Genetic Selection Barriers in Global Development of Rural Goat Production and a Simplified Approach in Identification of Proper Polymorphic Types

The World Bank estimated the rural fertile area of our planet is 38% [1]. The number of families living in rural areas of the world and involved in goat production is around 111 millions, with a total number of served rural goats equivalent to 833 million heads [2,3]. Most of the goat shepherds and their families are living below the poverty line [4,5], which is blamed mostly on the low productivity, poor veterinary services, and disease susceptibilities of their goats [6-8]. The genetic potential of most rural goats present on our planet is not yet characterized due to lack of funding, which is becoming more critical during the economic crisis of our time [9].

imported from France, were used in all optimization procedures of the Polymerase Chain Reaction (PCR), targeting the amplification of four inclusive genes namely, the two production genes of GH and αS1-Casein, the immunity gene of MHC Class II DRB, and the PrP gene for TSE susceptibility recognition [23,24]. Four major service rural male goats were selected for semen collection due to their wide-spread use in insemination of four largest herds reared in the rural areas at the Eastern side of the Mediterranean Sea, located at Latitude of 33° 00'N and Longitude of 35°50'E.
The semen samples were collected from the reference Saanen goat and from the rural goats in sterile urinary cups, using an electroejaculator (Standard Precision Electronics, Inc. Denver, Colorado). The samples were shipped on ice to the Animal and Veterinary Science Laboratories of the American University of Beirut, and stored at -20°C for genetic analysis.

DNA Extraction from Goat's Sperms
The QIAamp® DNA Mini Kits were used for DNA extraction from the goat's sperms, following the protocol of the manufacturer (Qiagen 2007 edition, Qiagen GmbH, D-40724, Hilden). Briefly, the enzyme Proteinase K was used for lyses of the sperms, and the QIAamp Mini spin columns were used for elution of the extracted DNA. The extracted DNA from the Saanen and the rural goats were stored at -40°C. The stored Saanen DNA was used to optimize the PCR for amplification of the four targeted genes, as described in our previous publications [23,24], and the stored rural goat DNA were subjected to the optimized PCR protocols, to produce amplicons for further polymorphism analysis.

PCR Optimization
The PCR optimization of the four genes was performed on reference Saanen sperms, as described previously in details [23,24]. Briefly, the optimization used a constant sperm-DNA concentration of 93.0 ng/50 µl in the PCR mixture. The variables used in the optimization were the primers concentration, thermal and cyclic conditions applied to the PCR reactions. The optimized protocols for the PCR of each of the multiple genes in the Saanen sperms are presented in Table 1. The same optimized protocols were applied on rural goat sperms to amplify the same four genes, enabling the study of their multiple polymorphisms.

Polymorphism in Rural Goat Genes
To detect polymorphism in rural goat genes, restriction endonuclease procedures were applied on the DNA amplicons of GH, αS1-Casein, and MHC Class II DRB genes, while a nucleotide sequencing procedure was applied on the amplicons of the PrP gene. The Saanen DNA was included in every analysis as a reference. Briefly, the restriction endonuclease procedures were applied on the amplicons of GH, αS1-Casein, and MHC Class II DRB using the following respective enzymes (20 units/PCR mix) namely, the HaeIII, XmnI, and TaqI or PstI (Fermentas International Inc., 830 Harrington Court, Burlington, Ontario, C7N3N4, Canada). The incubation temperature and time of the enzymatic reaction were 37 o C and 16 hours, respectively. The digested amplicons were banded on 2% agarose, visualized and documented by UV, using GelDoc system (Bio-Rad Laboratories, 1000 Alfred Nobel Prize, Hercules, CA, USA). The nucleotide sequencing was performed on the amplicons of the PrP gene. Briefly, the G2reverse primer (Table 1) was included at 5 pmol/µl, and the Dynamic ET (GE Health Care, 81050, USA) was added in 2 µl volume to PrP amplicon that is eluted from the Agarose Gel. The rest of the sequencing steps followed the instructions for sequencing by the ABI-Prism 3130, Applied Biosystems, UK. The coded amino acids from the determined PrP sequence were deduced by using the Basic Local Alignment Search Tool (BLAST) of the National Center for Biotechnology Information (Bethesda, MD 20894, USA).

