Establishing gene Amelogenin as sex-specific marker in yak by genomic approach

Abstract

Yak, an economically important bovine species considered as lifeline of the Himalaya. Indeed, this gigantic bovine is neglected because of the scientific intervention for its conservation as well as research documentation for a long time. Amelogenin is an essential protein for tooth enamel which eutherian mammals contain two copies in both X and Y chromosome each. In bovine, the deletion of a fragment of the nucleotide sequence in Y chromosome copy of exon 6 made Amelogenin an excellent sex-specific marker. Thus, an attempt was made to use the gene as an advanced molecular marker of sexing of the yak to improve breeding strategies and reproduction. The present study confirmed that the polymerase chain reaction amplification of the Amelogenin gene with a unique primer is useful in sex identification of the yak. The test is further refined with qPCR validation by quantifying the DNA copy number of the Amelogenin gene in male and female. We observed a high level of sequence polymorphisms of AMELX and AMELY in yak considered as novel identification. These tests can be further extended into several other specialized fields including forensics, meat production and processing, and quality control.

Introduction

Yak (Bos grunniens) is a semidomesticated, multifunctional bovid found in high altitudes of the Himalayan region. It provides milk, meat, wool, fuel, shelter and are also used as a pack animals for highlanders (Wang et al. 2014). Although similar chromosome number persist among yak and cattle, their survival capacity in extreme cold and harsh environments make yak economically viable animal. Here, we illustrated a reliable method for sex determination in the yak with X and Y chromosome-specific Amelogenin gene (AMELX and AMELY) and found AMELY gene as a signature point in the evolution of Y chromosome in the yak. The Amelogenin gene has undergone very less recombination throughout its evolution, thereby thought to represent the ancestral pseudoautosomal boundary (Sasaki and Shimokawa 2003) and thereby used in the study of chromosome evolution in mammals (Lahn and Page 1999; Pfeiffer and Brenig 2005; Gokulakrishnan et al. 2012). However, precise understanding and annotation of this gene in yak are yet to be studied. Few previous studies proposed that the mammalian sex chromosomes were evolved by four events and a series of inversions on the Y chromosome, and Amelogenin loci span ancient pseudoautosomal boundary in diverse mammalian species (Iwase et al. 2003; Grzybowski et al. 2006). In eutherian mammals, a copy of the Amelogenin gene is present in X and Y chromosomes with a significant sequence variation (Girondot and Sire 1998). The two copies of this gene appears due to its independent duplication from X to Y chromosome (Sire et al. 2007), whereas in some other vertebrate a single copy is present only on X chromosome (Lahn and Page 1999). Bovine Amelogenin gene consists of six exons and a portion of about 63 nucleotides is deleted in Y linked copy of the gene (Girondot and Sire 1998; Das et al. 2009). This deletion appears to be an excellent tool for sex determination marker with considerable distinction in AMELX and AMELY. Till date, the different choice of methodologies has been developed to determine the sex of animal and animal products by specific gene analysis or hormone assessment (Pfeiffer and Brenig 2005). However, the DNA based techniques of sex determination in both human and animal have turned out to be more prominent in recent times. With the aim of similar hypothesis, this study was conducted to establish Amelogenin gene, a reliable sex-specific marker in yak by the genomic approach through PCR, quantitative PCR (qPCR), and comparative sequence analysis.

Materials and methods

Procurement of blood was done in accordance with the approval of Institute Animal Ethics Committee of ICAR-NRC on Yak, Dirang, India. The approved animal use protocol number is 4(17)/NRCY/IAEC-02 dated 01.08.2013.

Table 1 Detail of primer sequences, annealing temperature and product size of AMELX (different fragments), AMLEY and AR.

Fig. 1

Agarose gel images showing PCR results. Amplification of product of (a) 393 bp with primer-1 (AMELX1) in lanes 2 and 3; (b) 200 bp with primer-2 (AMELX2) in lanes 2 and 3; (c) 298 bp with primer-3 (AMELX3) in lanes 2 and 3; (d) 195 bp with primer-4 (AMELX4) in lanes 2 and 3; (e) 279 and 216 bp with primer-5 (AMELX5) in lanes 2 and 3; (f) 94 bp with primer-6 (AMELX6) of in lanes 2 and 3; (g) 289 bp with primer-7 (AMELX7) in lanes 2 and 3 (h) 103 bp with primer-8 (AMELX8) in lanes 2 and 3. Lanes: 1 and 8, 100-bp marker; 2, female yak genomic DNA; 3, male yak genomic DNA; 4, AR gene as the positive control in female yak genomic DNA; 5, AR gene as the positive control in male yak genomic DNA; 6, negative control; 7, blank.

