Identification of candidate genes associated with milk yield trait in buffaloes ( <i>Bubalus bubalis</i> ) by restriction-site-associated DNA sequencing

Ye, Manhong; Xu, Mengting; Lu, Manran; Zhou, Bin; El-Kader, Heba Abd; Alam, Sally Said; Mahrous, Karima Fathy

doi:10.37496/rbz4920190267

Manhong Ye

College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China

Key Laboratory of Animal (Poultry) Genetics Breeding and Reproduction, Ministry of Agriculture and Rural Affairs, Beijing, China

http://orcid.org/0000-0002-1969-4810

Mengting Xu

College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China

Joint International Research Laboratory of Agricultural & Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China

Manran Lu

College of Bioscience and Biotechnology, Yangzhou University, Yangzhou, China

Joint International Research Laboratory of Agricultural & Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China

Bin Zhou ^* * Corresponding author: bzhou@yzu.edu.cn

College of Animal Science and Technology, Yangzhou University, Yangzhou, China

http://orcid.org/0000-0002-0824-964X

Heba Abd El-Kader

Department of Cell Biology, Genetic Engineering and Biotechnology Division, National Research Center, Dokki, Giza, Egypt

http://orcid.org/0000-0003-1092-9908

Sally Said Alam

Department of Cell Biology, Genetic Engineering and Biotechnology Division, National Research Center, Dokki, Giza, Egypt

http://orcid.org/0000-0002-4358-3627

Karima Fathy Mahrous

Department of Cell Biology, Genetic Engineering and Biotechnology Division, National Research Center, Dokki, Giza, Egypt

http://orcid.org/0000-0002-1662-1034

* Corresponding author: bzhou@yzu.edu.cn

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

Conceptualization: M. Ye and B. Zhou. Data curation: M. Ye, M. Xu and M. Lu. Formal analysis: M. Ye and M. Lu. Funding acquisition: B. Zhou. Methodology: M. Ye and M. Xu. Project administration: B. Zhou. Resources: M. Xu, M. Lu, H. Abd El-Kader, S.S. Alam and K.F. Mahrous. Software: M. Lu. Supervision: B. Zhou. Writing-original draft: M. Ye.

