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Genetic diversity and relationships among indigenous Borgou and White Fulani cattle breeds based on milk protein loci: Implications for breed improvement and conservation in Benin

Houaga Isidore, Chakirath F A Salifou1, Antoine Abel Missihoun2, Paulin Sedah2, Clément Agbangla2, Anne W T Muigai3, Souradjou O G Idrissou1, Kévin S Kassa1 and Issaka A K Youssao1

Research Unit "Vector-Borne Diseases and Biodiversity" (UMaVeB), Centre International de Recherche-Développement sur l’Elevage en zone Subhumide (CIRDES), 01 BP 454 Bobo-Dioulasso 01, Burkina Faso
ihouaga@gmail.com
1 Laboratory of Animal Biotechnology and Meat Technology, Department of Animal Health and Production, Polytechnic school of Abomey-Calavi University, 01 BP 2009 Cotonou, Benin Republic
2 Department of Genetics and Biotechnology, Faculty of Sciences and Technics (FAST), University of Abomey-Calavi, BP 526 Cotonou, Benin
3 Department of Botany, Jomo-Kenyatta University of Agriculture and Technology, P O Box 62000-200 Nairobi, Kenya

Abstract

Genetic diversity in a population is a prerequisite for implementing genetic selection. The aim of the study was to assess the genetic diversity, the gene flow and phylogenetic relationships among Borgou and White Fulani cattle breeds in Benin using the β-Lactoglobulin ( MBLG), α-Lactalbumin (LALBA), αS1-Casein (CSN1S1 ) and κ-Casein (CSN3) genetic polymorphisms. Thus, 94 Borgou and 96 White Fulani indigenous cattle breeds were genotyped using polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) and Sanger sequencing methods. The results showed that all the studied loci were polymorphic. Borgou presented higher (p <0.01) observed heterozygosity (Hob) and unbiased gene diversity (H ex) (0.33, 0.36) than White Fulani (0.31, 0.33). The mean number of observed (MNA) alleles per locus was 2±0.000 in both breeds. The means expected number of alleles (MNE) were similar (p>0.05) in Borgou (1.59 ± 0.27) and White Fulani (1.50 ± 0.33). The f (within population inbreeding) and F (total inbreeding) were respectively 0.11 and 0.13 and were not different from zero (p>0.05). The population differentiation (q) was 0.02 and different from zero (p <0.01). High level of gene flow (Nem=16.44), a low Nei’s DA genetic distance (0.014) and a very close phylogenetic relationship were observed between both breeds. The high genetic exchange observed between the two indigenous cattle breeds is a big concern. There is therefore a need to develop a sustainable breeding program aiming to improve cattle productivity as well as their conservation in Benin. However, further studies with high density SNP chips are needed to better understand the genome-wide diversity of the studied breeds.

Keywords: Borgou, gene flow, genetic diversity, White Fulani


Introduction

The cattle population of Benin was estimated at approximately 2.3 million head in 2016 (FAOSTAT 2018). The cattle population consists mainly of indigenous Bos taurus cattle breeds namely, Borgou, Somba, and Lagune and Bos indicus breeds namely, Gudali and White Fulani and their different crossbreds (Youssao 2015). Borgou cattle breed represents more than 50 % of the total cattle population in Benin (MAEP 2007) while White Fulani Breed is the most common Zebu cattle breed of the country. The two indigenous breeds are the main milk and meat producer in Benin. There are two breeding systems in Benin; these include a traditional breeding system and a semi-modern breeding system (Youssao et al 2009). The latter is practiced only in government farms. Thus the traditional breeding system which is practiced throughout the rest of Benin is the most common method used. These cattle breeders in the traditional breeding system do not have clear breeding objectives that are defined by production of milk or meat (Alkoiret et al 2011). Although Borgou and White Fulani breeds play important role in provision of milk and meat, there are no effective genetic improvement programmes in place for these traits hence their current low productivity.

The level of genetic diversity in a population is essential and has practical implication for implementing genetic selection (Brito et al 2017). In Benin, previous studies characterized the main indigenous cattle breeds using mainly microsatellite markers (Moazami-Goudarzi et al 2001; Koudandé et al 2009; Kassa et al 2019). From the last decade, there has been a lot of introduction of exotic cattle breeds in Benin hence different crossbreeding with local cattle breeds. There is therefore a need of new molecular characterization of Borgou and White Fulani, the main indigenous cattle breeds of Benin. Furthermore, Borgou and White Fulani cattle breeds are less characterized at major milk protein loci although the milk protein genetic polymorphism is an important tool for genetic diversity, breed characterization, gene evolutionary studies with many applications in human nutrition and animal breeding (Caroli et al 2009). The casein loci have been used to investigate genetic diversity, origin and phylogeny of taurine cattle (Jann et al 2004). Moreover, the polymorphisms in β-Lactoglobulin ( MBLG), α-Lactalbumin (LALBA), αS1-Casein (CSN1S1 ) and κ-Casein (CSN3) genes have been used to assess the genetic diversity and relationships between West and Central African indigenous cattle populations (Ibeagha-Awemu et al 2004; Ibeagha-Awemu and Erhardt 2006). High levels of zebu introgression have been reported in some African taurine breeds (Hanotte et al 2002; Ibeagha-Awemu et al 2004; Koudandé et al 2009; Kassa et al 2019). This situation is alarming and will lead to the loss of African taurines diversity should this trend continue. For the purposes of breed improvement and conservation in Benin, there is a need to assess the diversity at major milk protein loci in Borgou and White Fulani, the main the indigenous cattle breeds of the country.

