Identification of Single Nucleotide Polymorphism in Y-chromosome Specific DDX3Y Gene in Murrah Buffalo Bulls

The modern breeding program using frozen semen from genetically superior bulls through artificial insemination is extensively used to improve economically important traits. Semen production and fertility index of bulls are complex traits which are influenced by both genetic and environmental factors. Identifying bulls with high genetic merit is very important for quality frozen semen production, as bulls selected for semen production are often culled due to poor reproductive performance. The development of molecular markers based approaches is essential for the selection of breeding bulls, which accurately predicts the reproductive efficiency of the bulls at an early age.
        
The semen production is primarily governed by the clusters of genes in the male-specific region of Y- chromosome, which plays a significant role in reproduction, sexual development, sex differentiation, spermatogenesis and spermatogenic cells structural and functional integrity (Mukherjee et al., 2013). The Y-specific genes regulating the process of semen production could be considered as potential candidate genes to determine the bull’s fertility. Therefore, identifying single nucleotide polymorphisms in these candidate genes would help to develop potential genetic markers for selecting quality bulls through Marker Assisted Selection (MAS). However, only few Y-chromosome specific genes (SRY, TSPY, RBMY and ZNF280BY) have been investigated for the polymorphisms associated studies in buffaloes (Mukhopadhyay et al., 2011; Kaur et al., 2014; Revanasiddu et al., 2019), but still, Y-specific genes like DDX3Y, USP9Y, HSFY, PRAMEY, etc. are yet to be explored in buffaloes.
        
DEAD-box polypeptide 3 Y-linked (DDX3Y) gene (also known as DBY) belongs to the DEAD (Asp-Glu-Ala-Asp) box protein family, which is involved in ATP-dependent RNA unwinding and other cellular activities like slicing, ribosome biogenesis and RNA degradation (Gueler et al., 2012 and Ramathal et al., 2015). DDX3Y gene transcript functions are testis-specific, the proteins are expressed predominantly in male germ cells, indicating its role in the spermatogenic process and spermatocyte development (Liu et al., 2009). The mutations in this gene were associated with the reduced number of sperm cells, structural deformities, male subfertility and infertility in humans (Ramathal et al., 2015 and Liu et al., 2009). Hence, the present study was undertaken to identify single nucleotide polymorphism in the Y- chromosome specific DDX3Y gene in Murrah buffalo bulls.

Experimental animals
 
Blood samples were collected from 70 Murrah buffalo bulls maintained in the frozen semen production station, Exotic Cattle Breeding Farm, Thanjavur, Tamil Nadu, India. Bulls with normal seminal traits (42) and poor semen production performance i.e., <60% initial motility (17), <50% post-thaw motility (7) and poor libido (4) were selected for the study. The experiment was performed in 2016 at the Department of Animal Genetics and Breeding, Madras Veterinary College, Chennai.
 
DNA extraction
 
About 10 ml of blood was collected aseptically from the jugular vein and transferred to sterile Becton-Dickinson vacutainer precoated with 0.5% of 0.12 ml EDTA. The genomic DNA was extracted from the blood samples by organic method (Sambrook et al., 1989). The quality of DNA was checked in 0.8% agarose using horizontal gel electrophoresis with 0.5X TAE as running buffer. The concentration and purity were estimated with Nanodrop Bio-spectrophotometer using millipore water as blank reference. Based on concentration, the DNA samples were diluted with sterile nuclease-free water to working solution of 20 to 50 ng/μl and stored at -20°C.
 
Primer designing
 
The primers for the exons of DDX3Y gene were designed with “Primer3” online software (http://frodo.wi.mit.edu/primer3) using Bos taurus DDX3Y gene sequence (Gene ID with accession number: 783057 - NM_0011725951.1) as reference. The sequence of exons was selected along with the intronic regions for the proper amplification of the entire exons. The Oligo Analyzer 1.0.3 software was used to analyse the primers for self-annealing, GC content and primer loops to obtain the best pair of primers. The primers working concentration of 10 picomoles were prepared from the stock concentration of 100 pmol/µl using nuclease-free water. The primer sequences, their corresponding product sizes and annealing temperatures are presented in Table 1.
 

 
PCR amplification
 
The polymerase chain reaction (PCR) was performed in 15 μl reaction volume with 25-50 ng of genomic DNA, primers and PCR master mix using thermal cyclers. The annealing temperature of each primer was standardized by gradient PCR, based on the quality yield of specific PCR products. About 3 μl of PCR products along with 50 bp DNA ladder was checked in 2% agarose gel holding 0.5 µg/ml ethidium bromide. The gels were run at a voltage of 100V for 45 minutes and the product size was visualized under UV trans- illuminator and documented by gel documentation system. The composition of the PCR reaction mixture and thermocycle conditions are furnished in Tables 2 and 3.
 

 


Sequencing and analysis
 
The PCR amplicons were sequenced in both forward and reverse directions by M/s. SciGenom Labs Pvt. Ltd., Cochin. The “Seqman module” of DNASTAR LASERGENE software for multiple sequence analysis was used to identify the single nucleotide polymorphism within the sequences of Murrah buffalo bulls taking Bos taurus DDX3Y gene sequence as reference. Clustal W sequence alignment (MegAlign module) was used to determine the single nucleotide variations between the sequences of Murrah buffalo and Bos taurus. The phylogenetic tree and genetic distance were constructed for pairwise sequence alignment under Neighbour-Joining (NJ) with 1000 bootstrap using the MegAlign module.

