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Association between Microsatellite Polymorphism and Body Size Traits in Sonid Bactrian Camels

PJZ_53_2_515-520

Association between Microsatellite Polymorphism and Body Size Traits in Sonid Bactrian Camels

Jun Yan Bai1*, Ren Tao Di Wu2, Qiang Zhang2, Di Bao2, Le Ma Dao2 and Xing Hua Tian3

1College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471023,China,

2Institute of Animal, Alxa of Inner Mongolia, Bayanhaote 750306, China

3School of Life Sciences, Henan Universiry, Kaifeng 475000, China

ABSTRACT

In this study, 17 microsatellite markers were used to analyze the genetic diversity of Sonid bactrian camels, and the correlation between microsatellite markers and their body size was analyzed to find microsatellite markers associated with the body size traits of Sonid bactrian camels, so as to provide reference for marker-assisted selection of Sonid bactrian camels. The results showed that 64 alleles were detected in 17 microsatellite markers. The average number of alleles was 3.7647, the average heterozygosity was 0.6205, and the average polymorphism information content was 0.5602. It showed that the population of Sonid bactrian camel had high genetic diversity. Among 17 microsatellite markers, 4 microsatellites were found to be associated with body size traits of Sonid bactrian camels. The body length of AB genotype labeled LCA33 was significantly higher than that of AA genotype (P<0.05), and the chest circumference, tube circumference and body weight of AB genotype were significantly higher than those of AA genotype and AC genotype (P <0.05). The chest circumference and body weight of A A genotype marked LCA90 were significantly higher than those of AC genotype and BB genotype (P < 0.05). The body length of AB genotype marked CMS36 was significantly higher than that of AA genotype and BB genotype (P < 0.05). The body weight of CE genotype marked YWLL44 was significantly higher than that of AC genotype, BD genotype and BE genotype (P< 0.05), the circumference of CE genotype was significantly higher than that of AC genotype (P < 0.05), and the chest circumference of CE genotype was significantly higher than that of AC genotype and BD genotype (P< 0.05). The other 13 microsatellite markers had no significant effect on the body size of Sonid bactrian camels.


Article Information

Received 05 September 2019

Revised 22 September 2019

Accepted 01 October 2019

Available online 12 February 2021

Authors’ Contribution

JYB conceived and designed the study and conducted the lab work. RTDW and QZ analyzed the data and wrote the article. DB and LMD helped in sampling. XHT helped in analysis

of data.

Key words

Sonid bactrian camel, Microsatellite markers, Genetic diversity, Body size traits, Association analysis

DOI: https://dx.doi.org/10.17582/journal.pjz/20190905010927

* Corresponding author: junyanbai@163.com

0030-9923/2021/0002-0515 $ 9.00/0

Copyright 2021 Zoological Society of Pakistan


INTRODUCTION

Bactrian camels, which are special livestock species with double back humps and tall body and adapting to desert and semi-desert regions, mainly live in deserts, and their range of activity is mainly located in western and northern Asia. The region where bactrian camels live is very narrow and it is only restricted to arid areas, so they are precious livestock species in the world. In recent years, domestic and foreign researches regarding hair characteristics (Wuren et al., 2017), dairy products (Zhang et al., 2016), blood physiological properties (Bai et al., 2015), genetic diversity (Hedayat-Evrigh et al., 2018; Banerjee et al., 2012) and organization structure (Ye et al., 2014a, 2014b) of bactrian camels have achieved progress.

Microsatellite markers have been extensively applied to genetic diversity studies of cow (Ni et al., 2018), sheep (Bai et al., 2015) and poultry (Bai et al., 2016a, 2016b, 2016c, 2017) by virtue of high abundance, good repeatability, co-dominance marker and selective neutrality, It is also widely used in plant genetic diversity research (Yang et al., 2013; Li et al.,2017; Guo et al., 2018). Genetic diversity of 17 microsatellite markers in the Sonid Bactrian camel population was analyzed in this study, association analysis of these microsatellite markers with body weight and body size and other traits was conducted, and microsatellite markers associated with body size traits of Sonid bactrian camels was found out so as to provide a reference for marker assisted selection, protection and rational utilization of Sonid bactrian camels.