Therefore, the aim of the study was to assess the genetic diversity, the gene flow and phylogenetic relationships among Borgou and White Fulani cattle breeds using the MBLG, LALBA, CSN1S1 and CSN3 genetic markers. The findings of the study will guide for sustainable improvement and conservations decisions.


Materials and methods

Sampling
Description of study area

Borgou cattle were sampled from state owned farms (Samiondji Breeding Farm, Betecoucou Breeding Farm and Okpara Breeding Farm) and privately owned farms in Benin. It was critical that the government farms were included in the sampling because they are centers of conservation of the indigenous Borgou cattle breed. The White Fulani cattle were sampled from the privately owned farms in peri-urban area of Parakou District because there is no center of conservation of White Fulani cattle breed. Figure 1 shows locations where the sampling was conducted.

The Samiondji Breeding Farm (SBF) is located in the Department of Zou, District of Zangnanado (Benin). SBF has an area of 3,600 hectares and lies between latitudes 7/25°N and 7/30°N and longitudes 2/22°E and 2/25°E. The climate is intermediate between the subequatorial maritime and the Sudano-Guinean climate characterized by 4 seasons: a long dry season (November to March), a long rainy season (March to July), a short dry season (July to August) and a short rainy season (August to November). The average rainfall varies between 900 and 1100 mm per year. The average annual temperature is around 29 ° C. The types of fodders found areBrachiaria ruziziensis, Panicum C1,Moringa Oleifera, Andropogon gayanus and stylosanthes guineensis.

The Betecoucou Breeding Farm (BBF) is located in the Department of Collines and the District of Dassa (Benin). The BBF has an area of 11,127 hectares and lies between latitudes 7/45°N and 7/50°N and longitudes 2/20°E and 2/27°E. The climate is Sudano-Guinean with one rainy season from April to October and one dry season from November to March. The average rainfall is 1100 mm per year and the temperature between 24 and 25°C. The main fodders found are Brachiaria ruziziensis, Panicum C1 and Panicum maximum.

The Okpara Breeding Farm (OBF) is located in the Department of Borgou, District of Tchaourou. The farm has an area of 10,000 hectares and lies between latitudes 9/6°N and 9/21°N and longitudes 2/39°E and 2/53°E. The climate is Sudanian with one rainy season from May to October and one dry season from November to April. The average rainfall is 1125 mm per year and the temperature between 26 and 27°C. The different cattle breeds found are Borgou, Azawak, N’Dama, Girolando and Girolando X Borgou and Gir X Borgou crossbreds. The main fodders found are Brachiaria ruziziensis, Panicum maximum, Andropogon gayanus, Leucena leucocephala and stylosanthes sp.

Figure 1. Map of Benin showing the study area
Blood and milk sampling

A total of 94 Borgou (BO) and 96 White Fulani (WF) indigenous cows in lactation were sampled in this study between May-July 2016 during the rainy season. The sampling was done during rainy season because the White Fulani herdsmen are pastoralists and herd their animals in the forest during dry season. Blood samples were obtained from three to ten cows in lactation per herd from a total of 12 herds of Borgou and 9 herds of White Fulani. Pedigree information was obtained from farmers in order to avoid related animals. Blood samples were collected from the jugular vein into 10 ml EDTA vacutainer tubes, labelled and immediately transported to the laboratory in a cool box containing ice and stored at -20°C until further analysis. The use of animals and sample collection procedures were in accordance with the national ethical standards. The farmers gave a written consent prior to blood sample collection.

DNA extraction, quantity and quality check

Genomic DNA was isolated from blood samples using standard phenol-chloroform method (Sambrook and Russell 2001). The DNA extraction was carried out at the Molecular Genetics and Genome Analysis laboratory of Abomey-Calavi University (Benin). Briefly, the frozen 10ml blood sample was thawed at room temperature. The tube was inverted gently to homogenize the contents and 1ml of blood was transferred into 2ml Eppendorf tube.One ml of T10E1 washing buffer (10ml 1M tris HCL (pH= 8.5), 2ml 0.5M EDTA, 988 ml dH2O) was added. The tube was vortexed then centrifuged at 7235×g for 10 min at 4°C and the supernatant discarded. The white blood cells were washed a second time. One ml of SDS (Sodium Dodecyl Sulphate) lysis buffer (193g urea, 7 g NaCl, 8g SDS, T10E1 added to a 400 ml final volume) was added. The tube was then incubated at 37°C overnight. One ml of phenol chloroform isoamylic alcohol (25 volume phenol, 24 volume chloroform and 1 volume isoamylic alcohol) was added. The tube was mixed thoroughly by inverting the tube for 15 min and centrifuged at 7235×g for 15 min. The upper layer containing the DNA was transferred into a new clean 2ml Eppendorf tube. Two volumes of cold 96% ethanol was added and the tube mixed by inverting several times for precipitation. The tube was centrifuged at 7235×g for 10mn at 4°C and the supernatant discarded. The DNA pellet was washed twice with 500µl of 70% Ethanol. The DNA pellet was then air dried and 75µl of T 10 E1 buffer added and incubated at 65°C for 30mn. The stock DNA was stored at -20°C. The DNA quality was checked on 0.8% agarose gel and the quantity was determined using the NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., USA).

