGCCATGAAATGAACTCC

C26L1(20390)
AGGAGCCTACACTGGGTTTTC
60.0
250-268

TGTAGCTCTGCAATCAGCCT

3.1.3. Analysis of Population Microsatellite Loci

We inputted the results of STR scanning into Popgene 3.2 to analyze experimental chicken in the outbred groups and the haplotypes at 29 loci. In the outbred groups, 28 microsatellite loci show a high degree of polymorphism, and the average number of observed alleles is 4.571. The average number of effective alleles is 3.270, and the average Shannon’s information index is 1.198 (Table 5). Furthermore, the average effective heterozygosity is 0.492. The average polymorphism information content (PIC) is 0.610. All these data indicate a good genetic diversity of screening loci in the outbred groups and large heterozygosity difference among the laboratory experimental chicken populations.

Table 5: Number of alleles, effective alleles, effective heterozygosity, PIC, and Shannon’s index of the outbred colony chicken samples.

Loci
Observed number of alleles
Effective number of alleles
Shannon’s information index
Effective heterozygosity
PIC

MCW0029
4
2.931
1.209
0.579
0.603

GGNCAMZO
2
1.069
0.146
0.060
0.062

ADL0293
5
3.200
1.311
0.573
0.634

ADL0317
7
5.236
1.768
0.554
0.783

GGAVIR
3
1.916
0.796
0.456
0.408

ADL0201
5
2.103
1.013
0.429
0.482

GCT0016
5
3.042
1.274
0.337
0.618

ADL0304
6
4.641
1.627
0.666
0.751

MCW0402
8
6.042
1.881
0.702
0.813

MCW0063
7
4.319
1.626
0.568
0.736

ADL185
5
3.204
1.359
0.614
0.647

GGMYC
2
1.800
0.637
0.427
0.346

LEI0094
6
3.674
1.468
0.562
0.683

LEI0074
4
3.707
1.348
0.597
0.681

ADL328
3
2.785
1.058
0.526
0.565

GGVITC
1
1.000
0.000
0.000
1.000

GGANTECL
3
2.897
1.080
0.600
0.580

LEI094
6
4.444
1.579
0.690
0.738

MCW0330
4
3.232
1.269
0.577
0.637

LEI0141
4
3.162
1.229
0.341
0.623

ADL0292
3
2.793
1.061
0.475
0.568

GGVITIIG
2
1.965
0.684
0.460
0.371

MCW0087
8
5.930
1.898
0.544
0.810

MCW0347
3
1.948
0.815
0.447
0.419

ADL176
9
4.846
1.858
0.522
0.773

ADL166
5
3.729
1.380
0.574
0.682

MCW0014
5
4.342
1.543
0.592
0.735

GGCYMA
3
1.603
0.632
0.317
0.322

Mean
4.571
3.270
1.198
0.492
0.610

In the other 3 haplotype populations, 14 microsatellite loci showed monomorphism in each population but showed different lengths in different haplotype populations. The average number of observed alleles is 1.571. The average number of effective alleles, the average Shannon’s information index, and the average effective heterozygosity are 1.433, 0.316, and 0.207, respectively (Table 6). The specific data of each haplotype population is shown in Supplementary Tables 1–3.

Table 6: Number of alleles, effective alleles, effective heterozygosity, and Shannon’s index of the haplotype chicken samples.

Loci
Observed number of alleles
Effective number of alleles
Shannon’s information index
Effective heterozygosity

GGNCAMZO
1
1.000
0.000
0.000

GGAVIR
2
1.923
0.673
0.480

MCW0402
1
1.000
0.000
0.000

MCW0063
1
1.000
0.000
0.000

ADL185
3
2.174
0.898
0.540

GGMYC
1
1.000
0.000
0.000

LEI0094
3
2.778
1.055
0.640

GGVITC
1
1.000
0.000
0.000

ADL0292
2
1.471
0.500
0.320

GGVITIIG
2
2.000
0.693
0.500

ADL166
1
1.000
0.000
0.000

MCW0014
1
1.000
0.000
0.000

GGCYMA
1
1.000
0.000
0.000

STMSGGHU2-1A
2
1.724
0.611
0.420

Mean
1.571
1.434
0.316
0.207

In the outbred group of duck, 25 microsatellite loci show polymorphism. The average number of observed alleles is 7.520, and the average number of effective alleles in the population is 4.162. The average Shannon’s information index is 1.574, and the average effective heterozygosity is 0.683. The average PIC is 0.698. These data showed that in the outbred groups, the genetic diversity of microsatellite DNA loci is better, and the genetic diversity of each locus is quite different. The specific results are shown in Table 7.