MATERIALS AND METHODS

Test material

Random collection of 40 Sonid bactrian camels, blood was taken from jugular vein (10ml from each bactrian camel), ACD anticoagulant was added for anticoagulation, blood DNA was extracted using the whole-blood genomic DNA extraction kit method from Beijing Dingguo Changsheng, and it was preserved under -20. Seventeen microsatellite markers with high polymorphism were screened (Evdotchenko et al., 2003; Prasad et al., 2014),

 

Table I. Microsatellite marker information.

Name

Primer sequence

Fragment size (bp)

Annealing temperature

CVRL101

F:GAAGAGGTTGGGGCACTAC; R:CAGGCAGATATCCATTGAA

188-253

60

CMS15

F:AAAACTAAAGCCAGAAAGGCAAA; R:TTTTTCCAGATCTTGCACCAC

81-121

58

CMS18

F:GAACGACCCTTGAAGACGAA; R:AGCAGCTGGTTTTAGGTCCA

144-166

55

CMS36

F:TGCTTTCCAGTTGTTTGCTG; R:GCAAGGTGGTGTTGGAGATT

195-227

55

CMS104

F:CACTTAGGTCCCTGGGCTTT; R:GCATTCTCTTGCATCGTGTG

75-101

55

LCA33

F:GAGCACAGGGAAGGATATTCA; R:ACAGCAAAGTGATTCCATAATACA

122-167

55

LCA37

F:TAATTACCTCCCCCACCACA; R:TGGACCCAGGACTTGAAATG

143-183

55

LCA63

F:TTACCCAGTCCTTCGTGGG; R:GGAACCTCGTGGTTATGGAA

213-233

54

LCA66

F:GTGCAGCGTCCAAATAGTCA; R:CCAGCATCGTCCAGTATTCA

212-241

55

LCA71

F:CCGTATCTATCTATACACACACACACA; R:TCACCCTCCTCCTATTTTGG

127-167

55

LCA82

F:CGTGACACCAGGCTAAGTGA; R:TTTCAGATGGTAGCTTTAAAAATTG

85-129

55

LCA90

F:TATAACCCTGGTCTCGCCAA ; R:CCAAGTAGTATTCCATTATGCG

237-248

55

VOLP08

F:CCATTCACCCCATCTCTC ; R:TCGCCAGTGACCTTATTTAGA

142~172

53.3

VOLP32

F:GTGATCGGAATGGCTTGAAA ; R:CAGCGAGCACCTGAAAGAA

147–203

53.3

YWLL29

F:GAAGGCAGGAGAAAAGGTAG; R:CAGAGGCTTAATAACTTGCAG

200-228

55

YWLL36

F:CAAATGTTGTAAAATTGCACAGAAC; R:TTCAGGTTTGCCTAGTTTTAACTGT

149-189

55

YWLL44

F:CTCAACAATGCTAGACCTTGG; R:GAGAACACAGGCTGGTGAATA

95-111

55

 

and the sequence of primers was shown in Table I. The primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.

PCR amplification

Pre-denaturation at 94 for 4 min, then denaturation at 94 for 40 s, annealing at 60 for 1 min, annealing at 72 for 20 seconds, denaturation, annealing and elongation were carried out for 35 cycles, then elongation at 72 and finally the reaction was completed and cooled and preserved at 4 C.


 

Table II. Allele frequencies of microsatellite markers.

Microsatellite markers

Alleles

A

B

C

D

E

LCA33

0.6000

0.0875

0.2500

0.0625

LCA37

0.4750

0.5000

0.0250

LCA63

0.4250

0.1375

0.3625

0.0750

LCA66

0.1625

0.3375

0.1625

0.3375

LCA71

0.0625

0.4625

0.4750

LCA82

0.4500

0.0500

0.4500

0.0500

LCA90

0.3500

0.2750

0.1750

0.2000

CMS15

0.0375

0.2875

0.4625

0.2125

CMS18

0.5000

0.2875

0.1875

0.0250

CMS36

0.5875

0.4125

CMS104

0.5125

0.3750

0.1125

CVRL101

0.1375

0.3000

0.2000

0.2375

0.1250

YWLL29

0.6625

0.1125

0.2250

YWLL36

0.0250

0.4750

0.0250

0.4750

YWLL44

0.0250

0.4375

0.0625

0.2625

0.2125

VOLP08

0.2250

0.2750

0.2250

0.2750

VOLP32

0.2875

0.2125

0.2875

0.2125

 

The amplified product 5μl was detected by 2% agarose electrophoresis at 120 V for 30 min. After electrophoresis, the results were observed under ultraviolet light and photographed on the basis of DNA marker D2000.

