SEQUENCING, POLYMORPHISM AND PHYLOGENETIC CHARACTERISATION OF KISS-1 GENE IN TWO NIGERIAN INDIGENOUS GOAT BREEDS

: KiSS-1 gene encodes a protein product kisspeptin which are intense inducers of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion in various mammalian species through its receptor GPR54 (G protein-coupled receptor-54). A total of 100 goat compromising of Red Sokoto (n = 72) and Sahel (n = 28) breeds were used to detect single nucleotide polymorphisms (SNPs) in the intronic region of the KiSS-1 gene by sequencing and investigate their relationship with other goat breeds. Nucleotide sequence analysis revealed five novel SNPs (g.1745G>A present in Red Sokoto, g.1776G>A, g.1827A>G, g.1857T>C and g.2208T>C present in Red Sokoto and Sahel breeds). To obtain a correct phylogenetic relationship between goat breeds, nucleotide sequences were compared to other sequences in NCBI database using a BLASTn algorithm and retrieved for further analysis. Neighbour-joining phylogenetic relationship tree constructed revealed two distinct clusters with ancestral lineage of 100% identity. Nigerian goat breeds (Red Sokoto and Sahel) clustered into a clade with Indian goat breeds (Ganjam and Osmanabadi) while the second cluster involved eight other goat breeds. Genetic distance estimate revealed high genetic similarity between Red Sokoto and Sahel breeds as observed in their genetic distance value of 0.003. The nucleotide sequences of the two Nigerian goat breeds (Red Sokoto and Sahel) for KiSS-1 gene were submitted to GenBank database and have accession numbers: MN122316 and MN122317, respectively. The analysis of polymorphism in KiSS-1 gene indicates that genetic variation exists in the goat breeds studied. Therefore, attempts can be made to investigate the association of these polymorphism with reproductive traits in Nigerian goat breeds.


Introduction
Goats spread all over the world because of their ability to adapt to varying environmental conditions and different regimes under which they are subsequently maintained (Assan, 2014) with over 300 distinct breeds to available for different purposes (Hirst, 2018). In Nigeria, goats constitute the largest group of small ruminant livestock totaling about 73.8 million (FAOSTAT, 2016). Red Sokoto and Sahel goat breeds are two well adapted and predominantly found in the Northern part of Nigeria where they are majorly managed by traditional production system with an average flock size of 3-5 goats per small holder farmers constituting a good source of protein (Makun et al., 2006). The Red Sokoto (RS) goat is a highly prolific breed with high incidence of multiple births and twinning rate of 54% and 43% respectively (Akpa et al., 2010). However, the Sahel are known to have a short fine hair with different coat colours from plain white, grey, pied, dappled, black or brown goat is a multipurpose goat breed mostly reared for meat and skin production (Adebambo, 2012) although reproductive potentials of this breeds have not been fully explored. Both breeds have interestingly unique adaptive traits making them strive in the hot savanna such as long leggedness, long distance walking ability, feeding behavior, heat tolerance and remarkable recovery capacity from scarcity of feed resources (Muema et al., 2009). With the increasing population and demand always exceeding supply for chevon (Okewu and Iheanacho, 2015), it is necessary to exploit ways of increasing animal protein availability other than the traditional breeding method which is by evaluating genetic variation in genes relatively affecting prolificacy in goat. Genetic markers like single nucleotide polymorphisms of some genes have been reported to be significantly associated with litter size in goats such as POU class 1 homebox 1 (POU1F1) gene (Feng et al., 2011), KiSS-1 gene (Cao et al., 2010An et al., 2013a,b;Othman et al., 2015;Mekuriaw et al., 2017) Gonadotropin-releasing hormone receptor (GnRHR) gene (Yang et al., 2011;Huang et al., 2012;Bemji et al., 2018), KIT ligand gene (An et al., 2011;2015;, Inhibin alpha (INHA) gene Sharma et al., 2015;Isa et al., 2017). Although characterization of genetic variation in Nigerian goat breeds have been carried out using genetic markers like Microsatellite, Mitochondrial, Biochemical and SNPs (Shoyombo et al., 2015;Awotunde et al., 2015;Ojo et al., 2017;Bemji et al., 2018;Isa et al., 2019), there has been no published information on KiSS-1 gene variant of Nigerian goat breeds. Therefore, this study was intended to identify single nucleotide polymorphism, genetic distance and phylogenetic relationship among two Nigerian Northern goat breeds.