Genotyping
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of LALBA (A/B) variants

The LALBA A/B polymorphisms were determined by PCR-RFLP. The primer sequences 5’-TTGGTTTTACTGGCCTCTCTTGTCATC-3’ (forward) and 5’-TGAATTATGGGACAAAGCAAAATAGCAG-3’’ (reverse) (Mitra et al 1998) were used to amplify the 309 bp fragment in exon 1 of LALBA gene. The PCR reactions were carried out in a 30µL volume containing 45ng of template DNA, 15 µL of PCR Master Mix (Bioneer, Korea) and 4.5 pmol of each primer (3pmol/µL). The PCR conditions were as follows: an initial denaturation step at 94°C for 3 min, 35 cycles of 94°C for 45s, 64°C for 60s, 72°C for 60 s, and a final extension step of 72°C for 5 min. All the PCR reactions in this study were performed in the GeneAmp PCR System 9700 (Applied Biosystems, USA). Five µL of purified PCR products (309 bp) containing theLALBA A/B variants was digested overnight at 37°C with 10 U MspI restriction enzyme (New England Biolabs Inc., USA). The digestion patterns were recorded on 1.8% agarose gel as described by Mitra et al (1998).

CSN1S1, MBLG and CSN3 sequencing

The single nucleotide polymorphisms in CSN1S1, MBLG and CSN3 genes were assessed by PCR followed by Sanger Sequencing. Primers 5´- TGCATGTTCTCATAATAACC- 3´ (forward) and primer 5´- GAAGAAGCAGCAAGCTGG - 3 (reverse) (Koczan et al 1993) were used to amplify the polymorphic region (310bp) located between 5’end and exon 1 of CSN1S1 gene. The primers 5´-TGTGCTGGACACCGACTACAAAAAG-3´ (forward) and 5´- GCTCCCGGTATATGACCACCCTCT-3’ (reverse) (Medrano and Aguilar‐Cordova 1990) were used to amplify the partial exon 4 and intron 4 of MBLG gene. The primers 5’-CCAACTACCATGGCACGTCA-3’ (forward) and 5’-AGCCCATTTCGCCTTCTCTG-3’ (reverse) were designed to amplify partial exon 4 and flanking intronic sequences (386 bp) of CSN3 gene based on the reference sequence (GeneBank Accession N°AY380228). The NCBI Primer Blast tool was used for that purpose ( https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome ). The PCR conditions were as described above for LALBA gene except for the annealing temperature of 58°C (CSN1S1), 64°C ( MBLG) and 62°C (CSN3).

The PCR products of CSN1S1, MBLG and CSN3 were purified with the QIA quick PCR Purification Kit (Qiagen, Germany). 30µl (at least 25ng/µl) of the purified PCR products were sent for sequencing at Bioneer company (Bioneer, KOREA) using Sanger method (ABI machine, USA). The same forward and reverse primers used for the PCR amplification were used for the sequencing.

Statistical Analysis

Genetic polymorphisms in LALBA, CSN1S1, CSN3 and MBLG genes

The CSN1S1, MBLG, CSN3 sequencing data were analyzed using the CLC Main Workbench software version 7.8.1 (Qiagen Bioinformatics, Germany). Briefly, the sequencing reads were trimmed to eliminate reads of poor quality. Thereafter, the forward and reverse sequences were assembled to get a contiguous sequence (contig). The conflicts arisen from the assembling were found and edited manually. After editing, the consensus sequences were extracted for further analysis. The consensus sequences were aligned with the references sequences available in NCBI Genbank: X59856, Z48305 and AY380228 for CSN1S1, MBLG and CSN3 respectively. The positions of the SNPs were found in comparison to the reference sequences. The protein translation analysis was done to find the synonymous and nonsynonymous mutations using the CLC Main Workbench software version 7.8.1 (Qiagen Bioinformatics, Germany).

GENEPOP Program was used to estimate allele frequencies and test for Hardy-Weinberg Equilibrium (HWE) at all the studied loci (Raymond and Rousset 2001). Due to some failed genotyping in Borgou and White Fulani populations, data used for analysis is as follow: LALBA (85 BO and 96 WF), CSN1S1 (80 BO and 76 WF), CSN3 (94 BO and 96 WF) and MBLG (76 BO and 90 WF).

Genetic diversity, gene flow and phylogenetic relationship among breeds

The polymorphisms in all the studied genes were used in the genetic diversity analysis. The observed heterozygosity (Hob) and expected unbiased heterozygosity or unbiased gene diversity, (H exp) for each breed were determined using POPGEN Program (Yeh et al 1999). Hexp was estimated using algorithm of Levene (Levene 1949). The mean number of allele (MNA) occurred at the studied loci and the mean effective number of alleles (MNE) per population were assessed using the POPGEN program (Yeh et al 1999). The significance differences between H exp, MNA and MNE between the two breeds, and the Hob and Hexp within each breed were tested using paired t-test with IBM SPSS version 20 software package (SPSS Inc., USA). The extent of gene flow between breeds was estimated with GENEPOP program (Raymond and Rousset 1995 2001). The within population inbreeding (f), total inbreeding (F); and population differentiation θ were estimated according to the variance-based method (Weir and Cockerham 1984). The phylogenetic tree of relationship between breeds was constructed based on the CSN3 gene sequences (Prinzenberg et al 2008) using the Neighbor-Joining method (Saitou and Nei 1987) with MEGA7 software (Kumar et al 2016). The evolutionary distances were computed using the Maximum Composite Likelihood

method (Tamura et al 2004). CSN3 sequences from Indian Bos indicus (KF571745) and Ethiopian Bos Taurus (HQ5899208) indigenous cattle breeds were downloaded from NCBI database and used in the phylogenetic analysis for comparison purposes. The CSN3 sequence of Indian water Buffalo (D14370) was used as an outgroup.