The blood sample was collected from organized yak farm, and subsequently DNA was isolated using the standard protocol. Eight pairs of primers were designed from bovine genome spanning whole AMELX gene and one pair primer from X-linked androgen receptor (AR) as a positive control and four pairs from housekeeping gene using Primer3 software (http://www.primer3.ut.ee) (table 1). PCR was performed as per manufacturer protocol (Thermo Scientific) with a slight modification. All the eight amplified PCR products of female yak along with extra band amplified in male yak by primer-5 were extracted from agarose gel and purified with Qiagen gel extraction kit. A total of nine fragments, namely AMELX1-393bp, AMELX2-200bp, AMELX3-298bp, AMELX4-195bp, AMELX5-279bp and 216, AMELX6-94bp, AMELX7-289bp and AMELX8-103bp products size covering 9023 bp of AMELX were sequenced by standard double-stranded sequencing reaction and analysed in ABI3700 automated DNA Analyzer (ABI).

All the fragments of Amelogenin sequences of female yak and one male-specific Amelogenin sequence were submitted to NCBI databank (accession numbers KT826760 and KT835052), and these sequences were aligned with available cattle sequences (http://www.ncbi.nlm.nih.gov/). Phylogenetic and molecular evolutionary analyses were conducted using MEGA v6.0 (Thompson et al. 1994). The qPCR was performed using the Applied Biosystems StepOne Plus Real-Time PCR System (Life Technologies) to construct standard curve and estimation of $\Delta \Delta \hbox {CT}$ for quantification of copy number of AMELX gene. The DNAs from five male and five female yaks were amplified by primers 1, 2, 3, 4, 5, 6, 7 and 8 (table 1). Each reaction was run on triplicate at a final volume of $20\,\mu \hbox {L}$ per reaction with $2\,\mu \hbox {L}$ of 10-fold diluted DNA, 100 nM of each primer and $10\,\mu \hbox {L}$ of SYBR Select Master Mix in $58^{\circ }\hbox {C}$, 2 min; $95^{\circ }\hbox {C}$, 2 min; 40 cycles of $95^{\circ }\hbox {C}$,15 s and $60^{\circ }\hbox {C}$, 1 min; followed by a melting curve program. The copy number variation of the gene and comparative results were analysed in StepOne plus software.

Results

Except for primer-5 (additional amplicon of 216 bp is amplified), all other primers produced similar product size in the male and female yaks along with positive control AR. Primer-5 gave two distinct amplified products in male but a single amplicon in the female. The AR gene amplified in both male and female genomic DNA of yak (figure 1, a–h). The sequencing of all the eight amplicons of AMELX (AMELX1, AMELX2, AMELX3, AMELX4, AMELX5, AMELX6, AMELX7 and AMELX8) from female yak and male-specific AMELX5 fragments of the male were successfully retrieved with expected product size. The qPCR with primers 2, 3, 4, 6, 7 and 8 in five male and five female genomic DNA distinguish the copy number in X and Y chromosomes. The result showed a significant higher copy number, almost equivalent to double in the female, compared to male ($P>0.5$), in all the primers except primer-6.

A total 1851-bp sequence size covering 9023-bp cattle AMELX at different intervals were sequenced. Further, the sequencing data of yak AMELX was aligned with the cattle and other closely related eutherian mammals sequences using NCBI blast tools. Although uniformity prevails after alignments of AMELX with cattle for the fragments AMELX1 and AMELX8, few mismatches were observed in the fragments AMELX2, AMELX3, AMELX4 and AMELX5 at positions 2106.A>C, 3255.A>G, 6217.A>T, 6235.C>A, G.6657.C respectively. A deletion was found at position G. 6657.C in AMELX5 amplicon in the female yak, but not in cattle. A 63-bp deletion in male-specific fragments of AMELX5 at position C.6464-6525 is a novel finding in the yak. Further, eight sequence mismatches at positions 6416.T>C, 6431.C>T, 6442.A>G, C.6462-6525.A, 6555.G>C, 6562.A>G, 6583.A>G, 6598.A>G, 6610.TG>CA, 6637.C>T also observed when compared among Y and X specific fragment of AMELX5 in yak (figure 2, a&b).

Fig. 2

Chromatogram of sequenced yak. (a) AMELX isoform; (b) AMELY isoform confirmed that a 63 bp deletion in male-specific fragments at position C.81-143.C as well as eight sequence mismatch at positions G.31G>T, G.46T>C, C.57G>A, A.69G>A, C.170/106C>G, C.177/114G>A, C.225/163G>T, C.253/189T>C comparing between Y and X specific fragment of AMEL of with primer5. Vertical red arrows are indicating primer sitting site of both forward (F) and reverse primer (R), horizontal red arrows indicate deletion of single nucleotide comparing to cattle, brown dotted line indicated deleted sequences in Y specific AMEL, purple arrows indicates position of deletion in Y specific AMEL; green and red squares indicated mismatch position in Y and X specific fragment of AMEL, respectively.

