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India is home to a large and diverse buffalo population. The Murrah breed of North India is known for its milk production, and it has been used in breeding programs in several countries. Selection signature analysis yield valuable information about how the natural and artificial selective pressures have shaped the genomic landscape of modern-day livestock species. Genotype information was generated on six buffalo breeds of India, namely, Murrah, Bhadawari, Mehsana, Pandharpuri, Surti, and Toda using ddRAD sequencing protocol. Initially, the genotypes were used to carry out population diversity and structure analysis among the six breeds, followed by pair-wise comparisons of Murrah with the other five breeds through XP-EHH and
Water buffalo is considered as an important livestock resource in tropical and sub-tropical countries due to its high milk production ability along with adaptability to hot and humid environment, and high feed conversion efficiency (
Murrah is the most important buffalo breed of India, constituting about 44.3% of the total buffalo population of the country. The main breeding area of this breed is the northern states of India, namely Punjab, Haryana, and Western Uttar Pradesh. Due to its high milk potential in varied environmental conditions, the germplasm of the breed has been extensively used throughout the country. It has also been imported in several countries like China, Brazil, Vietnam, Egypt, Bangladesh, etc., due to its higher milk production potential (
This study aimed to assess the genetic diversity and population structure among the six Indian buffalo breeds using ddRAD data. Furthermore, we attempted to unravel signatures of positive selection in Murrah by comparing it with other reference Indian breeds (Surti, Mehsana, Bhadawari, Pandharpuri, and Toda) through cross-population extended haplotype homozygosity (XP-EHH) and cross-population fixation index (
Ninety-six samples were collected from six breeds of riverine buffalo from different parts of India. These breeds are diverse in terms of physical features, milk production, and adaptation. Selection of the animals was done in a way to cover the genepool of the respective breeds. So the animals of all the breeds in the present study were chosen randomly from their respective institutional farms (except animals of the Toda breed of buffalo for which random samples were collected from its breeding tracts in the Nilgiri Hills area of Tamilnadu state of India). As the Murrah breed is mainly found in the northern part of India, the random samples were collected from three institutional farms of the area, i.e., the Livestock Research Station (LRS) ICAR-IVRI situated in Izatnagar, Bareilly (Uttar Pradesh), the Buffalo Farm at livestock research station of GBPUA and T, Pantnagar (Uttarakhand), and the Livestock Farm, GADVASU Ludhiana. The samples of Bhadawari buffalo were collected from the Buffalo Farm, ICAR-IGFRI, Jhansi (Uttar Pradesh), Mehsana buffalo samples were collected from the Livestock Research Station, SDAU, SK Nagar (Gujarat), Surti buffalo samples were collected from the Livestock Research Station, CVAS, Udaipur (Rajasthan), and Pandharpuri buffalo samples were collected from the Buffalo Farm, Zonal Agriculture Research Station, Kolhapur (Maharashtra). All these farms are situated in their respective breeding tract, and animals were randomly selected from these institution farms as to cover substantially the genepool of the population. The breed-wise details of sample numbers and location are also provided in
The reads were quality checked using FastQC (
The paired reads were aligned to the
The structural and functional annotation of the retained SNPs was performed using SnpEff v4.3 (
Linkage disequilibrium (LD) pruning of the SNPs was carried out using the
Cross-population selection signatures between Murrah buffalo and five other Indian water buffalo breeds (Bhadawari, Surti, Mehsana, Pandharpuri, and Toda) were derived using XP-EHH (
The pairwise
The candidate genes in the selected regions were annotated using the GTF (gene transfer format) file supplied with the UOA_WB_1 assembly, using BEDTools (
In the present study, total 397.8 million paired-end reads of 150-bp length were obtained for the 96 buffalo breeds, averaging 4.14 million reads per sample. After initial quality control, a total of 367.2 million reads (92.3% of the total reads) of average 135-bp length were retained. The average alignment rate of the reads was 99.82% with the reference genome. Sample-wise alignment percentages are given in
Based on the sequence ontology terms, a greater number of identified SNPs were located within the intronic regions (66.57%), followed by the intergenic regions (22.13%), and 0.34% of SNPs were found to be located in the transcript region (
About 71.89% of the annotated SNPs were identified as transitions (Ts) while 28.10% as transversions (Tv) with a TS/TV ratio of 2.5578. The Ts/Tv ratio serves as a quality control indicator of high-throughput sequencing data. Our values are consistent with previous reports of targeted sequencing methods in buffalo (
For the genetic diversity and population structure analyses, we used a subset of 67,798 SNPs after pruning the SNPs in LD. The average observed heterozygosity (Ho) and expected heterozygosity (He) of all breeds in the study are presented in
Number of animals, means of observed (HO) and expected heterozygosity (HE), and differentiation (FST) between each breed and the Murrah.