The DDX3Y gene consists of 17 exons with a sequence length of 130 bp, 134 bp, 154 bp, 118 bp, 182 bp, 145 bp, 145 bp, 161 bp, 99 bp, 86 bp, 136 bp, 101 bp, 159 bp, 130 bp, 48 bp, 61 bp and 165 bp, respectively. Eleven pairs of primers were designed to amplify the entire exons by covering the intron regions associated with the exons (Fig 1). Out of the total gene length of 12263 bp, the nucleotide sequence of 6865 bp covering 17 exons was amplified, which comprised 55.98% of the gene.
 
 
        
The analysis of sequences evinced that the exons and associated intronic regions of DDX3Y gene were monomorphic in nature in the studied population of Murrah buffalo bulls. There were no nucleotide variations observed between the sequences of bulls with normal and poor semen production performance in any of the exons of the gene (Fig 2). Duplicate samples were sequenced and analysed to confirm the monomorphism, which also revealed the absence of variation in Murrah bulls. These results indicate that the sequences of DDX3Y gene are highly conserved among the Murrah buffalo bulls studied and hence, association studies could not be conducted with seminal parameters. However, when performed pairwise alignment with Bos taurus sequences, single nucleotide variations were observed in the sequences of Murrah buffalo.
 

        
At positions, 846 (C<T), 861 (T<C), 863 (C<T), 869 (C<A), 886 (G<C), 889, 891 (G<A), 912 (A<C), 915 (C<G), 919 (G<T), 922 (A<C), 961 (C<T), 971 (T<C), 993 (A<C), 1007 (T<C), 1013 (A<G), 1015 (G<C), 1026 (A<G), 1048 (T<C), 1051 (A<G), 1056-57 (C<G), 1083 (T<C), 1111 (A<G), 1140 (G<A), 1182 (C<A), 1256 (A<G), 1258 and 1278 (C<T), 3268 (T<A), 3315(A<C), 3355 (C<T), 3387 (A<G), 3428 (C<T), 3429 (A<G), 3439 (T<C), 3442 (A<G) and 3464 (T<C) nucleotide variations were observed in the exons 1-6 and its intronic regions (Fig 3). Similarly, the exons 7-10 and their partial intronic regions revealed a considerable number  of single nucleotide variations in the Murrah buffalo sequence compared with Bos taurus (Fig 4). The nucleotide regions covering exons 11-17 along with intronic regions unfolded variations at position, 7962, 7980, 8116, 8191 and 8196 (G<A), 8201 and 8221 (A<G), 8331 (G<A), 8337 (C<A), 8341 and 8443 (G<A), 8490 (A<G), 8498 (T<C), 8501 (G<A), 8503 (C<T), 8620 (G<A), 9020 (A<C), 9054 (T<C), 9190 (A<G), 9258 (G<C), 9274 (C<G), 9500 (T<G), 10685 (A<T), 10686 (T<C), 10710 (A<C), 10712 (A<G), 10716 (T<G), 10730 (C<T), 10813 (T<C), 10845 (C<T), 10861 (C<G), 10936 (T<C), 10940 T<G), 11018 (G<T), 11031 and 11068 (T<C) and 11079 (A<T), respectively (Fig 5). The phylogenetic tree was constructed by comparing the nucleotide sequences of DDX3Y gene of Murrah buffalo with those of other closely related species in the Bovidae family to find out the level of homology. The phylogenetic tree analysis of DDX3Y gene sequences revealed clusters of two separate clades, consisting of all bovinae species on one group and the other with Capra hircus, Ovis aries and Odocoileus virignianus (Fig 6). Within the second clade, Ovis aries and Odocoileus virignianus were sub-grouped closely than Capra hircus. With respect to bovinae clade, Murrah buffalo showed close genetic homology with Bos taurus, followed by Bison bison and Bos indicus, which indicates that these regions are highly conserved among bovinae species though they are structurally diversified. The DDX3Y gene sequence of buffalo, cattle and bison are more closely associated with each other than the caprinae and cervinae species.
 

 

 

 

        
On perusal of literature, there is dearth of information on the DDX3Y gene in Murrah buffalo to compare or contrast the findings of the present study. DDX3Y gene is one of the two genes located in the ‘azoospermia factor A’ (AZFa) region of the Y-chromosome and exerts its effect on spermatogenesis in combination with other genes of the region (Vineeth and Malini, 2011). Earlier studies reported that major changes like deletion or shortening in the DDX3Y gene resulted in disrupted spermatogenesis, causing subfertility and infertility (azoospermia or oligospermia or oligoasthenozoospermia) in humans (Foresta et al., 2000). However, in the present study, no such variation was observed in the studied population of bulls, the poor semen production performance might be attributed to other genetic, environmental and managemental factors. Further studies on AZFa region genes targeting bulls with severe subfertility problems like reduced motility, sperm abnormalities, azoospermia, could able to provide markers to differentiate bulls with normal semen production and subfertility problems. The highly conserved region of the DDX3Y gene in Murrah buffalo is in agreement with earlier reports in crossbred Jersey and crossbred Holstein Friesian bulls (Gopinathan, 2014). Similarly, Hellborg and Ellegren (2004), while studying nucleotide variation in the intronic region of mammalian Y-chromosome, reported lower level of genetic variability in Y-specific genes (DDX3Y, SRY, UTY and ZFY) compared to X-Chromosome in Swedish and Hereford cattle. However, Xu (2014) identified several mutation sites in the DBY gene of Chinese water buffalo, but no association studies were performed.

The molecular characterization of DDX3Y gene in identification of single nucleotide polymorphism revealed that the sequences of DDX3Y gene are highly conserved and no variation was observed among the studied population of Murrah buffalo bulls. However, single nucleotide variations were observed in the sequences of Murrah buffalo when compared with Bos taurus sequences. Further studies on DDX3Y and other Y-chromosome specific genes in the detection of polymorphisms in a large, diverse population of buffaloes could help to develop suitable candidate markers for the selection of quality bulls at an early age.