The phylogeny was analysed with yak AMELX fragment5 from both male and female. Sequence alignment of AMELX isoform (fragment5) of yak with bison, buffalo, cattle, goat and sheep showed a Bos specific deletion at position 126.C-135.C and yak specific deletion at position 278.A-280.C. The present study also revealed some distinctive deletions and substitutions in yak Amelogenin gene sequence when compared with its closely related eukaryotic species. Finally, a composite schematic diagram was constructed using all the available information on yak Amelogenin gene which confirms that the Amelogenin gene can be used as a molecular marker to determine the sex differences in yak (figure 3, a–f).

Fig. 3

A schematic diagram of the Amelogenin gene as a sex-specific marker in the yak. (a) Schematic diagram of complete AMELX gene of cattle showing different exons in blue squares and introns in the green square. Vertical arrows in both direction showing forward (F) and reverse (R) primer sitting site of primers 1, 2, 3, 4, 5, 6, 7 and 8. Exact start and end sites of all primers. (b) Showing PCR product size in male and female yak genomic DNA amplified by primers 1, 2, 3, 4, 5, 6, 7 and 8. The red square box indicates sex-specific PCR product size in male and female genomic DNA by primer5. (c) Indicating sequence mismatches between yak and cattle AMEL gene. Numbers indicate mismatches observed under each fragment of AMEL sequences. (d) Showing copy number variation in male and female genomic DNA using primers 2, 3, 4, 7, 8 confirming 2:1 ratio in male and female. Primer6 in the red box is presenting equal copy number in male and female genomic DNA. (e) Schematic representation showing 63 bp deletion in exon 5 in Y chromosome-specific AMEL detected by primer5. (f) Schematic representation of Y (red) and X (green) chromosome-specific fragment of AMEL gene, showing deletion of 63 bp in male-specific AMEL gene and exact sequenced product size 279 bp in female and 279 and 216 bp in male genomic DNA.

Discussion

Use of sophisticated tools for sex determination in an animal is always benefiting the breeding strategies and various scientific approaches. The present study revealed a sequence variation in Amelogenin gene could access as a phenomenal sex assurance marker in yak at the molecular level based on a sequence insertion and deletion attributes of X and Y-specific Amelogenin gene. It correlates with the previous study carried out in other vertebrates like cattle (Grzybowski et al. 2006), sheep (Pfeiffer and Brenig 2005), horse (Hasegawa et al. 2001), red deer (Gurgul et al. 2010) and human (Salido et al. 1992; Dutta et al. 2017). The differences in sequence length of X and Y copy of Amelogenin gene in yak were confirmed by the primer (AMELX-5), is first such report in the yak. Moreover, qPCR validation of DNA copy number in either sex enhances trials of the hypothesis. Double the copy number in female indicates X specific amplification. The sequencing data of yak AMELX may further facilitate the understanding of its biological importance, which provides the sequence differences with cattle AMELX genes. Sequencing results of few fragments of yak i.e., AMELX1 and AMELX8 showed uniformity with cattle AMELX whereas, others i.e., AMELX2, AMELX3, AMELX4, AMELX5, AMELX6 and AMELX7 of yak witnessed few mismatches with cattle seem to be unique, as of Amelogenin protein found highly conserved in placental mammals (figure 2, a&b). We also observed sequence variations within male and female as well as between species in the yak. A 63-bp deletion in male-specific fragments at position C.81-143.C as well as eight sequence mismatches at positions G.31G>T, G.46T>C, C.57G>A, A.69G>A, C.170/106C>G, C.177/114G>A, C.225/163G>T, C.253/189T>C in between Y and X specific fragments of AMELX5 reflects a unique identity of yak evolutionary lineage (figure 2, a&b). This deletion may occur in the Y chromosome, which subsequently lost most of its gene sequences in the course of evolution. The Amelogenin, on other hands, could not be completely lost or else may owe a transposon-based partial duplication to other chromosomes. The qPCR results of primer-6 showed same DNA copy number in both male and female yak since primer-6 was designed from the identical sequence in X and Y chromosomes (figure 2, a&b). Further, the DNA copy number variation in Amelogenin gene for sex determination is established for the first time in yak along with its mapping and unique sequence information on the yak is considered as a novel phenomenon.