Sample ID		Raw data					High-quality data					Mapping%	Sequencing depth
Sample ID		Read number	Total bases	GC%	Q20	Q30	Read number	Total bases	GC%	Q20	Q30	Mapping%	Sequencing depth
Chinese swamp buffalo (CHB)	CHB1	23483262	3345991045	51.55	98.22	95.24	22666712	3178681630	51.50	99.36	97.34	99.94	1.11
	CHB10	13660992	1946820967	51.21	98.38	95.55	13212758	1856249624	51.16	99.39	97.45	99.93	0.63
	CHB11	22154648	3156451570	51.31	98.35	95.49	21428862	3008829863	51.26	99.38	97.42	99.94	1.03
	CHB12	20794648	2962766393	50.63	97.03	92.56	19269706	2673377743	50.56	98.85	95.94	99.93	0.97
	CHB13	17116116	2438646583	50.49	97.12	92.73	15898504	2207722249	50.42	98.86	95.98	99.93	0.79
	CHB14	19141986	2727510678	50.71	97.09	92.65	17749796	2465049223	50.64	98.86	95.97	99.93	0.89
	CHB15	18829828	2683148163	51.22	97.05	92.58	17471544	2424334962	51.15	98.85	95.92	99.93	0.87
	CHB16	19117758	2724260348	50.65	97.09	92.68	17747388	2464321459	50.58	98.86	95.98	99.92	0.89
	CHB17	19352994	2757680662	49.68	96.91	92.31	17793190	2470113665	49.60	98.81	95.84	99.92	0.92
	CHB18	14601084	2080212343	50.43	97.01	92.55	13503522	1874404644	50.36	98.85	95.96	99.93	0.69
	CHB19	15824808	2254549289	50.90	97.10	92.72	14688264	2039401837	50.83	98.88	96.04	99.92	0.75
	CHB2	20021100	2852797991	51.52	98.25	95.30	19330432	2711810289	51.46	99.37	97.36	99.93	0.95
	CHB20	15554564	2216323698	50.62	97.05	92.59	14414186	2000938996	50.55	98.85	95.95	99.93	0.72
	CHB21	13565544	1932895934	50.64	96.94	92.38	12532968	1737769606	50.56	98.84	95.90	99.92	0.63
	CHB22	16781692	2391529428	50.67	97.01	92.53	15520054	2154991876	50.60	98.86	95.96	99.93	0.78
	CHB23	14087196	2007059441	50.85	97.33	93.21	13163216	1832286401	50.79	98.93	96.21	99.92	0.66
	CHB24	25602402	3647828885	50.84	97.04	92.57	23686928	3288141585	50.78	98.85	95.95	99.92	1.18
	CHB25	20004772	2850526487	50.89	97.11	92.71	18566866	2579046583	50.82	98.87	95.98	99.93	0.93
	CHB3	22209640	3164493855	51.16	98.17	95.11	21404240	3000276126	51.10	99.35	97.28	99.93	1.08
	CHB4	21184982	3018436318	51.06	98.33	95.45	20479252	2875244683	51.01	99.38	97.41	99.94	1.01
	CHB5	21044612	2998680228	51.56	98.19	95.16	20291230	2844179769	51.50	99.35	97.30	99.93	1.00
	CHB6	14901502	2122978529	51.39	98.28	95.36	14403006	2020465701	51.33	99.37	97.39	99.93	0.71
	CHB7	18503152	2636571988	51.25	98.20	95.19	17850416	2502434069	51.20	99.35	97.32	99.93	0.89
	CHB8	17669184	2517594782	51.06	98.26	95.32	17067010	2394258215	51.01	99.36	97.35	99.93	0.86
	CHB9	22367020	3187119649	51.01	98.36	95.52	21650772	3040402821	50.96	99.39	97.42	99.92	1.07
Egyptian river buffalo (EGB)	EGB1	19954660	2842865676	50.99	98.50	95.83	19364892	2721824674	50.95	99.42	97.57	99.92	0.91
	EGB10	13094116	1865663266	52.41	98.80	96.69	12794148	1802969450	52.38	99.53	98.06	99.92	0.60
	EGB11	17304894	2465864004	52.98	98.72	96.5	16873112	2377092551	52.94	99.51	97.97	99.94	0.79
	EGB12	18741238	2670639359	52.84	98.77	96.61	18296674	2578620130	52.80	99.52	98.02	99.92	0.83
	EGB13	16020408	2282435287	52.48	98.75	96.59	15642202	2203598697	52.45	99.51	98.01	99.94	0.74
	EGB14	21578934	3074784478	55.86	98.66	96.36	21011628	2958560798	55.82	99.49	97.90	99.94	0.99
	EGB15	19704988	2807575460	53.25	98.80	96.68	19249162	2713243995	53.21	99.52	98.03	99.92	0.87
	EGB16	20921572	2981120627	52.75	98.75	96.57	20420138	2877061205	52.72	99.51	97.99	99.95	0.94
	EGB17	19359784	2758187364	51.23	98.66	96.36	18860878	2654363628	51.19	99.49	97.91	99.95	0.92
	EGB18	18513462	2637721803	52.85	98.75	96.57	18063298	2544782377	52.82	99.52	98.02	99.93	0.82
	EGB19	14351804	2044907188	50.79	98.34	95.69	13875576	1948100342	50.76	99.39	97.63	99.94	0.69
	EGB2	14196368	2022727385	51.51	98.28	95.34	13697516	1922456015	51.47	99.33	97.32	99.94	0.65
	EGB20	19359496	2758848086	53.79	98.57	96.18	18818842	2648032249	53.75	99.47	97.84	99.92	0.90
	EGB3	17859996	2544885691	51.09	98.34	95.46	17276294	2425544690	51.03	99.38	97.41	99.93	0.83
	EGB4	13471634	1919720732	53.07	97.30	93.29	12689384	1766386475	53.04	99.16	96.65	99.94	0.63
	EGB5	17726002	2526040780	50.18	97.32	93.35	16692040	2324816545	50.15	99.21	96.77	99.94	0.86
	EGB6	20876454	2974128637	56.28	98.62	96.26	20319050	2858790994	56.24	99.47	97.84	99.89	0.97
	EGB7	20921442	2980137664	56.10	98.68	96.39	20387808	2869809003	56.06	99.49	97.88	99.90	0.95
	EGB8	20532550	2924949190	51.17	98.65	96.36	20009496	2815285014	51.14	99.49	97.92	99.94	0.99
	EGB9	21615612	3079790305	51.77	98.75	96.59	21097104	2971753216	51.74	99.52	98.03	99.95	0.99