Results

Alleles and genotypes frequencies
Polymorphisms in LALBA gene

Three restrictions patterns were detected corresponding to AA, AB and BB genotypes. The genotypic frequencies of AA, AB and BB in White Fulani and Borgou breeds were 0.03, 0.52, 0.45 and 0.07, 0.87, 0.06 respectively. Frequencies of A and B alleles were 0.29 and 0.71, and 0.51 and 0.49 in White Fulani and Borgou breeds respectively (Table 1). The LALBA genotype frequencies deviated from Hardy-Weinberg equilibrium in Borgou and White Fulani cattle breeds (p <0.05) (Table 1).

Table 1. Genotypes and allelic frequencies of LALBA polymorphisms in Borgou and White Fulani cattle breeds

Gene

Breed

Genotype frequencies

Allele
frequencies

Heterozygosity

p value
c
HWE

AA

AB

BB

B

Hoba

Hexb

LALBA (A/B)

Borgou (85)

0.07 (6)

0.87 (74)

0.06 (5)

0.49

0.87

0.50

0.00

White Fulani (96)

0.03 (3)

0.52 (50)

0.45 (43)

0.71

0.52

0.41

0.01

a Hob= observed heterozygosity; bH ex= expected heterozygosity c <0.05, genotypes deviated from Hardy-Weinberg Equilibrium. Numbers in brackets are actual animal numbers

CSN1S1 genotypes and alleles frequencies

Three different SNPs (g.10331 A>G, g.10359 T>C and g.10430 G>A) were discovered in CSN1S1 5’ flanking region. The SNPs genotypes and allelic frequencies are presented in Table 2. At the CSN1S1 g.10331 A>G locus, the AA, AG and GG genotypes were observed in Borgou while the AG genotype was absent in White Fulani. The A allele was the most frequent with frequencies of 0.96 and 0.99 in Borgou and White Fulani respectively (Table 2). At the CSN1S1 g.10359 T>C locus, the CC, CT and TT genotypes were observed in Borgou and White Fulani. The T allele was the most frequent with frequencies of 0.74 and 0.59 in Borgou and White Fulani respectively (Table 2). At the CSN1S1 g.10430 G>A, The G allele was the most frequent with frequencies of 0.67 and 0.56 in Borgou and White Fulani respectively (Table 2). All the genotypic frequencies at the CSN1S1 loci deviated from Hardy–Weinberg Equilibrium in Borgou and White Fulani populations (p <0.05).

Table 2. CSN1S1 genotypes and allelic frequencies in Borgou and White Fulani cattle breeds

Polymorphisms CSN1S1

Borgou (BO)

White Fulani (WF)

p value HWEa

Genotypic
frequency

Allelic
frequency

Genotypic
frequency

Allelic
frequency

BO

WF

g.10331 A>G
(5’flanking region)

AA

AG

GG

A

G

AA

AG

GG

A

G

76

1

3

0.96

0.04

75

0

1

0.99

0.01

<0.001

<0.001

g.10359 T>C
(5’flanking region)

CC

CT

TT

C

T

CC

CT

TT

C

T

12

17

51

0.26

0.74

18

27

31

0.41

0.59

<0.001

0.02

g.10430 G>A
(5’flanking region)

AA

AG

GG

A

G

AA

AG

GG

A

G

18

17

45

0.33

0.67

20

27

29

0.44

0.56

<0.001

0.01

The SNPs positions were based on the bovine CSN1S1 gene reference sequence GenBank accession No.: X59856. BO: Borgou, WF: White Fulani. a p <0.05 population deviated from Hardy-Weinberg Equilibrium

CSN3 genotypes and alleles frequencies

Through screening of exon IV and partial intronic sequences of bovine CSN3 gene, a total of six SNPs were found in this study. Five of these SNPS (g.13065 C>T, g.13068 C>T, g.13104 A>C, g.13111 A>G and g.13165 A>G) were found in exon IV while the g.13173 A>T was found in intron IV (Table 3). . The SNP genotypes and allelic frequencies are presented in Table 3. At the CSN3 g.13065 C>T locus, the CC, CT and TT genotypes were observed in Borgou and White Fulani. The C allele was the most frequent with frequencies of 0.65 and 0.55 in Borgou and White Fulani respectively (Table 3). At the CSN3 g.13068 C>T locus, the CC, CT and TT genotypes were observed in Borgou and White Fulani. The C allele was the most frequent with frequencies of 0.67 and 0.76 in Borgou and White Fulani respectively (Table 3). At the CSN3 g.13104 A>C, the AA, AC and CC genotypes were observed in Borgou and White Fulani. The A allele was the most frequent with frequencies of 0.66 and 0.75 in Borgou and White Fulani respectively (Table 3). At the CSN3 g.13111 A>G locus, the AA, AG and GG genotypes were observed in Borgou while the GG genotype was not found in White Fulani. The A allele was the most frequent with frequencies of 0.85 and 0.89 in Borgou and White Fulani respectively (Table 3). At the CSN3 g.13165 A>G locus, the AA, AG and GG genotypes were observed in Borgou and White Fulani. The A allele was the most frequent with frequencies of 0.67 and 0.75 in Borgou and White Fulani respectively (Table 3). At the CSN3 g.13173 A>T locus, the AA, AT and TT genotypes were observed in Borgou and White Fulani. The A allele was the most frequent with frequencies of 0.68 and 0.75 in Borgou and White Fulani respectively (Table 3).