Materials and Methods
Animal sampling and DNA isolation A total of 100 goat belonging to two Nigerian indigenous breeds respectively managed semi-intensively at National Animal Production Research Institute (NAPRI), Shika-Zaria were included in this study. About 5 ml of blood sample was collected from 72 Red Sokoto goats and 28 Sahel via jugular venipuncture into vacutainer tubes containing ethylene diamine tetra acetic acid (EDTA) anticoagulant. Genomic DNA extraction was isolated from whole blood samples using Zymo Research quick-gDNA TM Miniprep kit adhering to manufacturer's protocol. The concentration of DNA was evaluated by the Nanodrop spectrophotometer (ND1000; NanoDrop Technologies, USA) while the quality was virtually assessed by agarose gel electrophoresis.  (Table 1) was designed to amplify and sequence a 1088 bp fragment of intron 1 in Stab Vida genetics laboratory, Portugal. Polymerase chain reaction (PCR) amplification was carried out in SureCycler 8800 Thermal Cycler (Agilent Technologies, USA) with a total reaction volume of 20μL containing 50ng genomic DNA was added to a reaction mix containing 12.8μL of H2O MQ, 2.5μL of 1XPCR reaction buffer, 1μL dNTP's, 1.5μL MgCl2, pH 9, 1μL each of forward and reverse primers and 0.2 μL of Taq DNA polymerase. PCR cycling protocol was accomplished by an initial denaturation at 96°C for 15 minutes followed by 40 cycles of denaturing at 95°C for 30 seconds, annealing at 58°C for 30 seconds, extension at 70°C for 1 minute and 40 seconds and final extension at 70°C for 5 minutes. The PCR products were subjected to 1% agarose gel stained with GelRed TM nucleic acid stain in a Thermo EC Midicell Primo EC-330 gel system containing 1XTBE electrophoresis buffer, scored using a standard 100 bp molecular ladder and viewed under UV light and photographed using a Vilber lourmat gel documentation system.

DNA sequencing and sequence analysis
The PCR products were purified from the gel using a Carboxylate Magnetic Beads technology (MCLab, USA) and eventual sequencing was done with BigDye ® terminator cycle sequencing kit on the ABI 3730XI (Applied Biosystems, USA). The sequences were viewed and edited using Bioedit software (Hall, 1999). Multiple sequence alignment of the sequences were performed using ClustaW in MEGA-X software package (Kumar et al., 2018). Identification of single nucleotide polymorphism in KiSS-1 gene was carried out using codon code aligner (Codon code Corporation Dedham, MA, USA) and MEGA-X software packages. A BLAST algorithm (Altschul et al., 1990) search was carried out to retrieve homologous nucleotide sequences from the NCBI GenBank (http://www.ncbi.nlm.nih.gov/BLAST) database. Nucleotide sequence showing 97-100 % similarity for KiSS-1 gene of two Nigerian goat breeds (MN122316 and MN122317), ten goat published sequences viz. San Clemente (NC_030823.1), Barki (KP835800.1), Zaraibi (KP835799.1), Xinong Saanen (JQ806381.1), Guanzhong (JQ806382.1), Ganjam (KJ425411.1), Osmanabadi (KJ425412.1), Gaddi (MH397145.1), Lezhi Black (KR065750.1), Jining Grey (GU142847.1), sheep (Barki = KP835798.1) and a Japanese medaka fish as an out-group (NC_019863.2) were assembled. Genetic distance and phylogenetic analysis of the KiSS-1 gene were carried out for Nigerian goat breeds, other goat breeds and fish sequences applying p-distance model. A neighbor joining tree method was used to infer phylogenetic relationship between the populations following alignment of the sequence of Nigerian goat breeds and the published sequences. The reliability of the phylogenetic tree branching was estimated using a bootstrap confidence level of 1000 replications.