Reference sequence (Chromosome)	Gene	Position in reference	Exon	Reference → Alteration		Allele frequency (Reference/alteration)		P-value
Reference sequence (Chromosome)	Gene	Position in reference	Exon	Codon	Amino acid	CHB (n = 25)	EGB (n = 20)	P-value
XM_006050687.2 (1)	FAM90A26	380	3	C T G → C C G	Leu → Pro	1.00/0.00	0.90/0.10	0.033
XM_006057766.2 (2)	NEDD9	421	2	A CT → G CT	Thr → Ala	1.00/0.00	0.83/0.17	0.002
XM_025279334.1 (3)	CEP112	2579	22	T C T → T T T	Ser → Phe	1.00/0.00	0.78/0.22	0.000
XM_025281542.1 (3)	TPD52L3	2440	1	G A C → G G C	Asp → Gly	1.00/0.00	0.83/0.17	0.003
XM_006052150.2 (6)	CLCC1	1752	11	A C G → A T G	Thr → Met	1.00/0.00	0.10/0.90	0.035
XM_006063939.2 (7)	FAM184B	2331	9	G AC → A AC	Asp → Asn	0.64/0.36	1.00/0.00	0.000
XM_006046572.2 (11)	PNMA1	1031	1	C C G → C T G	Pro → Leu	1.00/0.00	0.90/0.10	0.035
XM_025295940.1 (11)	RASGRP1	1482	1	A AA → G AA	Lys → Glu	1.00/0.00	0.60/0.40	0.000
XM_006051359.2 (14)	SPATA25	1633	2	C G G → C C G	Arg → Pro	1.00/0.00	0.78/0.23	0.000
XM_025266585.1 (16)	PPFIBP2	1863	16	G CA → A CA	Ala → Thr	1.00/0.00	0.83/0.17	0.002
XM_025271460.1 (20)	MTMR10	400	3	T G G → T C G	Trp → Ser	0.14/0.86	0.65/0.35	0.000
XM_025271849.1 (20)	OTUD7A	447	4	CA C → CA G	His → Gln	1.00/0.00	0.78/0.22	0.000
XM_025272296.1 (21)	SLC22A14	3016	10	G CC → A CC	Ala → Thr	0.50/0.50	1.00/0.00	0.000
XM_025274028.1 (23)	TACC2	4719	5	G AG → A AG	Glu → Lys	0.22/0.78	0.93/0.07	0.000
XM_025275895.1 (X)	CACNA1F	5237	43	A C G → A T G	Thr → Met	0.00/1.00	0.75/0.25	0.000