 

Table III. Polymorphic information of microsatellite markers.

Microsatellite

markers

Na

Ne

O_Hom

O_Het

E_Hom

E_Het

Ave_Het

PIC

LCA33

4.0000

2.3038

0.2500

0.7500

0.4269

0.5731

0.5356

0.5111

LCA37

3.0000

2.0997

0.0000

1.0000

0.4696

0.5304

0.5248

0.4103

LCA63

4.0000

2.9712

0.0000

1.0000

0.3282

0.6718

0.6756

0.6004

LCA66

4.0000

3.5635

0.0000

1.0000

0.2715

0.7285

0.7330

0.6680

LCA71

3.0000

2.2551

0.0500

0.9500

0.4364

0.5636

0.5727

0.4566

LCA82

4.0000

2.4390

0.0000

1.0000

0.4025

0.5975

0.6045

0.5039

LCA90

4.0000

3.7209

0.2500

0.7500

0.2595

0.7405

0.7270

0.6823

CMS15

4.0000

2.9144

0.0000

1.0000

0.3348

0.6652

0.6782

0.5938

CMS18

4.0000

2.7142

0.0000

1.0000

0.3604

0.6396

0.6292

0.5664

CMS36

2.0000

1.9406

0.8750

0.1250

0.5092

0.4908

0.3795

0.3672

CMS104

3.0000

2.4042

0.1250

0.8750

0.4085

0.5915

0.5728

0.5000

CVRL101

5.0000

4.5262

0.0000

1.0000

0.2111

0.7889

0.7464

0.7437

YWLL29

3.0000

1.9913

0.5500

0.4500

0.4959

0.5041

0.4025

0.4410

YWLL36

4.0000

2.2099

0.0000

1.0000

0.4456

0.5544

0.5993

0.4446

YWLL44

5.0000

3.2258

0.0000

1.0000

0.3013

0.6987

0.6963

0.6373

VOLP08

4.0000

3.9604

0.0000

1.0000

0.2430

0.7570

0.7413

0.7003

VOLP32

4.0000

3.9120

0.0000

1.0000

0.2462

0.7538

0.7298

0.6968

Mean

3.7647

2.8913

0.1235

0.8765

0.3618

0.6382

0.6205

0.5602

 

SSCP

15% non denaturing polyacrylamide gels were used to detect the products, silver nitrate dyeing method is used for dyeing, mainly through fixation, oxidation, dyeing, color rendering, photography and other links.

Statistical analysis

Popgene32 software was used to calculate numbers of effective alleles, allele frequencies and heterozygosity of microsatellites. SPSS software was used to conduct association analysis of microsatellites with body weight, and the following was the analytical model: yijkl=µ+Si+Mj+Gk+eijkl, where yijkl is trait phenotypic value, µ is ensemble average and Si is the i (th) sex effect; Mj is the j (th) age effect; Gk is the k (th) genotype effect; eijkl is residual error effect.

RESULTS AND DISCUSSION

Polymorphism detection of microsatellite markers

The polyacrylamide gel electrophoresis results of PCR products of some markers are presented in Figure 1. The allele frequencies of microsatellite markers in Sonid bactrian camel population are shown in Table II. The maximum number of alleles detected by CVRL101 is 5, the minimum number of alleles detected by CMS36 is 2, the number of alleles detected by other markers is between 3 and 4.

The polymorphism of microsatellite markers can be seen in Table III, 64 alleles were detected in 17 microsatellite markers. The average number of alleles was 3.7647, the average heterozygosity was 0.6205, and the average polymorphism information content was 0.5602. It can be seen that the Sonid bactrian camel has high genetic diversity. The study carried out by Gao et al. (2009) showed that the average polymorphic information content value of microsatellite markers detected in hundreds of bactrian camels from 13 areas of China and Mongolia was 0.5414. Tian et al. (2012) studied the genetic diversity of bactrian camels from 6 places of Xinjiang by microsatellite markers, and showed that all of the used microsatellite markers presented high polymorphism, with polymorphic information content values ranging from 0.6099 to 0.6551. Vijh et al. (2007) conducted a microsatellite marker analysis of 4 dromedary species in India, and results indicated that a large number of alleles were under a low frequency. So the polymorphic level of microsatellite markers in this study was moderate, intermediate between Gao et al. (2009) and Tian et al. (2012).