Table 3. CSN3 genotypes and allelic frequencies in Borgou and White Fulani cattle breeds

Polymorphisms CSN3 (Exon 4-Intron 4)

Borgou (BO)

White Fulani (WF)

p valuea HWE

Genotypic
frequency

Allelic
frequency

Genotypic
frequency

Allelic
frequency

BO

WF

g.13065 C>T
Non synonymous (Thr135Ile)

CC

CT

TT

C

T

CC

CT

TT

C

T

40

43

11

0.65

0.35

30

45

21

0.55

0.45

0.74

0.56

g.13068 C>T
Non synonymous (Thr136Ile)

CC

CT

TT

C

T

CC

CT

TT

C

T

44

38

12

0.67

0.33

55

35

6

0.76

0.24

0.25

0.85

g.13104 A>C
Non synonymous (Asp148Ala)

AA

AC

CC

A

C

AA

AC

CC

A

C

44

37

13

0.66

0.34

54

36

6

0.75

0.25

0.14

0.96

g.13111 A>G
synonymous (Pro150Pro)

AA

AG

GG

A

G

AA

AG

GG

A

G

68

24

2

0.85

0.15

74

22

0

0.89

0.11

0.88

0.22

g.13165 A>G
synonymous (Ala168Ala)

AA

AG

GG

A

G

AA

AG

GG

A

G

44

39

11

0.67

0.33

54

36

6

0.75

0.25

0.40

0.96

g.13173 A>T
intron IV

AA

AT

TT

A

T

AA

AT

TT

A

T

45

39

10

0.68

0.32

54

36

6

0.75

0.25

0.51

0.96

The SNPs positions were based on the bovine CSN3 gene reference sequence GenBank accession No.: AY380228. Thr: Threonine, Ile: Isoleucine; Asp: Aspartic acid, Ala: Alanine, Pro: Proline. a p >0.05 population did not deviate from Hardy-Weinberg Equilibrium

MBLG genotypes and Alleles frequencies

Two SNPs were found through screening of partial exon IV and intron IV of MBLG gene. The SNPs MBLG g.5864C>T was detected in exon IV and the MBLG g.5940G>A in intron IV. The MBLG g.5864C>T mutation led to the change of amino acid alanine by valine at position 118 in the resulting protein. The genotypic and allelic frequencies of MBLG polymorphism are presented in Table 4. At the MBLG g.5864C>T locus, the CC, CT and TT genotypes were observed in Borgou while the TT genotype was not found in White Fulani. The C allele was the most frequent with frequencies of 0.84 and 0.94 in Borgou and White Fulani respectively (Table 4). At the MBLG g.5940G>A locus, the AA, AG and GG genotypes were observed in Borgou and White Fulani The G allele was the most frequent with frequencies of 0.92 and 0.88 in Borgou and White Fulani respectively (Table 4). Only the MBLG g.5940G>A genotypes deviated from Hardy-Weinberg Equilibrium and in Borgou only (P<0.05).

Table 4. MBLG genotypic and allelic frequencies

Polymorphisms MBLG

Borgou

White Fulani

p valuea HWE

Genotypic frequency

Allelic frequency

Genotypic frequency

Allelic frequency

BO

WF

MBLG g.5864C>T (Ala118Val)-Exon IV

CC

CT

TT

C

T

CC

CT

TT

C

T

52

23

1

0.84

0.16

79

11

0

0.94

0.06

0.40

0.56

MBLG g.5940G>A (intron IV)

GG

AG

AA

G

A

GG

AG

AA

G

A

66

8

2

0.92

0.08

70

18

2

0.88

0.12

0.01

0.48

The SNPs positions were based on the bovine MBLG gene reference sequence GenBank accession No.: Z48305. BO: Borgou, WF: White Fulani.a p >0.05 population did not deviate from Hardy-Weinberg Equilibrium. a p >0.05 population did not deviate from Hardy-Weinberg Equilibrium

Genetic diversity and gene flow

The genetic diversity estimates within and between Borgou and White Fulani using 4 milk protein genes are presented in Table 5 and Table 6. Differences were found between the observed and expected heterozygosities of Borgou (p <0.01) and White Fulani (p <0.001) (Table 5). The observed heterozygosity values per locus ranged from 0.000 (White Fulani) to 0.87 (Borgou) and the expected heterozygosity from 0.03 (White Fulani) to 0.50 (Borgou) (Table 5). The 0 heterozygosity was observed only at the CSN1S1 g.10331 A>G locus and in White Fulani breed. Borgou presented higher (p <0.01) means of observed heterozygosity and expected heterozygosity (0.33, 0.36) than White Fulani (0.31, 0.33) (Table 5). The mean number of observed (MNA) alleles per locus was 2±0.000 in both breeds. Moreover, the mean expected numbers of alleles (MNE) were similar (p >0.05) in Borgou (1.59 ± 0.27) and White Fulani (1.50 ± 0.33) (Table 6).

The Multi-locus estimates of Wright’s F-statistics genetic differentiation, f (within population inbreeding), F (total inbreeding) and q (genetic differentiation), and gene flow between Borgou and White Fulani populations are shown in Table 7. The f and F were respectively 0.06 and 0.074 and were not different from zero (p>0.05) (Table 7). Although the within population and total inbreeding coefficients were not significant, within population heterozygosity deficiency was observed at the studied loci except the LALBA, MBLG g.5864C>T and CSN3 g.13111 A>G (Table 7). The population differentiation (q) was 0.02 and different from zero (p <0.01) (Table 7). The highest q (0.05) was observed at LALBA and the lowest q (0.00) were observed at CSN3 g.13111 A>G and CSN3 g.13173 A>T loci (Table 7). High level of gene flow (Nem=16.44) was observed between Borgou and White Fulani cattle populations at the typed loci (Table 7). The highest movement of genes was observed at the CSN3 g.13111 A>G locus (Nem=88.4) and the lowest at the LALBA locus (Nem= 4.98).