BBU	Gene	Associated trait	Novel SNP identified
1	BDH1	Expression patterns in mammary epithelial cells during lactation positively correlated with milk yield in Murrah buffaloes ( Yadav et al., 2015Yadav, P.; Kumar, P.; Mukesh, M.; Kataria, R. S.; Yadav, A.; Mohanty, A. K. and Mishra, B. P. 2015. Kinetics of lipogenic genes expression in milk purified mammary epithelial cells (MEC) across lactation and their correlation with milk and fat yield in buffalo. Research in Veterinary Science 99:129-136. https://doi.org/10.1016/j.rvsc.2015.01.003 https://doi.org/10.1016/j.rvsc.2015.01.0... )	An upstream BDH1 SNP (G/A at position 116814955)
2	INSIG2	305-d milk yield in Murrah and crossbred breeds ( Deng et al., 2016bDeng, T.; Pang, C.; Ma, X.; Lu, X.; Duan, A.; Zhu, P. and Liang, X. 2016b. Four novel polymorphisms of buffalo INSIG2 gene are associated with milk production traits in Chinese buffaloes. Molecular and Cellular Probes 30:294-299. https://doi.org/10.1016/j.mcp.2016.09.003 https://doi.org/10.1016/j.mcp.2016.09.00... )
2	STAT1	305-d milk yield and protein percentage in crossbred buffaloes ( Deng et al., 2016aDeng, T. X.; Pang, C. Y.; Lu, X. R.; Zhu, P.; Duan, A. Q. and Liang, X. W. 2016a. Associations between polymorphisms of the STAT 1gene and milk production traits in water buffaloes. Journal of Animal Science 94:927-935. https://doi.org/10.2527/jas.2015-0139 https://doi.org/10.2527/jas.2015-0139... )	Intronic SNP (C/T at position 132437140)
2	PRL	Milk yield, peak milk yield, and the contents of protein and fat in milk in Italian Mediterranean river buffalo ( Li et al., 2017Li, J.; Liang, A.; Li, Z.; Du, C.; Hua, G.; Salzano, A.; Campanile, G.; Gasparrini, B. and Yang, L. 2017. An association analysis between PRL genotype and milk production traits in Italian Mediterranean river buffalo. Journal of Dairy Research 84:430-433. https://doi.org/10.1017/S0022029917000693 https://doi.org/10.1017/S002202991700069... )
3	STAT5A	Milk yield trait in the Binlangjiang buffalo ( Ji et al., 2013Ji, M.; Liu, X. H.; Yu, C. L.; Yu, X. F.; Liu, D. J. and Shi, X. W. 2013. Association of STAT5A polymorphism and milk production traits in Binlangjiang buffalo. China Cattle Science 39:29-35. )
3	SREBF1	305-d milk yield in crossbred Chinese buffaloes ( Deng et al., 2017Deng, T.; Pang, C.; Ma, X.; Duan, A.; Liang, S.; Lu, X. and Liang X. 2017. Buffalo SREBP1: molecular cloning, expression and association analysis with milk production traits. Animal Genetics 48:720-721. https://doi.org/10.1111/age.12587 https://doi.org/10.1111/age.12587... )
3	ACACA	Expression patterns in mammary epithelial cells during lactation negatively correlated with milk yield in Murrah buffaloes ( Yadav et al., 2015Yadav, P.; Kumar, P.; Mukesh, M.; Kataria, R. S.; Yadav, A.; Mohanty, A. K. and Mishra, B. P. 2015. Kinetics of lipogenic genes expression in milk purified mammary epithelial cells (MEC) across lactation and their correlation with milk and fat yield in buffalo. Research in Veterinary Science 99:129-136. https://doi.org/10.1016/j.rvsc.2015.01.003 https://doi.org/10.1016/j.rvsc.2015.01.0... )	Intronic SNP (G/A at position 49760383 and 49760609, respectively)
8	LEP	High milk yield in Egyptian buffaloes ( Nasr et al., 2016Nasr, M. A. F.; Awad, A. and El Araby, I. E. 2016. Associations of leptin and pituitary-specific transcription factor genes’ polymorphisms with reproduction and production traits in dairy buffalo. Reproduction in Domestic Animals 51:597-603. https://doi.org/10.1111/rda.12726 https://doi.org/10.1111/rda.12726... )
12	LIPIN1	Expression patterns in mammary epithelial cells during lactation positively correlated with milk yield in Murrah buffaloes ( Yadav et al., 2015Yadav, P.; Kumar, P.; Mukesh, M.; Kataria, R. S.; Yadav, A.; Mohanty, A. K. and Mishra, B. P. 2015. Kinetics of lipogenic genes expression in milk purified mammary epithelial cells (MEC) across lactation and their correlation with milk and fat yield in buffalo. Research in Veterinary Science 99:129-136. https://doi.org/10.1016/j.rvsc.2015.01.003 https://doi.org/10.1016/j.rvsc.2015.01.0... )
14	ACSS2	Expression patterns in mammary epithelial cells during lactation positively correlated with milk yield in Murrah buffaloes ( Yadav et al., 2015Yadav, P.; Kumar, P.; Mukesh, M.; Kataria, R. S.; Yadav, A.; Mohanty, A. K. and Mishra, B. P. 2015. Kinetics of lipogenic genes expression in milk purified mammary epithelial cells (MEC) across lactation and their correlation with milk and fat yield in buffalo. Research in Veterinary Science 99:129-136. https://doi.org/10.1016/j.rvsc.2015.01.003 https://doi.org/10.1016/j.rvsc.2015.01.0... )	A 3’UTR ACSS2 SNP (G/A at position 19375891)
14	OXT	Milk yield in Italian buffaloes ( Pauciullo et al., 2012Pauciullo, A.; Cosenza, G.; Steri, R.; Coletta, A.; Jemma, L.; Feligini, M.; Di Berardino, D.; Macciotta, N. P. and Ramunno, L. 2012. An association analysis between OX T genotype and milk yield and flow in Italian Mediterranean river buffalo. Journal of Dairy Research 79:150-156. https://doi.org/10.1017/s0022029911000914 https://doi.org/10.1017/s002202991100091... )
15	VPS13B	Total milk yield, fat yield, and protein yield ( Liu et al., 2018aLiu, J. J.; Liang, A. X.; Campanile, G.; Plastow, G.; Zhang, C.; Wang, Z.; Salzano, A.; Gasparrini, B.; Cassandro, M. and Yang, L. G. 2018a. Genome-wide association studies to identify quantitative trait loci affecting milk production traits in water buffalo. Journal of Dairy Science 101:433-444. https://doi.org/10.3168/jds.2017-13246 https://doi.org/10.3168/jds.2017-13246... )	Intronic SNP (C/T at position 17737368)
15	RGS22	Total milk yield, fat yield, and protein yield ( Liu et al., 2018aLiu, J. J.; Liang, A. X.; Campanile, G.; Plastow, G.; Zhang, C.; Wang, Z.; Salzano, A.; Gasparrini, B.; Cassandro, M. and Yang, L. G. 2018a. Genome-wide association studies to identify quantitative trait loci affecting milk production traits in water buffalo. Journal of Dairy Science 101:433-444. https://doi.org/10.3168/jds.2017-13246 https://doi.org/10.3168/jds.2017-13246... )	Intronic SNP (C/T at position 17857096)
22	MC4R	Milk yield trait in the Binlangjiang buffalo ( Deng et al., 2016cDeng, T. X.; Pang, C. Y.; Liu, M. Q.; Zhang, C. and Liang, X. W. 2016c. Synonymous single nucleotide polymorphisms in the MC4R gene that are significantly associated with milk production traits in water buffaloes. Genetics and Molecular Research 15:gmr.15028153. https://doi.org/10.4238/gmr.15028153 https://doi.org/10.4238/gmr.15028153... )
23	SCD	Expression patterns in mammary epithelial cells during lactation positively correlated with milk yield in Murrah buffaloes ( Yadav et al., 2015Yadav, P.; Kumar, P.; Mukesh, M.; Kataria, R. S.; Yadav, A.; Mohanty, A. K. and Mishra, B. P. 2015. Kinetics of lipogenic genes expression in milk purified mammary epithelial cells (MEC) across lactation and their correlation with milk and fat yield in buffalo. Research in Veterinary Science 99:129-136. https://doi.org/10.1016/j.rvsc.2015.01.003 https://doi.org/10.1016/j.rvsc.2015.01.0... )