Polymorphism in growth hormone (GH) gene
The polymorphism in GH gene of rural goats sperms is deduced from the electrophoretic patterns of DNA fragments resulting from the HaeIII endonuclease digestion of amplified exons 2 and 3 ( Figure 1) and exon 4 ( Figure 2).

Polymorphism in MHC Class II DBR gene
The polymorphism in MHC Class II DRB gene in sperms of rural and reference Saanen goats is deduced from restriction by two enzymes, the PstI and the TaqI (Figure 4). The digestion of MHC Class II DRB gene in all rural and Saanen goats' sperms by PstI endonuclease enzyme resulted in one band at 285, preventing the uncovering of polymorphism (lanes 3-7); however, the digestion of the same gene by the TaqI showed two polymorphic types: a homozygous genotype resulting in an undigested 285 bp band (lanes 8 and 10 for rural goats, and lane 12 for the reference Saanen goat), and a heterozygous genotype resulting in two fragments banded at 150 and 285 bp (lanes 9 and 11 for rural goats).

Polymorphism in Prion (PrP) gene
The polymorphism in the amino acid sequence at defined positions in the PrP protein-coding exon of rural goat, in reference to Saanen goat sperms, is presented in Table 2

Polymorphism in Growth Hormone (GH)
The HaeIII endonuclease used in digestion of the GH gene amplicons (Figures 1 and 2) of rural and reference Saanen sperms had a 5`GG/CC3` restriction site [25]. According to previously reported restriction banding patterns produced by HaeIII applicability on GH gene of goats [18,26], the obtained pattern by the sperms of all rural goats belongs to the reported CD genotype, while that obtained by the Saanen belongs to the reported CC genotype. It is worth noting that the CC genotype resulted in better growth traits and performance, and significantly higher prolificacy and superovulation response in goats [18,26]; actually, a five times higher performance in production of milk is reported in Saanen compared to these rural goats, a data that is documented in our previous work [27]. It is advised to combine the polymorphism in exon 2 and 3 to that of exon 4 of GH gene, to get a more profound genetic-model for future selection. Actually, Hua et al. [18] in 2009 recommended excluding the Boer with a genotype AACD for GH gene, since it was associated with poor growth performance. In addition, Zhang et al. [26] reported that both the Matou and Boer dams with ABCD genotype for GH gene had the largest litter size, while those with AACD genotype were associated with the lowest litter size, a marker in the genotype that should be included in future selection models for better performance. The search for CC and/or ABCD genotype for GH gene in rural goats should be continued to include in future model for rural goat selection.