Table 7: Number of alleles, effective alleles, effective heterozygosity, PIC, and Shannon’s index of outbred colony duck samples.

Loci
Observed number of alleles
Effective number of alleles
Shannon’s information index
Effective heterozygosity
PIC

CMO211
8
4.628
1.698
0.764
0.752

CAUD011
9
5.024
1.835
0.799
0.775

CAUD027
9
3.698
1.588
0.654
0.696

APH09
8
4.840
1.728
0.756
0.763

AY314
12
7.285
2.165
0.806
0.848

AY258
9
3.503
1.586
0.700
0.684

CAUD018
4
2.941
1.194
0.640
0.596

CAUD031
8
4.459
1.711
0.730
0.746

CAUD026
7
4.674
1.697
0.750
0.757

CAUD023
7
2.725
1.315
0.584
0.591

CMO212
8
4.154
1.642
0.739
0.724

CAUD006
4
3.333
1.280
0.440
0.645

CAUD004
7
5.556
1.834
0.720
0.798

CAUD001
6
5.000
1.696
0.600
0.772

CAUD034
10
3.943
1.742
0.730
0.723

CAUD007
8
3.894
1.639
0.714
0.713

APL579
7
3.068
1.412
0.635
0.636

CAUD010
6
4.655
1.630
0.768
0.753

CAUD028
5
3.549
1.378
0.541
0.668

CAUD012
7
3.122
1.354
0.652
0.630

CAUD035
10
5.768
1.922
0.759
0.804

CAUD014
9
3.600
1.448
0.696
0.672

CAUD032
14
6.159
2.120
0.797
0.821

APH11
2
1.923
0.673
0.479
0.365

APL2
4
2.556
1.067
0.609
0.529

Mean
7.520
4.162
1.574
0.683
0.698

In 4 haplotype populations, 15 microsatellite loci show monomorphism in each population. The average number of observed alleles is 4.133, the average number of effective alleles is 2.863, and the average Shannon’s information index is 1.153, indicating that the genetic diversity of the loci in these haplotype populations is poor; the average effective heterozygosity is 0.500, indicating that the heterozygosity difference is small and the genetic information of the selected loci is relatively single. See Table 8 for more detailed information, and the specific data in each haplotype population is shown in Supplementary Tables 4–7.

Table 8: Number of alleles, effective alleles, effective heterozygosity, and Shannon’s index of haplotype duck samples.

Loci
Observed number of alleles
Effective number of alleles
Shannon’s information index
Effective heterozygosity

CAUD002
3
2.020
0.857
0.360

CAUD006
4
2.740
1.142
0.540

CAUD018
3
1.802
0.746
0.400

CAUD005
5
3.945
1.490
0.551

APL579
5
2.632
1.205
0.500

APH18
7
4.301
1.655
0.640

CAUD010
3
2.597
1.010
0.420

CAUD028
2
1.980
0.688
0.360

CAUD012
3
2.597
1.010
0.420

CAUD035
4
3.756
1.353
0.605

CAUD014
4
3.509
1.306
0.580

CAUD026
4
2.740
1.142
0.520

CMO212
5
3.774
1.458
0.640

AY258
4
2.353
1.063
0.500

CAUD034
6
2.198
1.164
0.460

Mean
4.133
2.863
1.153
0.500

In the outbred colony of experimental goose, 14 loci were selected. The average number of observed alleles, the average number of effective alleles, the average Shannon’s information index, the average effective heterozygosity, and the PIC are 4.714, 3.038, 1.195, 0.528, and 0.582, respectively. The microsatellite loci have large interindividual differences within the population, and the population has high gene stability (Table 9).

Table 9: Number of alleles, effective alleles, effective heterozygosity, PIC, and Shannon’s index of outbred colony goose samples.