 

Table IV. Association between microsatellite markers and body size of Sonid bactrian camels.

Name

Genotype

Body height/cm

Body length/cm

Bust/cm

Round tube/cm

Weight/ kg

LCA33

AA

161.000 ±02.0330 a

139.6000 ±2.9257 b

201.6000 ±3.3373 b

16.8000 ±0.2494 b

445.2900 ±17.6812 b

AB

168.2000 ±2.7459 a

154.0000 ±3.3015 a

225.4000 ±4.2614 a

18.4000 ±0.5099 a

568.9090 ±22.5901 a

AC

163.3889 ±2.2854 a

145.8889 ±2.5112 ab

211.4444 ±4.2994 ab

17.2778 ±0.3110 ab

498.6865 ±22.2293 ab

AD

165.8000 ±0.9695 a

146.4000 ±2.5416 ab

218.6000 ±5.9883 ab

17.2000 ±0.3741 ab

521.3083 ±26.7245 ab

BC

157.5000 ±7.5000 a

141.0000 ±9.0000 ab

198.5000 ±6.5000 b

16.5000 ±0.5000 b

437.5177 ±41.8083 b

LCA37

AB

163.2105 ±1.3303 a

144.8947 ±1.6611 a

210.8421 ±2.7096 a

17.2895 ±0.1919 a

493.0276 ±13.8334 a

BC

167.0000 ±2.0000 a

150.0000 ±4.0000 a

213.5000 ±2.5000 a

16.5000 ±0.5000 a

510.1657 ±1.2928 a

LCA63

AB

164.0000 ±2.7928 a

148.0000 ±6.2928 a

210.6000 ±7.0964 a

17.8000 ±0.6633 a

500.1861 ±40.2844 a

AC

163.4483 ±1.5604 a

144.9655 ±1.7281 a

212.0690 ±3.0681 a

17.1379 ±0.2089 a

497.0368 ±15.3422 a

BD

162.6667 ±3.5839 a

143.6667 ±4.7935 a

206.0000 ±7.1039 a

17.3333 ±0.4944 a

473.3970 ±37.8698 a

LCA66

AC

165.8462 ±2.6234 a

145.1538 ±2.9566 a

213.5385 ±5.8677 a

17.4615 ±0.3859 a

505.7870 ±29.4103 a

BD

162.2222 ±1.3788 a

145.1481 ±1.9235 a

209.7407 ±2.6287 a

17.1481 ±0.2046 a

488.1536 ±13.7101 a

LCA71

AC

161.8000 ±4.1641 a

141.4000 ±3.6551 a

202.8000 ±5.7740 a

16.8000 ±0.3741 a

453.7243 ±27.2104 a

BB

162.0000 ±5.0000 a

142.5000 ±1.5000 a

208.0000 ±5.0000 a

17.0000 ±0.0000 a

471.2891 ±13.4620 a

B

163.7273 ±1.4123 a

145.8788 ±1.8431 a

212.3939 ±2.9538 a

17.3333 ±0.2161 a

501.3388 ±15.1859 a

LCA82

AC

162.9167 ±1.2359 a

145.1944 ±1.6382 a

210.6944 ±2.5054 a

17.2222 ±0.1695 a

492.1925 ±12.4821 a

BD

167.7500 ±6.5494 a

144.7500 ±6.9447 a

213.5000 ±14.1745 a

17.5000 ±1.1902 a

509.1126 ±77.6066 a

LCA90

AA

166.0000 ±1.4960 a

150.1429 ±2.9230 a

221.5714 ±5.0749 a

17.8571 ±0.5084 a

544.7257a ±27.4807 a

AC

163.7857 ±1.6811 a

144.6429 ±2.4147 a

210.5000 ±3.4668 b

17.2857 ±0.1941 a

488.7325 ±17.2819 b

BB

163.6667 ±8.6474 a

146.6667 ±9.8375 a

207.0000 ±9.0185 b

17.6667 ±0.8819 a

483.2152 ±52.1286 b

BD

161.8750 ±2.3977 a

143.1250 ±2.6690 a

207.5000 ±4.8140 b

16.8750 ±0.3275 a

478.1499 ±24.5348 b

CMS15

AB

165.3333 ±2.6666 a

146.3333 ±4.1766 a

218.3333 ±11.2891 a

17.0000 ±0.5773 a

522.6098 ±51.1865 a

BC

163.6000 ±1.5481 a

144.6500 ±1.7877 a

210.2000 ±3.1217 a

17.1500 ±0.1956 a

487.9703 ±14.9405 a

CD

162.8235 ±2.3890 a

145.5294 ±3.1096 a

210.5882 ±4.5794 a

17.4118 ±0.3644 a

495.7731 ±24.6920 a

CMS18

AB

164.0000 ±1.7286 a

144.5652 ±1.9819 a

213.1739 ±3.6930 a

17.2609 ±0.2608 a