Identification of single nucleotide polymorphism in KiSS-1 gene of Nigerian indigenous goat breeds
The sequence result analysis of this study among two Nigerian indigenous goat breeds revealed novel polymorphism of KiSS-1 intron 1 gene. Five single nucleotide polymorphisms (g.1745G>A, g.1776G>A, g.1827A>G, g.1857T>C, g.2208T>C) was identified within Intron 1 of goat KiSS-1 gene. The breed, SNPs type and the position along the reference sequence and chromosome position are presented in Table 2. Several similar studies on KiSS-1 (intron 1) gene have identified several polymorphism in different goat populations (Cao et al., 2010;An et al., 2013b;Maitra et al., 2014a;El-Tarabany et al., 2017). Although these studies are focused on the non-coding regions of the gene it has been proven that mutations in intragenic (intron) regions may be involved in alternative splicing/regulation, transcript processing, gene expression and protein function, chromosomal rearrangement (Guey-Shin and Thomas, 2007;Sjakste et al., 2011) of varying phenotypes and have been found to affect economic traits (Jiang et al., 2010;Ibeagha-Awemu et al., 2014).

Genetic distance between Red Sokoto, Sahel and other goat breeds
The pair-wise estimation of genetic distance between Red Sokoto, Sahel and other goat breeds was based on sequence analysis of KiSS-1 gene intron 1 and shown in Table 3. Among the twelve breeds sequences used for this analysis Guanzhong and Xinong Saanen had 100 percent nucleotide identity having the closest value of 0.000 and the highest was observed at 0.574 between Red Sokoto and Guanzhong, Xinong Saanen, Lehzi black. Based on the polymorphisms earlier reported in this study we observed a low pair-wise distance of 0.003 between Red Sokoto and Sahel goat breeds which compares favourable with findings of other studies estimate for Red Sokoto and Sahel (Murital et al., 2015;Udeh, 2015;Ajibike et al., 2016;Ajayi et al., 2016;Ojo et al., 2017). The low genetic distance estimates obtained between Red Sokoto and Sahel from different molecular markers could be attributed to migration, high degree of miscegenation or high gene flow, geographical adaptation. Similarly, low genetic distance observed in this study would be due to shared alleles, origin and geographical adaptation between the two breeds.

Phylogenetic relationship
The Neighbor-joining phylogenetic tree used to infer relationship between Red Sokoto, Sahel goats, some selected goat breeds and other species (Ovis aries, Oryzias lapites) shown in Figure 3 revealed three major clusters for which the small ruminant population had separate common ancestors at 100% reliability. First cluster estimate comprised eight exotic goat breeds and an ovine species, second cluster comprised Red Sokoto, Ganjam, Osmanabadi and Sahel, goat breeds which is indicative of these breeds having common alleles being shared and could reflect a common origin. The third clade comprised the Japanese rice (medaka) fish which from the tree had similar nucleotide sequence but branched separately showing that it is the most genetically divergent.

Conclusion
In this study, the analysis of KiSS-1 gene of Nigerian goat breeds showed to be polymorphic. Phylogenetic relationship analysis revealed that Nigerian goat breeds (Red Sokoto and Sahel) clustered into a clade with low genetic distance (0.003) for KiSS-1 (Intron 1) gene. The presence of genetic variation permits for genetic conservation and improvement programme of these breeds having better understanding on their genome architecture, breed relationship and phenotypic performance. It is recommended that the number of animals used in this study be increased in further studies in other to estimate genetic diversity indices and explore the significant association of KiSS-1 gene polymorphism with litter size in Nigerian indigenous goat breeds.