Loci
Observed number of alleles
Effective number of alleles
Shannon’s information index
Effective heterozygosity
PIC

G-Ans17
4
1.843
0.775
0.441
0.388

G-TTUCG1
3
2.255
0.943
0.381
0.494

G-APH13
4
1.605
0.752
0.315
0.352

G-Ans02
8
5.389
1.837
0.749
0.790

G-Ans07
4
3.073
1.220
0.634
0.613

G-Ans18
3
2.208
0.922
0.309
0.481

G-Ans25
4
3.333
1.282
0.629
0.647

G-Hhiμ1b
4
2.965
1.147
0.471
0.594

G-CKW47
4
3.143
1.238
0.573
0.623

G-Bcaμ5
3
2.728
1.051
0.469
0.562

G-Bcaμ7
6
2.731
1.158
0.455
0.562

G-Bcaμ8
7
2.845
1.290
0.635
0.599

G-CAUD006
4
3.704
1.344
0.602
0.680

G-APH20
8
4.713
1.772
0.734
0.761

Mean
4.714
3.038
1.195
0.528
0.582

The selected microsatellite loci all show good polymorphism in the experimental outbred pigeon populations. A total of 16 loci were selected. The average number of observed alleles is 7.875. The average effective allele number is 4.554; the average Shannon’s information index and the average effective heterozygosity are 1.559 and 0.649. The average PIC is 0.674 (Table 10).

Table 10: Number of alleles, effective alleles, effective heterozygosity, PIC, and Shannon’s index of outbred colony pigeon samples.

Loci
Observed number of alleles
Effective number of alleles
Shannon’s information index
Effective heterozygosity
PIC

UU-Cli02
5
3.613
1.374
0.694
0.672

UU-Cli06
4
2.921
1.163
0.383
0.593

PG5
2
1.681
0.595
0.397
0.323

C26L9(1265223)
4
2.576
1.076
0.602
0.533

UU-Cli14
10
5.144
1.923
0.787
0.784

C12L1(532572)
4
2.810
1.118
0.487
0.575

C12L4(906353)
11
6.375
2.052
0.766
0.825

CliμD11
7
4.541
1.682
0.734
0.750

C26L10(1404758)
11
9.118
2.281
0.860
0.880

C26L4(568923)
13
5.854
2.062
0.807
0.812

PG4
10
6.847
2.017
0.767
0.836

UU-Cli12
8
2.825
1.364
0.623
0.599

CliμT47
7
3.492
1.413
0.658
0.666

CliμD32
9
6.695
1.991
0.807
0.833

UU-Cli07
5
1.352
0.592
0.252
0.251

C26L1(20390)
16
7.014
2.244
0.759
0.844

Mean
7.875
4.554
1.559
0.649
0.674

3.1.4. Population Genetic Structure Analysis

Among the three outbred chicken groups, the mean number of observed alleles, the mean number of effective alleles, the mean Shannon’s information index, and the mean effective heterozygosity are shown in Table 11. All these data are the highest in the Beijing oil chicken, indicating the best gene diversity.

Table 11: Comparison of mean observed allele number, mean effective allele number, mean Shannon’s index, and mean effective heterozygosity among the outbred colonies of chickens.

Colonies
Mean observed number of alleles
Mean effective number of alleles
Mean Shannon’s information index
Mean effective heterozygosity

BWEL
2.857
2.024
0.730
0.424

BM
2.857
2.132
0.802
0.485

Beijing oil chicken
4.464
2.821
1.088
0.569

In the haplotype chicken populations, the highest mean observed number of alleles is observed in G7groups. Haplotype G7 has the highest mean effective allele number and the highest mean Shannon’s information index. The mean effective heterozygosity of haplotype G7 is 0.364. The genetic heterozygosity of the 3 populations is very low, and the consistency is good (Table 12).

Table 12: Comparison of mean observed allele number, mean effective allele number, mean Shannon’s index, and mean effective heterozygosity among the haplotype chickens.

Colonies
Mean observed number of alleles
Mean effective number of alleles
Mean Shannon’s information index
Mean effective heterozygosity

G1
1.571
1.434
0.316
0.207

G2
1.643
1.409
0.335
0.224

G7
2.000
1.626
0.548
0.364

In the two outbred groups of duck, the mean number of observed alleles, the mean effective number of alleles, the mean Shannon’s index, and the mean effective heterozygosity of outbred group 1 are higher than those of outbred group JD, indicating that outbred group 1 had better diversity. The results are shown in Table 13. Among the 4 haplotype populations, the highest mean number of alleles is observed in haplotype A. Haplotype A has the highest mean Shannon’s information index. The highest mean effective heterozygosity in the duck groups is 0.489 in haplotype A (Table 14). The genetic heterozygosity of 4 populations is in good agreement.

Table 13: Comparison of mean observed allele number, mean effective allele number, mean Shannon’s index, and mean effective heterozygosity among the outbred colonies of ducks.

Colonies
Mean observed number of alleles
Mean effective number of alleles
Mean Shannon’s information index
Mean effective heterozygosity

1
6.320
3.518
1.410
0.685

JD
5.280
3.466
1.335
0.680

Table 14: Comparison of mean observed allele number, mean effective allele number, mean Shannon’s index, and mean effective heterozygosity among the haplotype ducks.