500.9518 ±18.9254 a

AC

162.0000 ±2.1224 a

145.4667 ±3.0282 a

207.2000 ±3.8436 a

17.1333 ±0.2905 a

480.7806 ±20.0080 a

AD

167.0000 ±1.0000 a

149.5000 ±0.5000 a

214.0000 ±1.0000 a

18.0000 ±0.0000 a

510.8888 ±2.3896 a

CMS36

AA

162.7619 ±1.6314 a

144.5714 ±1.9130b

211.7143 ±3.4751 a

17.2381 ±0.2478 a

494.6097 ±17.3440 a

AB

167.4000 ±1.8055 a

154.0000 ±4.3243a

214.6000 ±3.0757 a

17.2000 ±0.3741 a

525.4455 ±16.1662 a

BB

162.9286 ±2.6195 a

142.8571 ±2.9442b

208.5714 ±5.2043 a

17.2857 ±0.3695 a

481.5249 ±26.8202 a

CMS104

AA

166.0000 ±3.7859 a

151.6667 ±6.1191 a

210.6667 ±4.7022 a

17.0000 ±0.5773 a

505.8133 ±33.0667 a

AB

163.4615 ±1.7639 a

144.2308 ±2.0140 a

211.8077 ±3.6089 a

17.3077 ±0.2403 a

495.8371 ±18.4025 a

AC

162.0000 ±2.2173 a

143.6667 ±2.9154 a

208.3333 ±4.7900 a

17.1111 ±0.4231 a

478.9616 ±22.6887 a

BB

165.0000 ±3.0000 a

154.0000 ±9.0000 a

212.5000 ±2.5000 a

17.5000 ±0.5000 a

517.7604 ±33.1562 a

CVRL101

AB

165.6000 ±1.0295 a

144.2000 ±1.5937 a

217.0000 ±5.9916 a

17.0000 ±0.0000 a

509.7066 ±26.4570 a

AC

163.1667 ±1.7966 a

142.0000 ±4.0166 a

207.6667 ±7.1305 a

17.0000 ±0.4472 a

473.8557 ±34.8770 a

BD

162.8421 ±1.5844 a

146.3158 ±2.2530 a

211.6842 ±3.3113 a

17.3684 ±0.2560 a

498.6778 ±17.5233 a

CE

163.5000 ±4.1048 a

145.3000 ±4.2165 a

208.6000 ±6.7019 a

17.3000 ±0.5174 a

488.8834 ±34.9227 a

YWLL29

AA

164.2727 ±1.7434 a

146.2273 ±2.1370 a

211.2727 ±3.5144 a

17.2273 ±0.2626 a

497.7660 ±18.5661 a

AC

161.0000 ±2.7487 a

141.6667 ±2.7436 a

209.1111 ±5.7019 a

17.1111 ±0.2605 a

477.8839 ±26.1093 a

BC

163.6667 ±2.6510 a

146.0000 ±4.0276 a

212.1111 ±5.5987 a

17.4444 ±0.4746 a

500.3969 ±28.4474 a

YWLL36

BD

156.0000 ±9.0000 a

137.5000 ±8.5000 a

202.5000 ±13.5000 b

16.0000 ±0.0000 a

444.7873 ±64.0855 a

CE

163.7895 ±1.2639 a

145.5526 ±1.6207 a

211.4211 ±2.6445 a

17.3158 ±0.1889 a

496.4685 ±13.5022 a

YWLL44

AC

164.0000 ±5.0000 a

144.0000 ±10.0000 a

205.5000 ±5.5000 b

16.5000 ±0.5000 b

467.9481 ±43.5104 b

BD

162.5714 ±1.8510 a

144.6190 ±2.2860 a

208.4762 ±3.2038 b

17.0476 ±0.2233 ab

482.9272 ±16.3426 b

BE

162.5000 ±1.5468 a

144.6429 ±2.4798 a

212.5714 ±4.4987 ab

17.4286 ±0.2911 ab

498.0232 ±21.9750 b

CE

173.0000 ±7.0237 a

152.0000 ±7.0000 a

224.6667 ±15.8359 a

18.3333 ±1.3333 a

568.5623 ±86.5378 a

VOLP08

AC

161.0000 ±1.8185 a

142.6111 ±2.2825 a

206.5556 ±3.8866 a

17.0556 ±0.2056 a

471.9134 ±18.9111 a

BD

165.3636 ±1.6922 a

147.2273 ±2.1601 a

214.5909 ±3.3183 a

17.4091 ±0.2917 a

511.8608 ±17.6782 a

VOLP32

AC

164.6087 ±1.8657 a

145.8261 ±2.2419 a

209.4348 ±3.3242 a

17.4348 ±0.2654 a

490.2826 ±17.8966 a

BD

161.7647 ±1.5846 a

144.2353 ±2.2630 a

213.0588 ±4.1253 a

17.0000 ±0.2425 a

498.7577 ± 19.8198 a

 