Table 5. Observed (Hob) and expected (Hex) heterozygosities per locus and breed

Locus

Borgou

White Fulani

Hoba

Hexb

Hob

Hex

LALBA (A/B)

0.87

0.50

0.52

0.42

MBLG g.5864C>T

0.30

0.28

0.12

0.12

MBLG g.5940G>A

0.11

0.15

0.20

0.22

CSN3 g.13065 C>T

0.47

0.45

0.47

0.50

CSN3 g.13068 C>T

0.39

0.44

0.37

0.37

CSN3 g.13104 A>C

0.37

0.45

0.38

0.377

CSN3 g.13111 A>G

0.25

0.26

0.23

0.20

CSN3 g.13165 A>G

0.398

0.44

0.38

0.38

CSN3 g.13173 A>T

0.40

0.43

0.38

0.38

CSN1S1 g.10331 A>G

0.01

0.08

0.00

0.026

CSN1S1 g.10359 T>C

0.21

0.384

0.36

0.49

CSN1S1 g.10430 G>A

0.21

0.45

0.36

0.49

Mean (SDc)

0.33** (0.15)

0.36 (0.11)

0.31 (0.12)

0.33*** (0.13)

a Hob= observed heterozygosity; bHex= expected heterozygosity or gene diversity; cSD= standard deviation.
** p <0.01; *** p <0.001



Table 6. Significance of genetic diversity indices (Hob, Hex, MNA and MNE) between Borgou and White Fulani populations (Mean ± Standard deviation)

Borgou

White Fulani

Significance

Hob

0.33 ± 0.15

0.31 ±0.12

**

Hex

0.36 ± 0.11

0.33 ± 0.13

***

MNA

2.000 ± 0.000

2.00 ± 0.00

NS

MNE

1.59 ± 0.27

1.50 ± 0.33

NS

Hex= expected heterozygosity; MNA=Mean observed number of alleles; MNE= mean effective number of alleles *** p < 0.001; NS= not significant



Table 7. Multi-locus estimates of F-statistics and gene flow in Borgou and White Fulani

 

f

F

q

Nem

LALBA (A/B)

-0.52

-0.45

0.05

4.98

MBLG g.5864C>T

-0.09

-0.06

0.03

9.12

MBLG g.5940G>A

0.15

0.16

0.01

48.06

CSN3 g.13065 C>T

0.01

0.02

0.01

19.65

CSN3 g.13068 C>T

0.07

0.08

0.01

31.19

CSN3 g.13104 A>C

0.09

0.09

0.01

30.92

CSN3 g.13111 A>G

-0.05

-0.05

0.00

88.40

CSN3 g.13165 A>G

0.05

0.05

0.01

42.18

CSN3 g.13173 A>T

0.04

0.04

0.00

61.12

CSN1S1 g.10331 A>G

0.89

0.89

0.01

28.48

CSN1S1 g.10359 T>C

0.34

0.36

0.03

9.12

CSN1S1 g.10430 G>A

0.39

0.40

0.01

21.34

Mean (SEa)

0.11±0.09NS±0.04

0.13± 0.09NS

0.02±0.00**

16.44

f = within population inbreeding estimate; F= total inbreeding estimate; q = measure of population differentiation; Ne m=gene flow a Standard error; ** p < 0.01. NS: Not significant

Nei’s DA genetic distance and Phylogenetic relationship between Borgou and White Fulani

The Nei’s DA genetic distance between Borgou and White Fulani cattle populations was 0.014 (Table 8). The Phylogenetic relationships among Borgou (Bos taurus), White Fulani ( Bos indicus), Indian zebu (Bos indicus) and Ethiopian Sheko (Bos taurus) indigenous cattle breeds based on CSN3 gene is shown in Figure 2. The water buffalo (Bubalus bubalis) was used as an outgroup in the phylogenetic tree construction. According to the tree, the Beninese Borgou and White Fulani were closely related. The Ethiopian Sheko separated from Borgou and White Fulani while the Indian zebu was diverged more from all other breeds (Figure 2).

Table 8. Nei's DA genetic distance (below diagonal) and genetic identity (above diagonal) estimates between breed using 4 genes loci related to milk production

Borgou

White Fulani

Borgou

-

0.986

White Fulani

0.014

-



Figure 2. Phylogenetic tree constructed using the Neighbor-Joining method (Saitou and Nei 1987) showing genetic relationships
among Borgou, White Fulani, Indian zebu and Ethiopian Sheko indigenous cattle breeds based on CSN3 gene