Colonies
Mean observed number of alleles
Mean effective number of alleles
Mean Shannon’s information index
Mean effective heterozygosity

A
2.400
2.022
0.760
0.489

B
2.333
2.029
0.745
0.484

C
2.400
1.912
0.726
0.459

D
2.333
1.944
0.701
0.442

In the two outbred groups of goose, the mean number of observed alleles, the mean effective number of alleles, and the mean Shannon’s index of Guangdong Wuzong goose are higher than those of Yangzhou goose, indicating that Guangdong Wuzong goose has a better diversity (Table 15).

Table 15: Comparison of mean observed allele number, mean effective allele number, mean Shannon’s index, and mean effective heterozygosity among the outbred colonies of geese.

Colonies
Mean observed number of alleles
Mean effective number of alleles
Mean Shannon’s information index
Mean effective heterozygosity

Guangdong Wuzong
4.000
2.769
1.112
0.618

Yangzhou
3.714
2.155
0.802
0.439

The analysis of the two main experimental pigeon populations used for scientific research shows that the mean effective heterozygosity of two populations is 0.647 and 0.651, respectively. The mean number of observed alleles, the mean effective number of alleles, and the mean Shannon’s index are higher in white king pigeons than in silver king pigeons. The comparison of the data is shown in Table 16.

Table 16: Comparison of mean observed allele number, mean effective allele number, mean Shannon’s index, and mean effective heterozygosity among the outbred colonies of pigeons.

Colonies
Mean observed number of alleles
Mean effective number of alleles
Mean Shannon’s information index
Mean effective heterozygosity

Silver king
6.125
3.260
1.307
0.647

White king
7.375
4.247
1.435
0.651

4. Discussion

Poultries are widely used and are indispensable supporting conditions for the life sciences and biomedicine industries. Specific pathogen-free (SPF) chicken embryos are used in the manufacture and quality control of biological product [4]; ducks play an important role in the research of avian influenza, fatty liver, duck hepatitis A, and duck hepatitis B [5–7]; goose blood contains a higher concentration of immunoglobulin, which is often used in pharmacology and toxicology research [8]; pigeons belong to the class of birds and are considered as important animal model in avian influenza research [9]. With the increasing demand for experiment poultry, people are paying more attention to the genetic structure analysis and genetic quality control. However, the current methods of genetic structure analysis and genetic quality control for experimental poultry animals are insufficient.

Coat colour gene testing method, biochemical marker gene testing method, immune marker gene testing method, and DNA molecular marker method are popular methods for genetic monitoring. Microsatellite DNA, mitochondrial DNA (mtDNA), restriction fragment length polymorphism (PCR-RFLP), single-stranded conformation polymorphism (PCR-SSCP), and specific gene polymorphisms are commonly used DNA molecular marker methods [24–27]. Among them, microsatellite DNA has become valuable tools for evaluating population genetic diversity due to their unique virtue.

Microsatellite DNA is characterized by short tandem repeats (STRs) of 1 to 6 nucleotides in eukaryotic genome with a random manner [28]. It has rich polymorphism and large genetic information. Microsatellite can be used to distinguish heterozygous from homozygous because of their codominant inheritance feature [29]. In previous studies, microsatellites have been used as biomarkers for monitoring rodent genetic traits [30, 31]. With the deep understanding of microsatellites, it plays a more important role in genetic monitoring for being simple, clear, and stable in operation. In this research, we screened out microsatellite loci with suitable length and high specificity as candidate loci by gel electrophoresis firstly. Then, we performed STR scanning on these candidate loci. Microsatellite loci with good polymorphism, abundant alleles in the outbred groups, and good monomorphism in the haplotype populations were selected to form the microsatellite marker system. We analyzed the average effective allele number, average Shannon’s index, average effective heterozygosity, and other analytical indices to estimate genetic variation in different groups.

The mean effective number of alleles is an indicator of genetic variation and mutation drift balance. In our study, Beijing oil chicken has the highest mean effective allele number of three outbred chicken populations; outbred duck group 1 has higher mean effective allele number than outbred duck group JD. The outbred goose group Guangdong Wuzong and outbred pigeon group white king have the highest mean number of effective alleles in outbred goose populations and outbred pigeon populations, respectively. The higher mean effective number of alleles indicates that the population can maintain the original gene and avoid new variations under the pressures from genetic drift and artificial selection. The results show that Beijing oil chicken, outbred duck group 1, Guangdong Wuzong goose, and white king pigeon are the most stable strains in the outbred group of experiment chicken, duck, goose, and pigeon groups in this research, respectively.