Chromosome	Number of SNP	Average coverage depth
Chromosome	Number of SNP	CHB	EGB
1	132	20.01	15.86
2	127	20.09	15.38
3	96	18.67	15.30
4	81	19.18	15.67
5	101	19.14	16.11
6	58	20.16	15.15
7	70	20.70	15.68
8	66	19.69	14.52
9	52	17.73	15.57
10	51	19.66	14.53
11	44	20.54	14.81
12	74	19.95	17.44
13	34	17.33	14.54
14	51	19.35	15.89
15	41	20.05	14.90
16	49	20.63	16.44
17	45	19.85	15.68
18	41	19.50	15.92
19	48	20.34	14.84
20	44	19.72	16.02
21	37	19.82	15.22
22	45	19.43	16.87
23	46	19.24	14.93
24	40	21.30	16.84
X	17	19.96	16.95
Average	59.6	19.68	15.64

Region	Associated gene	BBU	Position	Reference (Ref)	Alteration (Alt)	Frequency of Ref allele
Region	Associated gene	BBU	Position	Reference (Ref)	Alteration (Alt)	CHB	EGB
Intergenic	gene- DIAPH3 (dist = 110981), gene- TDRD3 (dist = 249397)	13	88461660	A	G	0.040	0.825
Intergenic	gene- DIAPH3 (dist = 110637), gene- TDRD3 (dist = 249741)	13	88461316	C	G	0.040	0.975
Intronic	gene- FSTL4	9	66157381	C	G	0.080	0.600
Intergenic	gene- GMDS (dist = 95662), gene- MYLK4 (dist = 121469)	2	1453971	A	G	0.940	0.550
Intergenic	gene- KCNMA1 (dist = 29841), gene- DLG5 (dist = 108913)	4	152780961	T	C	0.920	0.600
Intergenic	gene- KCNMA1 (dist = 30087), gene- DLG5 (dist = 108667)	4	152781207	T	C	0.080	0.400
Intronic	gene- SLC44A5	6	68703492	A	G	0.980	0.350
Intronic	gene- SLC44A5	6	68703703	G	A	0.980	0.400

[1] * Corresponding author: bzhou@yzu.edu.cn

Brasil

Brasil

Identification of candidate genes associated with milk yield trait in buffaloes ( Bubalus bubalis ) by restriction-site-associated DNA sequencing

ABSTRACT