Discussion

Genetic diversity

All the studied loci were polymorphic in both breeds. High genetic diversity was reported in African indigenous Bos taurus and Bos indicus cattle breeds at microsatellites, milk proteins and blood proteins loci (MacHugh et al 1997, Ibeagha-Awemu et al 2004, Ibeagha-Awemu and Erhardt 2006). Thus, Ibeagha-Awemu and colleagues (2004) reported expected heterozygosity of 0.719, 0.453 and 0.376 at microsatellites, blood proteins and milk proteins loci respectively in Nigerian White Fulani. The expected heterozygosity of 0.302 and 0.353 in Beninese White Fulani and Borgou cattle in the present study are similar to the reported expected heterozygosity in Nigerian White Fulani (0.376) and Cameroonian White Fulani (0.370) at milk protein loci (Ibeagha-Awemu and Erhardt 2006). Although the White Fulani and Borgou cattle breeds play important role in the provision of milk and meat at the community level, there is no effective genetic improvement program for these traits in Benin. The high diversity observed in Borgou breed compared to White Fulani, would be an opportunity for a breed improvement and conservation program in Benin. In order to put a genetic improvement program in Benin, the loss of genetic diversity must be avoided as experienced in some Western breeds through high selection pressure and also controlled gene flow between breeds (Ibeagha-Awemu and Erhardt 2006).

Gene flow and phylogenetic relationship among Borgou and White Fulani cattle breeds

Despite the observed diversity at the studied loci, a high rate of gene flow (Nem=16.439) and a very close relationship were found between Borgou and White Fulani breeds. The genetic exchange between the two breeds is higher than the reported gene flow of 5.655 between Nigerian White Fulani and Cameroonian Red Bororo (Ibeagha-Awemu and Erhardt 2006). High zebu introgression was reported in West and Central African cattle breeds (Ibeagha-Awemu et al 2004). Moreover, a high zebu introgression in the taurine population was observed in Benin and the Lagune breed was the most pure taurine breed (Koudandé et al 2009). The authors stated that it is because Lagune cattle breed is located on an island in Benin and has not gone through crossbreeding with zebu cattle. In a recent study on indigenous cattle breeds from Benin, high zebu introgression was observed at microsatellite loci (Kassa et al 2019). On the other hand, the high level of genetic exchanges between African indigenous cattle breeds was postulated to be due to the production system in use (Ibeagha-Awemu and Erhardt 2005). Indeed, the cattle management system in Benin is mainly traditional and pastoral. The system is characterized by pool management where different breeds are kept together or in the same area with movement from place to place in search of forage and water. The high level of gene flow and a closer phylogenetic relationship observed between Borgou and White Fulani may be explained by the cattle management system in Benin.

Implications for cattle improvement and conservation in Benin

The high genetic diversity observed in Borgou cattle breed offers opportunity for breed improvement. However, the high genetic exchange observed between the main two indigenous cattle breeds from Benin is a big concern. If the present situation continues unchecked and prevented, Borgou and White Fulani breeds will merge in the near future to form a single breed. This will lead to a reduction of variety of cattle breeds in Benin and consequently a loss in breed diversity at the African continent level. Therefore, to allow sustained genetic improvement, care must be taken to prevent the gene flow and loss of genetic diversity. This can be achieved by implementing an effective cattle management system with the aims of improving productivity and conserving the current within breed diversity. The smallholder farmers should be sensitized in Benin about the herd’s management system in order to avoid uncontrolled cross breeding between the different indigenous cattle breeds leading to loss of genetic diversity. Proper and sustainable breeding program needs to be developed in Benin to improve the meat, milk production and fatty acids traits in Borgou and White Fulani cattle breeds. The breeding program should involve all the stakeholders such as governments, farmers, scientists and non-governmental organizations. The roles of these parties should be a concerted effort to improve cattle productivity to meet the current and future needs while maintaining the genetic diversity. An ex-situ conservation plan should be put in place for the indigenous White Fulani cattle breed due to the observed low genetic diversity.


Conclusion


Acknowledgement

The authors are grateful to the Molecular Genetics and Genome Analysis Laboratory of Abomey-Calavi University (Benin Republic) for offering the facilities and the technical supports towards the DNA extraction. We also thank the field assistants as well as the cattle herdsmen for facilitating the sampling. The study was supported by the BecA-ILRI Hub through the Africa Biosciences Challenge Fund (ABCF) program. The ABCF Program is funded by the Australian Department for Foreign Affairs and Trade (DFAT) through the BecA-CSIRO partnership; the Syngenta Foundation for Sustainable Agriculture (SFSA); the Bill & Melinda Gates Foundation (BMGF); the UK Department for International Development (DFID) and the Swedish International Development Cooperation Agency (Sida). Sample collection and DNA extraction were supported by the Pan African University Institute of Basic Sciences, Technology and Innovation (PAUSTI) based at Jomo-Kenyatta University of Agriculture and Technology (JKUAT), Kenya.


References

Alkoiret I T, Akouedegni G C, Toukourou Y, Bosma R H and Mensah G A 2011 Effects of protein supplementation during the dry season on feed intake and performance of Borgou cows in Benin Republic. Journal of Animal and Veterinary Advances, 10(21), 2879–2884.

Brito L F, Kijas, J W, Ventura R V, Sargolzaei M, Porto-neto L R, Cánovas A and Schenkel F S 2017 Genetic diversity and signatures of selection in various goat breeds revealed by genome-wide SNP markers. BMC Genomics, 18:229. http://doi.org/10.1186/s12864-017-3610-0

Caroli A M, Chessa S and Erhardt G J 2009 Invited review: Milk protein polymorphisms in cattle: Effect on animal breeding and human nutrition. Journal of Dairy Science, 92(11), 5335–5352. http://doi.org/10.3168/jds.2009-2461

FAOSTAT 2018 Food and Agriculture Organization Statistical database. Available at: http://www.fao.org/faostat/en/#data/TP. Accessed on 13th February 2018.