S.No | Breeds | Number of animals | Ho | He |
|
---|---|---|---|---|---|
1 | Murrah | 30 | 0.2372 | 0.2462 | - |
2 | Bhadawari | 15 | 0.2343 | 0.2366 | 0.11 |
3 | Mehsana | 15 | 0.2314 | 0.2239 | 0.17 |
4 | Surti | 15 | 0.2361 | 0.2255 | 0.09 |
5 | Pandharpuri | 15 | 0.2366 | 0.2390 | 0.15 |
6 | Toda | 6 | 0.2150 | 0.2111 | 0.13 |
The population structure of the Indian water buffalo breeds was identified using PCA. The first and second principal component (PC) explained 3.4 and 2.86% of the total variance. PC1 separated the crossbred Mehsana individuals from the rest of the breeds, while PC2 separated the Pandharpuri, Surti, and Toda from the Murrah and Bhadawari (
(
The maximum-likelihood phylogram constructed with Treemix also displayed a similar tree (
As seen with PC1, the Mehsana was separated from the rest of the breeds at K = 2 in the admixture analysis. K = 3 separated the Pandharpuri as a distinct population from the rest of the breeds, which gives credence to the results of the phylogenetic analyses. The Toda samples in our study showed a mixture of Pandharpuri and Murrah inheritance. At K = 6, all the breeds were assigned to their own clusters, with varying levels of Murrah ancestry appearing in other breeds (Bhadawari, Mehsana, Surti, and Toda) (
The distribution of XP-EHH scores for the Murrah buffalo (positive values) against other water buffalo breeds in the study is visualized in
Cross-population extended haplotype heterozygote (XP-EHH) plot of the Murrah in comparison with the Bhadawari, Mehsana, Surti, Pandharpuri, and Toda.
Common selection sweeps identified by cross-population extended haplotype homozygosity (XP-EHH) in two or more pairwise comparisons involving the Murrah.
S.No | References breeds | Chr | Start | End | Annotated gene |
---|---|---|---|---|---|
1 | Bhadawari | 1 | 192,319,897 | 192,322,098 |
|
Mehsana | |||||
2 | Bhadawari | 2 | 56,674,658 | 56,740,551 |
|
Pandharpuri | |||||
3 | Bhadawari | 3 | 143,278,931 | 143,620,455 |
|
Surti | |||||
4 | Surti | 4 | 41,323,382 | 41,449,515 |
|
Toda | |||||
5 | Bhadawari | 7 | 28,640,078 | 30,146,985 |
|
Toda | |||||
Pandharpuri | |||||
6 | Mehsana | 9 | 64,216,990 | 64,326,407 |
|
Pandharpuri | |||||
7 | Bhadawari | 10 | 84,290,283 | 84,562,847 |
|
Pandharpuri | |||||
8 | Toda | 12 | 86,340,919 | 86,501,726 |
|
Bhadawari | |||||
9 | Toda | 20 | 49,776,417 | 49,968,750 |
|
Bhadawari | |||||
10 | Pandharpuri | 23 | 48,880,371 | 49,056,564 |
|
Bhadawari |
The Manhattan plot for pairwise
Manhattan plot for FST between the Murrah in comparison with the Bhadawari, Mehsana, Surti, Pandharpuri, and Toda.