Polymorphism in αS1-Casein gene
The restriction site of XmnI endonuclease used in digestion of the sperms-αS1-Casein gene is 5`GAANN/NNTTC3` [28]. The digestion resulted in four different banding patterns of the sperm's αS1-Casein gene fragments of four respective individual bucks (Figure 3). According to Ramunno et al. [28], the XmnI endonuclease digestion banding patterns of the goat αS1-Casein gene were correlated with a cytosine presence or absence at 23 nucleotide of the 9 th exon of the goat αS1- The obtained digestion fragments-patterns of αS1-Casein gene allow to assign a genotype to each rural and reference Saanen goat included in this study [28,29]. Thus, rural goats in Figure 3 lanes 2-5, conform to respective genotypes c/c, b/b, a/c, and a/a, while the reference Saanen agrees with the b/b genotype. The 'd' genotype genotype was absent in the goats included in this research. Da Silva et al. [30] reported the absence of the 'a' genotype in Mexican goats, whereas all genotypes (ad) were present in Egyptian, Italian, and Indian goats [28,29,31].
It is worth noting that some amplicons of the αS1-Casein gene had an eleven bp insertion (I+), while other didn't (I-). The respective size of amplicons for αS1-Casein of rural goats present in Figure 3, lanes 2-5 were 217(I-), 230 (I+), 217(I-), and that for reference Saanen was 230 bp (I+) [23].This 11 nucleotide insertion is characterized before as CGTAATGTTTC, located at 73 nucleotides downstream of 5` splice site of the 9 th intron of the αS1-Casein [28]. The assignment of alleles to the goat αS1-Casein is linked in literature to D (presence or absence of cytosine deletion at 23 rd nucleotide of the 9 th exon) and I (presence or absence of the 11 nucleotide insertion) [28].
Accordingly, the rural goats presented in lanes 2, 3, 4, and 5 ( Figure  3), and having respective D and I as D+I-, D-I+, D-I-/D+I-, D-I-, will be assigned the following respective αS1-Casein alleles: D, B or E, A or O1/D, and A or O1. It is worth noting that the ' A' allele is proven to be associated with high level of αS1-Casein in milk while the 'B' and 'E' allele are associated with high and medium levels of αS1-Casein in milk, respectively [28]. Moreover, the 'D' allele is associated with medium levels of αS1-Casein in milk [29,31], which could be selected in future programs to produce milk with lower αS1-Casein level, targeting allergic consumers, and developments of special infant diets [29]. The high polymorphism in the αS1-Casein gene is helpful to use as genetic markers in the future model for selection of goats with high or low Casein protein levels in milk, depending on the demand of the targeted consumers.

Polymorphism in MHC Class II DRB Gene
The polymorphism in MHC Class II DRB gene in sperms of rural and reference Saanen goats, created from the restriction by PstI and the TaqI is documented in Figure 4. The respective restriction sites of PstI and TaqI are 5`CTGCA/G3`-5`G/ACGTC3` and 5`T/CGA3`-5`AGC/ T3`, as confirmed by Amills et al. [32].
The uniform result of one band of 285 bp size, by the digestion of MHC Class II DRB amplicons by PstI of all rural and reference Saanen goat-sperms, reveals the absence of a CTGCAG or GACGTC nucleotide sequence in exon 2 of this gene in all included goats (Figure 4, lanes  3-7). The absence of PstI restriction site reflects the presence of a GTG (valine) or TGT (cytosine) codons at position 78 of exon 2 [32,33]. The Spanish caprine breeds showed two restriction patterns, following a digestion by the PstI restriction enzyme, namely two fragments of 15 and 270 bp, and three fragments pattern of 15, 44, and a 226 bp [5]. However, the PstI restriction of the MHC Class II DRB gene of Egyptian goat genome resulted in respective homozygosity and heterozygosity equivalent to 29.5 and 70.5% [33].
The electrophoretic pattern of TaqI digested amplicons of the MHC Class II DRB showed two restriction patterns, a one undigested 285 bpband ( Figure 4, lanes 8 and 10 for rural goats, and lane 12 for reference Saanen goat), and a two bands pattern at 150 and 285 bp (Figure 4, lanes 9 and 11 for rural goats). The presence of one-band pattern is indicative of a homozygous genotype in two out of the four rural goats, and in the reference Saanen goat; on the other hand, the presence of a two bands-pattern confirms the presence of a heterozygous genotype in the other two rural goats. It is worth noting that Ahmed and Othman [33] documented in Egyptian goat genome the presence of a homozygous tt genotype (9.1%) in MHC Class II DRB gene resulting in one bandpattern (285 bp), a homozygous TT genotype (29.5%) with two bandspattern (122 and 163 bp), and a heterozygous Tt genotype that had a three bands-pattern at 122, 163, and 385 bp. Moreover, the MHC Class II DRB gene of Spanish goats subjected to TaqI restriction had the following respective percentages of Tt and tt genotypes in MHC Class II DRB gene namely, 65 and 35%.
The relationship of such polymorphism in the MHC Class II DRB gene to humoral and cell-mediated immunity to economic diseases of goats will be the subject of the future investigation, to allow for selections of highly immune individuals.