The mean effective heterozygosity of a population is an important indicator of population genetic diversity and can reflect the richness of the detected genes. It is generally believed that when the mean effective heterozygosity of the population is less than 0.5, it indicates that the individual differences in the population are small and the genetic heterozygosity is low, which does not conform to the genetic characteristics of an outbred group animal. When the mean effective heterozygosity of the population is higher than 0.7, its genetic diversity is high [32].

Hence, we found that the mean effective heterozygosity of BWEL, BM, and Beijing oil chicken groups is all greater than 0.5, which conforms to the characteristics of the outbred group. The mean effective heterozygosity of BWEL and BM chicken groups is nearly 0.5. The average effective heterozygosity of G1, G2, and G7 groups is all less than 0.5. It is also consistent with the background that BWEL, BM, and Beijing oil chickens are outbred colonies; Beijing oil chicken has abundant genetic diversity and high selection potential for it has the highest mean effective heterozygosity among the outbred chicken groups in this study. This may be due to the large population. Duck group 1 and JD duck all have a mean effective heterozygosity greater than 0.680 which indicates a high genetic diversity. The mean effective heterozygosity of Guangdong Wuzong goose group, silver king pigeon group, and white king pigeon group is all greater than 0.5 which reflects abundant genetic diversity. The mean effective heterozygosity of three haplotype chicken groups and four haplotype duck groups is 0.207 and 0.500, respectively. The result indicates a good consistency in haplotype chickens and ducks. This may be the result of long-term full-sib and half-sib reproduction. Chickens and ducks are more widely used in biological research, and the breeding standards are stricter, while geese and pigeons are more useful in agriculture. Haplotype chickens have lower mean effective heterozygosity than haplotype duck populations, which is consistent with a longer history of breeding in experimental chickens.

When measuring the degree of gene variation, PIC is often used as a variation index. It is generally believed that when PIC is between 0.25 and 0.5, it is moderately polymorphic, and <0.25 shows a low level of polymorphism, when PIC is greater than 0.5, it means a high level of polymorphism [33]. In our microsatellite marker system, most of the microsatellite sites have a PIC greater than 0.5 that show high polymorphism. All these data prove that our microsatellite marker system provides rich genetic information, which can be used as effective genetic markers. In our study, highly polymorphic microsatellite marker systems showed powerful markers for quantifying genetic variations within and between poultry populations. We will collect more samples to make a more accurate description of genetic structure of the Chinese experimental chickens, ducks, geese, and pigeons in the future [34].

5. Conclusions

In conclusion, we identified appropriate microsatellite marker systems for native experimental chickens, ducks, geese, and pigeons in China. The combination of loci selected in our research provides a good choice for genetic monitoring of the quality and the population genetic diversity of poultry stocks.

Data Availability

All data, models, and code generated or used during the study appear in the submitted article.

Conflicts of Interest

The authors declare that there is no conflict of interest regarding the publication of this paper.

Acknowledgments

We are very grateful to the Institute of Animal Science, Chinese Academy of Agricultural Sciences, Harbin Veterinary Research Institute, Southern Medical University and Yangzhou University for providing animal samples for this study. This work was supported by the Beijing Municipal Science and Technology Projects (No. D181100000518002), Support Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year Plan (Grant Number IDHT20170516), and the National Key Research and Development Plan of China (No. 2017YFD0501602).

Supplementary Materials

Supplementary Table 1: number of alleles, effective alleles, effective heterozygosity, and Shannon’s index of the G1 haplotype chicken population. Supplementary Table 2: number of alleles, effective alleles, effective heterozygosity, and Shannon’s index of the G2 haplotype chicken population. Supplementary Table 3: number of alleles, effective alleles, effective heterozygosity, and Shannon’s index of the G7 haplotype chicken population. Supplementary Table 4: number of alleles, effective alleles, effective heterozygosity, and Shannon’s index of the A haplotype duck population. Supplementary Table 5: number of alleles, effective alleles, effective heterozygosity, and Shannon’s index of the B haplotype duck population. Supplementary Table 6: number of alleles, effective alleles, effective heterozygosity, and Shannon’s index of the C haplotype duck population. Supplementary Table 7: number of alleles, effective alleles, effective heterozygosity, and Shannon’s index of the D haplotype duck population. (Supplementary Materials)

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