A total of six fully or partially overlapping selection sweeps were identified from both the approaches XP-EHH and
Selection signatures in the Murrah identified by both XP-EHH and
S. No | Test | Chr | Start | End | Genes |
---|---|---|---|---|---|
1 | XP-EHH (Surti); |
1 | 187,322,925 | 187,600,000 |
|
2 | XP-EHH (Pandharpuri, Bhadawri, Toda); |
7 | 28,553,887 | 29,108,103 |
|
3 | XP-EHH (Surti); |
8 | 109,432,200 | 1,117,495,711 |
|
4 | XP-EHH(Pandharpuri); |
13 | 23,401,830 | 24,977,050 |
|
5 | XP-EHH (Pandharpuri); |
15 | 22,545,641 | 22,557,701 |
|
6 | XP-EHH (Toda); |
18 | 14,622,913 | 14,929,335 |
|
In the present study, ddRAD sequencing was used to identify genetic variants in six water buffalo breeds of India. The average heterozygosity levels ranged from 0.215 to 0.237, which were lower compared with a previous study (
The second objective of this study was to identify positive signatures of selection in the Murrah buffaloes. Humans have exerted strong artificial selection on different breeds of buffalo for similar traits since domestication (
The positive signatures of selection in the Murrah buffaloes were identified using XP-EHH and
On chromosome 1, a region was identified around the 192.2 Mb position against the Bhadawari, Mehsana, and Toda, which was in agreement with
A selection sweep (28.5–29.1 Mb) on chromosome seven in comparisons of the Murrah with the Pandharpuri, Toda, and Bhadawari also confirms a previously reported selection sweep (chromosome 7, 26.5–30.5 Mb) in the Murrah genome by
In agreement with
In addition, several novel regions of positive selection were also identified. These regions contain candidate genes, which are associated with the phenotypes that are under selection in the Murrah buffalo, including milk production and fat metabolism (
The genetic diversity and population structure analysis revealed varying levels of the Murrah inheritance in the Bhadawari, Mehsana, Surti, and Toda buffalo breeds. The selection signature analysis provides several genomic regions as selection signature in the Murrah, which is the prominent milch breed in India. Using reduced representation ddRAD data, our results confirm many regions, which have been previously identified as selection sweeps in the Murrah genome using WGS data. In addition, novel regions were also identified, which are involved in several biological pathways. The candidate genes, found to be positively selected, are involved in milk production (
The genotypes of the 96 individuals under study have been uploaded to Figshare under the DOI
The study was carried out in accordance with recommendation of Institute Animal Ethics Committee of ICAR-IVRI, Bareilly India.
AP and AK conceived and designed the experiments. ST performed the experiments. AM and AS analyzed the data and wrote the manuscript. TD and BM contributed reagents/materials/analysis tools. ST, AK and AP edited the manuscript.
This project work was supported by the CAAST-ACLH project of NAHEP, and institute funding of ICAR-IVRI.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
The authors acknowledge the logistic and laboratory support rendered by the Director ICAR-IVRI. We also thank Dr. P.P Dubey and Dr. Puneet Malhotra (GADVASU), Dr. B.N. Sahi (GBPUA&T), Dr. B.P. Kushwaha (ICAR-IGFRI, Jhansi), Dr. Mitesh Gaur (LRS, Vallabhnagar), Director of Research (SDAU, SK Nagar, Gujarat), Dr. R.S. Kataria (ICAR-NBAGR), Dr. Jayakumar S. (ICAR-Directorate of Poultry Research), and Dr. A. P. Fernandes (MPKV Rahuri) for their contribution in providing the samples used in this study. We also wish to acknowledge Prof. Hubert Pausch (Animal Genomics, ETH Zurich), for his inputs regarding the SNP calling pipeline.
The Supplementary Material for this article can be found online at: