Detection of polymorphisms in six genes and their association analysis with litter size in sheep

Figures & data

Table 1. Basic information on ewes used in this study.

Breed	Group	Number	District
Small Tail Han sheep	Polytocous	384	Yuncheng, Shandong Province, China
Hu sheep	Polytocous	96	Xuzhou, Jiangsu Province, China
Cele Black sheep	Polytocous	96	Cele, Xinjiang Uygur Autonomous Region, China
Sunite sheep	Monotocous	96	Wulateqianqi, Bayannaoer, Inner Mongolia Autonomous Region, China
Bamei Mutton sheep	Monotocous	96	Linhe, Bayannaoer, Inner Mongolia Autonomous Region, China

Table 2. Primers used in the present study.

Gene	Locus	Primer sequence	Tm (°C)
APAF1	rs416244410	F: ACGTTGGATGTCTCTCCTGTTTCAGCTTTG R: ACGTTGGATGATGATGACCCATGGGTAAGC	48.9
	rs426974615	F: ACGTTGGATGGGTCACAGAACAAGAGAGAC R: ACGTTGGATGGGTGTTATGTATCCTCTGGG	45.9
CLSTN2	rs399534524	F: ACGTTGGATGTGACCAGCTGGGACATAACG R: ACGTTGGATGGAAACCATCCTCTGCAACTC	59.2
CTH	rs407142552	F: ACGTTGGATGTATTGAGGATGAGAAGGCAG R: ACGTTGGATGTGCAGTTGACTTAAACGAAG	50.6
PLCB1	rs414162829	F: ACGTTGGATGGGAACTGTTAGATCTCAGCC R: ACGTTGGATGATGCTCCACTTCCTACCTTG	51.8
PLCB4	chr13_1651705	F: ACGTTGGATGGGTGTGTGTGAAGCACATTC R: ACGTTGGATGGACCAAAGTGTACTATGTGAC	45
CHST11	rs1092455701	F: ACGTTGGATGACCAGGTCACCGACACCTGC R: ACGTTGGATGAATAGATGAGCTCGTGGTCC	62

Figure 1. Genotyping results of candidate SNPs in six genes using MassARRAY® SNP system.

Table 3. Population genetic analysis of SNPs with different genes in five sheep breeds.

Gene	Locus	Breed	Genotype frequency			Allele frequency		PIC	HE	NE	Chi-square test(p-value)
APAF1	rs416244410		GG	GA	AA	G	A
		Small Tail Han sheep	0.86	0.14	0.00	0.93	0.07	0.13	0.14	1.16	0.97
		Hu sheep	0.81	0.18	0.01	0.90	0.10	0.16	0.18	1.22	0.95
		Cele Black sheep	0.94	0.06	0.00	0.97	0.03	0.06	0.06	1.06	0.75
		Sunite sheep	0.90	0.10	0.00	0.95	0.05	0.09	0.10	1.11	0.59
		Bamei Mutton sheep	0.98	0.02	0.00	0.99	0.01	0.02	0.02	1.02	0.92
	rs426974615		GG	GA	AA	G	A
		Small Tail Han sheep	0.01	0.09	0.90	0.05	0.95	0.09	0.10	1.11	0.32
		Hu sheep	0.03	0.17	0.80	0.11	0.89	0.18	0.20	1.25	0.08
		Cele Black sheep	0.00	0.04	0.96	0.02	0.98	0.04	0.04	1.04	0.83
		Sunite sheep	0.00	0.08	0.92	0.04	0.96	0.08	0.08	1.09	0.67
		Bamei Mutton sheep	0.00	0.02	0.98	0.01	0.99	0.02	0.02	1.02	0.92
CLSTN2	rs399534524		CC	CT	TT	C	T
		Small Tail Han sheep	0.69	0.22	0.09	0.80	0.20	0.27	0.32	1.48	0.00
		Hu sheep	0.59	0.24	0.17	0.71	0.29	0.33	0.41	1.69	0.00
		Cele Black sheep	0.31	0.43	0.26	0.53	0.47	0.37	0.50	1.99	0.16
		Sunite sheep	0.45	0.35	0.20	0.62	0.38	0.36	0.47	1.88	0.02
		Bamei Mutton sheep	0.43	0.25	0.32	0.55	0.45	0.37	0.49	1.98	0.00
CTH	rs407142552		CC	CT	TT	C	T
		Small Tail Han sheep	0.41	0.45	0.14	0.64	0.36	0.35	0.46	1.86	0.61
		Hu sheep	0.46	0.44	0.10	0.68	0.32	0.34	0.44	1.78	1.00
		Cele Black sheep	0.62	0.34	0.04	0.79	0.21	0.28	0.34	1.51	0.82
		Sunite sheep	0.46	0.38	0.16	0.65	0.35	0.35	0.46	1.84	0.08
		Bamei Mutton sheep	0.88	0.12	0.00	0.94	0.06	0.11	0.12	1.13	0.51
PLCB1	rs414162829		CC	CT	TT	C	T
		Small Tail Han sheep	0.06	0.40	0.54	0.26	0.74	0.31	0.38	1.62	0.38
		Hu sheep	0.04	0.29	0.67	0.19	0.81	0.26	0.30	1.44	0.68
		Cele Black sheep	0.15	0.47	0.38	0.38	0.62	0.36	0.47	1.89	0.96
		Sunite sheep	0.03	0.42	0.55	0.24	0.76	0.30	0.36	1.57	0.16
		Bamei Mutton sheep	0.09	0.43	0.48	0.31	0.69	0.34	0.43	1.74	0.98
PLCB4	chr13_1651705		GG	GT	TT	G	T
		Small Tail Han sheep	0.54	0.40	0.06	0.74	0.26	0.31	0.39	1.63	0.54
		Hu sheep	0.30	0.44	0.26	0.52	0.48	0.37	0.50	2.00	0.23
		Cele Black sheep	0.42	0.42	0.16	0.62	0.38	0.36	0.47	1.88	0.28
		Sunite sheep	0.40	0.42	0.18	0.61	0.39	0.36	0.48	1.91	0.31
		Bamei Mutton sheep	0.61	0.34	0.05	0.78	0.22	0.29	0.35	1.53	0.91
CHST11	rs1092455701		CC	CT	TT	C	T
		Small Tail Han sheep	0.71	0.27	0.02	0.84	0.16	0.23	0.27	1.37	0.71
		Hu sheep	0.80	0.19	0.01	0.90	0.10	0.17	0.19	1.23	0.96
		Cele Black sheep	0.95	0.05	0.00	0.97	0.03	0.05	0.05	1.05	0.79
		Sunite sheep	0.84	0.16	0.00	0.92	0.08	0.13	0.14	1.17	0.41
		Bamei Mutton sheep	0.95	0.05	0.00	0.97	0.03	0.05	0.05	1.05	0.79

NE: number of effective alleles; HE: heterozygosity; PIC: polymorphism information content; PIC > 0.5 is high polymorphism, 0.25 ≤ PIC ≤ 0.5 is moderate polymorphism, PIC < 0.25 is low polymorphism; p > 0.05 is in Hardy-Weinberg equilibrium; p < 0.05 is not in Hardy-Weinberg equilibrium.

Table 4. Genotype and allele frequencies in sheep populations of different fecundity.