Association between polymorphism of microsatellite markers and body size traits of bactrian camels

The association between microsatellite marker polymorphism and body size traits of bactrian camels is shown in Table IV. From Table IV, it can be seen that the body length of AB genotype marked LCA33 is significantly higher than that of AA genotype (P<0.05), and the chest circumference, tube circumference and body weight of AB genotype marked LCA33 are significantly higher than those of AA genotype and AC genotype (P<0.05). The chest circumference and body weight of A A genotype marked LCA90 were significantly higher than those of AC genotype and BB genotype (P<0.05). The body length of AB genotype marked CMS36 was significantly higher than that of AA genotype and BB genotype (P<0.05). The body weight of CE genotype marked YWLL44 was significantly higher than that of AC genotype, BD genotype and BE genotype (P<0.05), the tube circumference of CE genotype marked YWLL44 was significantly higher than that of AC genotype (P<0.05), and the chest circumference of CE genotype marked YWLL44 was significantly higher than that of AC genotype and BD genotype (P<0.05). The other 13 microsatellite markers had no significant effect on the size traits of Sonid bactrian camels(P>0.05). Therefore, microsatellite markers LCA33, CMS36, LCA90 and YWLL44 can be used in assistant selection of body size and weight markers for Sonid bactrian camels. Which was similar to the study results of Wuren et al. (2018a, 2018b) . By now, very few studies on bactrian camels association analysis and genetic mapping were reported, which limited the superior alleles exploring and marker assisted selection breeding developing. The4 microsatellite markers associated with body size or body weight traits in this study would be valuable in improving bactrian camels by molecular polymerization breeding, which could gather all of the superior alleles from 4 microsatellite markers into one individual.

There are more than 280,000 camels in China, of which Inner Mongolia Autonomous Region is the largest, accounting for about 67% of the total number of camels in China, followed by Xinjiang Uygur Autonomous Region, accounting for about 20%. To protect the genetic diversity of bactrian camel resources in Inner Mongolia Autonomous Region is not only to rationally manage and utilize the existing resources, but also to maintain a certain resource potential for future needs.

ACKNOWLEDGEMENT

Sincere gratitude goes to the sponsor of Alxa League Science and Technology Project in Inner Mongolia (2012-12).

Statement of conflict of interest

The authors declare there is no conflict of interest.

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Pakistan Journal of Zoology

February

Vol. 53, Iss. 1, Pages 1-400

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