Amplification, Manipulation, and digestion
This research note concerns mainly the differentiation between and within different livestock species for IGFBP-3 gene. It, also spotlights the association between polymorphism of IGFBP-3 and the performance of different species of farm animals for some economical traits. PCR amplification for the tested animals produced an amplified 654 bp fragment for Egyptian sheep IGFBP-3 gene comprised of part of exon 2, complete intron 2, exon 3 and a part of intron 3 (Fig. 1A), an amplified 651 bp fragment for Egyptian cattle (Fig. 1B) and an amplified 655 bp fragment for El-Beheiry buffalo (Fig. 1C), while the amplified 316 bp fragment for Falahi goat IGFBP-3 gene was comprised of part of exon 2 (Fig. 1D). The PCR products of IGFBP-3 gene obtained for sheep after digestion with HaeIII (Fig. 2A) showed a digestion profile revealing only one pattern for eight DNA fragments sized 201, 201, 87, 67, 57, 18, 16 and 7 bp and indicated absence of polymorphism in sheep IGFBP-3 gene. The restriction fragments with sizes; 18, 16 and 7 bp were not seen on the gel.
The present results agree with those of Kumar et al [3] who studied the genetic diversity among the Indian breeds of sheep; Marwari, Mandya, Madras, Red Muzaffarnagari and Banur based on sequencing and digestion profile of IGFBP-3 gene with HaeIII and reported that; the digestion profile revealed only one pattern with eight DNA fragments sized 201, 201, 87, 67, 56, 19, 16 and 7 bp for the tested animals and, consequently, no polymorphism was detected. Also, Choudhary et al [11] reported that; all sheep possess intact HaeIII restriction site (GG↓CC) at the base number 300 of IGFBP-3 gene sequence indicating also, an absence of polymorphism at this site.
For Egyptian cattle, three genotypes were identified: (1) Lanes 1 and 2; homozygous (BB) genotype with 8 restricted fragments at 199, 164, 154, 56, 36, 18, 16 and 8 bp, (2) Lanes 3 and 4; heterozygous (AB) genotype with 5 restricted fragments at 199, 194, 164, 56 and 36 bp and (3) Lanes 5 and 6; homozygous (AA) genotype with 8 restricted fragments at 194, 164, 159, 56, 36, 18, 16 and 8 bp (Fig. 2B). The restriction fragments with sizes; 18, 16 and 8 bp were not seen on the gel. The polymorphism in cattle was due to C→A (GG↓CC to GG AC) transition/mutation in exon (3) of the gene at the 451st base position of sequence for allele (A), while was due to (GG↓CC to AG AC) transition/mutation in exon (3) of the gene at the 456th base position of cattle sequence for allele (B), )Supplementary Figure 3(. These results were in agreement with those of Shukla [12]; Kumar et al [3], and Choudhary[11] who detected three genotypes identified in Jersey and exotic Holstein Friesian cattle. Moreover, Othman et al [13] found that; the digestion of PCR products with the restriction enzyme of HaeIII revealed three genotypes in some local cattle;(AA), (CC), and (AC) with frequencies nearly 22%, 22%, and 56 %, respectively.
As for the buffalo, the presence of TaqI site characterized by a single homozygous genotype possessing two fragments of sizes 415 and 240 bp were observed (Supplementary Fig. 1A). As for HaeIII restriction enzyme, all screened buffaloes showed only one genotype (AA) with restriction fragments of sizes 201, 165, 154, 56, 36, 19, 16 and 8 bp (Supplementary Fig. 1B) in accordance with those reported by Choudhary [11] on six breeds of buffalo, though the sizes of restriction fragments were different (201, 165, 154, 56, 36, 19, 16 and 8 bp). The above results mean the lack of detected polymorphism among studied buffalo breeds with respect to IGFBP-3 gene. Similarly, results of Padma et al [14] on 157 Indian Murrah, Surti, Jaffarabadi and Nagpuri riverine buffaloes, revealed that; the digestion with HaeIII yielded single restriction pattern of 8 fragments of sizes 201, 165, 154, 56, 36, 19, 16 and 8 bp for all animals and with TaqI and MspI also produced single restriction pattern yielding fragments of sizes 240, 415 bp and 145, 510 bp, respectively. This again shows the non-polymorphic nature of restriction sites in buffalo.
Finally, the PCR profiles of tested goats IGFBP-3 gene digested with HaeIII revealed one pattern only for three DNA fragments sized 263, 58 and 8 bp (Supplementary Fig. 2), the restriction fragment with size 8 bp was not seen on the gel. However, the study of Lan et al [5] on goats detected polymorphisms in the IGFBP-3 gene by PCR-SSCP and DNA sequencing methods. Though the associations of HaeIII and XspI PCR-RFLPs for goat IGFBP-3 locus with milk traits were analyzed, no significant statistical results were found.
Supplementary Fig.3 shows the diagrammatic representation of exon-intron regions of the tested animals of different species and restriction enzymes sites (HaeIII and TaqI) of the amplified IGFBP-3 gene fragments.
Nucleotide sequence comparison
Nucleotide sequencing of the amplified fragments of the IGFBP-3 gene of sheep, goats, cattle, and buffaloes were submitted to the NCBI GenBank (Accession no. MG738671.1, MG738672.1, MG738673.1 and MG738674.1, respectively) (Supplementary Fig. 4). The nucleotide sequence analysis performed by Blastn (https://blast.ncbi.nlm.nih.gov/Blast) indicated that the similarity percentages of IGFBP-3 gene fragment between (sheep and cattle was 88.54 %), between (sheep and buffalo was 89.63 %), while between (cattle and buffalo was 95.06%) (Supplementary Fig. 5).
Protein sequence comparison
The partial part of exon 2, complete intron 2, exon 3 and a part of intron 3 present in the sequence of the amplified IGFPB-3 gene fragments of tested animals as generated by ExPASy program (http://web.expasy.org/translate) and the comparison of amino acids obtained by MEGA-6 VERSION 4 are in (Supplementary Fig. 6). The protein sequence of sheep is different from that of cattle and buffalo by 18 amino acids. The display of amino acids (Table 1) accounted for approximately 70 % similarity in sequence between sheep groups and bovine species vs. cattle and buffalo. However, the study of Kumar et al [3] indicated approximately 93% similarity in the amino acid sequence for sheep with cattle and buffalo.
Table 1. The different amino acids in sheep as compared with cattle and buffalo, which were obtained from a part of exon 2, complete exon 3 for each species.
10
|
8
|
7
|
6
|
5
|
4
|
3
|
2
|
1
|
No.
|
K
lysine
|
T
Threonine
|
Q
Glutamine
|
L
Leucine
|
L
Leucine
|
Q
Glutamine
|
Q
Glutamine
|
S
Serine
|
D
Aspartic
|
Sheep
|
S
Serine
|
G
Glycine
|
S
Serine
|
S
Serine
|
Y
Tyrosine
|
S
Serine
|
S
Serine
|
Q
Glutamine
|
T
Threonine
|
Cattle & Buffalo
|
18
|
17
|
16
|
15
|
14
|
13
|
12
|
11
|
10
|
No.
|
Q
Glutamine
|
L
Leucine
|
F
Phenylalanine
|
R
Arginine
|
L
Leucine
|
C
Cysteine
|
A
Alanine
|
L
Leucine
|
Q
Glutamine
|
Sheep
|
P
Proline
|
R
Arginine
|
S
Serine
|
S
Serine
|
R
Arginine
|
Y
Tyrosine
|
P
Proline
|
C
Cysteine
|
S
Serine
|
Cattle & Buffalo
|