Polymorphism in Prion (PrP) gene
The polymorphism in sequences of the PrP gene in rural and reference Saanen goat-sperms, between amino acid positions of 101-194 and 201-240, having specific codons affecting the susceptibility in goats to Transmissible Spongiform Encephalopathy (TSE), is presented in Table 2. Four PrP haplotypes were found based on the detected amino acid sequences. Amino acids dimorphisms were observed in codons 101, 102, 107, 151, 154, 171, 202, 207, 211, and 240, and three different mutations at codon 222. The following three polymorphisms in PrP amplicons were not reported previously for the following codons: codon 101, glutamine was substituted by serine; codon 107, lysine was substituted by serine; codon 222, glutamine was substituted by proline or leucine.
These aforementioned polymorphisms were found in PrP amplicons of rural goats, while the remaining five polymorphisms were present all together in one haplotype of the reference Saanen goat, mainly at codon 151, in which arginine was substituted by proline, at codon 202, in which proline replaced theronine, at codon 207, where lysine was substituted by arginine, at codon 211, in which arginine was substituted by glutamic acid, while at codon 222, the glutamic acid was replaced by glutamine.
No polymorphisms were found at codons 142, 143, 146, 168 that are associated with differences in phenotypic expression of TSE in goats [35]. In fact a substitution of isoleucine by methionine at codon 142 has altered the TSE-incubation period in goats [34,38]; in addition, a substitution of asparagine by serine or aspartic acid at codon 146 has been associated with resistance to TSE [35]. However, the simultaneous presence of isoleucine at codon 142 and asparagine at codon 146, as obtained in our study (Table 2), were found to be associated with an atypical TSE in Swiss goats [39]. In addition, the replacement of arginine by histidine in one rural goat at codon 154 (Table 2) has a variable protective effect against TSE, depending on the goat breed [40,41]; actually this mutation (R→H) provided some TSE-protection in Dman and Chaouni goats [42] leading to less frequency in TSE of goats [43]; on the contrary, this substitution (R→H) at codon 154 created a risk factor of atypical TSE in goats [44].
The dimorphism shown in codon 240 (serine→proline) is not reported in other ruminants [34], and its presence is documented in PrP gene of mink, ferret, domestic dog, and dingo [45,46]. The role of substitution of serine by proline at codon 240, in relation to TSE susceptibility, is controversial. Bouzalas et al. [35] reported that such a substitution creates a partial protection from clinical TSE, without an increase in resistance to prion infection in goats. The dual presence of proline in codon 240 and histidine at position 154, as documented for rural goat L3 in Table 2 is reported to confer a partial resistance to TSE [34,47], while others [40] claimed a positive correlation between presence of proline at codon 240 with TSE manifestation. Several studies showed no association between the codon 240 polymorphism in PrP and the occurrence of TSE [34,38,43,47,48].
The presence of a similar PrP-amino acid sequence at codons 136 (alanine), 154 (arginine), and 171 (glutamine) in each of three rural goats and in the reference Saanen goat (Table 2) indicates a similarity to scrapie susceptibility in sheep [49]. Fortunately, one rural goat (L3 in Table 2) showed a novel haplotype of alanine (codon 136), histidine (codon 154), and arginine (codon 171). It is important to study in the future the correlation of such novel patterns of amino acid sequences in PrP gene to susceptibilities to TSE in goats, to help in selection for resistance to this economic zoonotic disease.
In conclusion, the search for CC and/or ABCD genotype for GH gene in rural goats is needed to be used in the future selection model for production improvements. The polymorphism in the αS1-Casein gene detected in this research is helpful in selection of goats for high or low casein protein level in the produced milk. The TaqI endonuclease succeeded in resulting with a polymorphism in MHC class II DRB gene, while PstI failed; the relationship of polymorphism created by the TaqI enzymes to selection for improvement of humoral and cellmediated immunity will be the subject of future investigation. The relationship of the novel polymorphism present in the specific amino acid sequences of the PrP gene of the rural goats to susceptibility to TSE needs to be investigated to allow for future selection programs to result in goats that are resistant to this economic disease and that provide safe foods to human consumers. The presence of multiple polymorphism in the studied 4 genes allow for assigning genetic markers in selection