Gene	Locus	Types of fecundity	Genotype frequency(count)			Chi-square test (p-value)	Allele frequency		Chi-square test (p-value)
APAF1			GG	GA	AA		G	A
	rs416244410	Polytocous sheep	0.86(497)	0.13(75)	0.01(3)	0.02	0.93	0.07	0.01
		Monotocous sheep	0.94(180)	0.06(12)	0.00(0)		0.97	0.03
	rs426974615	Polytocous sheep	0.01(5)	0.10(56)	0.89(515)	0.06	0.06	0.94	0.01
		Monotocous sheep	0.00(0)	0.05(10)	0.95(182)		0.03	0.97
CLSTN2	rs399534524		CC	CT	TT		C	T
		Polytocous sheep	0.61(351)	0.26(149)	0.13(76)	0.00	0.74	0.26	0.00
		Monotocous sheep	0.44(84)	0.30(58)	0.26(50)		0.59	0.41
CTH	rs407142552		CC	CT	TT		C	T
		Polytocous sheep	0.46(262)	0.43(247)	0.11(66)	0.00	0.67	0.33	0.00
		Monotocous sheep	0.67(128)	0.25(48)	0.08(16)		0.79	0.21
PLCB1	rs414162829		CC	CT	TT		C	T
		Polytocous sheep	0.07(40)	0.39(226)	0.54(310)	0.76	0.27	0.73	0.76
		Monotocous sheep	0.06(12)	0.42(81)	0.52(99)		0.27	0.73
PLCB4	chr13_1651705		GG	GT	TT		G	T
		Polytocous sheep	0.48(276)	0.41(235)	0.11(65)	0.85	0.68	0.32	0.73
		Monotocous sheep	0.50(96)	0.39(74)	0.11(22)		0.69	0.31
CHST11	rs1092455701		CC	CT	TT		C	T
		Polytocous sheep	0.76(439)	0.22(125)	0.02(12)	0.00	0.87	0.13	0.00
		Monotocous sheep	0.90(172)	0.10(20)	0.00(0)		0.95	0.05

Table 5. Least squares mean and standard error of litter size in Small Tail Han sheep with different genotypes.

Gene	Locus	Genotype	1st parity litter size(n)	2nd parity litter size(n)	3rd parity litter size(n)
APAF1	rs416244410	GG	2.22 ± 0.05(296)	2.43 ± 0.06(230)	2.91 ± 0.12(81)
		GA	2.18 ± 0.12(49)	2.61 ± 0.14(38)	3.06 ± 0.19(16)
		AA	2.00 ± 0.00(2)	2.50 ± 0.50(2)	–
	rs426974615	GG	2.50 ± 1.50(2)	–	–
		GA	2.12 ± 0.13(34)	2.57 ± 0.14(28)	3.00 ± 0.25(12)
		AA	2.22 ± 0.05(311)	2.43 ± 0.06(240)	2.93 ± 0.12(84)
CLSTN2	rs399534524	CC	2.15 ± 0.05^a (240)	2.42 ± 0.06(187)	2.91 ± 0.14(64)
		CT	2.46 ± 0.10^b (76)	2.63 ± 0.12(57)	3.08 ± 0.17(26)
		TT	2.03 ± 0.11^a (31)	2.28 ± 0.23(25)	2.71 ± 0.42(7)
CTH	rs407142552	CC	2.20 ± 0.07(142)	2.40 ± 0.08^ab (108)	2.87 ± 0.14(38)
		CT	2.22 ± 0.06(161)	2.58 ± 0.09^a (120)	3.01 ± 0.18(45)
		TT	2.18 ± 0.15(44)	2.24 ± 0.13^b (42)	2.73 ± 0.27(15)
PLCB1	rs414162829	CC	2.05 ± 0.15(21)	2.73 ± 0.15(15)	2.33 ± 0.33(3)
		CT	2.26 ± 0.07(137)	2.57 ± 0.09(114)	3.10 ± 0.16(47)
		TT	2.19 ± 0.06(189)	2.33 ± 0.08(140)	2.81 ± 0.14(47)
PLCB4	chr13_1651705	GG	2.24 ± 0.06(196)	2.42 ± 0.07(156)	2.79 ± 0.12(57)
		GT	2.16 ± 0.08(130)	2.56 ± 0.10(95)	3.22 ± 0.21(32)
		TT	2.19 ± 0.18(21)	2.17 ± 0.23(18)	2.88 ± 0.35(8)
CHST11	rs1092455701	CC	2.21 ± 0.05(245)	2.44 ± 0.07(190)	2.99 ± 0.13(68)
		CT	2.20 ± 0.09(91)	2.48 ± 0.10(73)	2.88 ± 0.18(25)
		TT	2.36 ± 0.28(11)	2.50 ± 0.22(6)	2.50 ± 0.65(4)

Figure 2. Interactions between proteins including CTH (A) and CLSTN2 (B).