Hanotte O, Bradley D G, Ochieng J W, Verjee Y, Hill E W and Rege J E O 2002 African pastoralism: Genetic imprints of origins and migrations. Science, 296(5566), 336–339. http://doi.org/10.1126/science.1069878

Ibeagha-Awemu E M, Jann O C, Weimann C and Erhardt G 2004 Genetic diversity , introgression and relationships among West / Central African cattle breeds. Genetics Selection Evolution, 36, 673–690.

Ibeagha-Awemu EM and Erhardt G 2005 Genetic structure and differentiation of 12 African Bos indicus and Bos taurus cattle breeds , inferred from protein and microsatellite polymorphisms. Journal of Animal Breeding and Genetics, 122, 12–20.

Ibeagha-Awemu E M and Erhardt G 2006 An evaluation of genetic diversity indices of the Red Bororo and White Fulani cattle breeds with different molecular markers and their implications for current and future improvement options. Tropical Animal Health and Production, 431–441. http://doi.org/10.1007/s11250-006-4347-y

Jann O C, Prinzenberg E M, Luikart G, Caroli A and Erhardt G 2004 High polymorphism in the κ-casein (CSN3) gene from wild and domestic caprine species revealed by DNA sequencing. Journal of Dairy Research, 71(2), 188–195. http://doi.org/10.1017/S0022029904000093

Kassa KS, Dayo G K, Yapi-Gnaoré V, Sylla S, Konkobo M and Youssao A K I 2019 Genetic diversity of Benin cattle populations using microsatellite markers. International Journal of Animal Science and Technology, 3(1), 7–19. http://doi.org/ 10.11648/j.ijast.20190301.12

Koczan D, Hobom G and Seyfert H M1993 Characterization of the bovine αs1 -casein gene C-allele based on a MaeIII polymorphism. (n.d.). Animal Genetics, 24, 74.

Koudandé O D, Dossou-Gbété G, Mujibi F, Kibogo H, Mburu D, Mensah G A, Hanotte O and van Arendonk J A M 2009 Genetic diversity and zebu genes introgression in cattle population along the coastal region of the Bight of Benin. Animal Genetic Resources Infomation, 44, 45–55.

Kumar S, Stecher G and Tamura K 2016 MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33, 1870–1874.

Levene H 1949 On a matching problem in genetics. Annals of Mathematical Statistics, 20, 91–94.

MacHugh D E, Shriver M D, Loftus R T, Cunningham P and Bradley D G 1997 Microsatellite DNA variation and the evolution, domestication and phylogeography of taurine and zebu cattle (Bos taurus and Bos indicus). Genetics, 146(3), 1071–1086.

MAEP 2007 Programme de Relance des Productions A nimales au Bénin. Ministère de l’Agriculture, de l ’Elevage et de la Pêche : Cotonou, 205 p.

Medrano J F and Aguilar‐Cordova E 1990 Polymerase chain reaction amplification of bovine β‐lactoglobulin genomic sequences and identification of genetic variants by RFLP analysis. Animal Biotechnology, 1(1), 73–77. http://doi.org/10.1080/10495399009525730

Mitra A, Sashikant and Yadav B R 1998 Alpha-lactalbumin polymorphism in 3 breeds of Indian zebu cattle. Journal of Animal Breeding and Genetics, 115, 403–405.

Moazami-Goudarzi K, Belemsaga D M A, Ceriotti G, Laloë D, Fagbohoun F, Kouagou N’T, Sidibé I, Codjia V, Crimella M C, Grosclaude F, Touré S M 2001. Caracterisation de la race bovine Somba à l’aide de marqueurs moléculaires. Revue d’élevage et de Médecine Vétérinaire Des Pays Tropicaux, 54(2), 129–138. Retrieved from http://agritrop.cirad.fr/489759/1/ID489759.pdf

Raymond M and Rousset F 2001 GENEPOP (version 3.2). Population genetics software for exact tests and ecumenicism (http:/wbiomed.curtin.edu.au/genepop/).

Raymond M and Rousset F 1995 GENEPOP . Population genetics software and ecumenicism, http:/wbiomed.curtin.edu.au/genepop /. J. Hered, 86, 248–249.

Saitou N and Nei M 1987 The neighbor-joining method: A new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4, 406–425.

Sambrook J F and Russell DW 2001 Molecular cloning: A Laboratory Manual.3rd Ed. Vol.1. Cold Spring Harbor Laboratory Press, New York, USA.

Tamura K, Nei M and Kumar S 2004 Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences (USA), 101, 11030–11035.

Weir B S and Cockerham C C 1984 Estimating F-statistics for the analysis of population structure. Evolution, 38, 1358–1370.

Yeh FC, Yang R-C and Boyle T 1999 POPGENE Version 1.31. Microsoft windows-based freeware for population genetics analysis (ftp://ftp.microsoft.com/Softlib/MSLFILES/HPGL.EXE).

Youssao A K I, Koutinhouin G B, Kpodekon T M, Agnandjo H, Toure Z and Ahissou A 2009 Influence d’une sélection phénotypique sur les performances de croissance et les caractères de développements musculaire et squelettique de jeunes bovins de race Borgou à la Ferme d’Elevage de l’Okpara (Bénin). Annales de Medecine Veterinaire, 153(2), 105–111.

Youssao A K I 2015 Programme National d’Amélioration Génétique (Projet d’Appui aux Filières Lait et Viande (PAFILAV)). Cotonou (Bénin),362p.


Received 11 October 2019; Accepted 8 December 2019; Published 2 January 2020

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