大熊猫遗传多样性评估的微卫星分型体系优化

doi:10.16288/j.yczz.21-348

遗传 ›› 2022, Vol. 44 ›› Issue (3): 253-266.doi: 10.16288/j.yczz.21-348

• 技术与方法 • 上一篇

大熊猫遗传多样性评估的微卫星分型体系优化

寇洁^1,²(), 李严^1,²(), 王鹏³, 刘红^1,², 刘佳文^1,², 王涓^1,², 王也^1,², 张亮^1,², 沈富军^1,²()

1. 成都大熊猫繁育研究基地, 成都 610081
2. 四川省濒危野生动物保护生物学重点实验室,成都 610081
3. 西南科技大学生命科学与工程学院,绵阳 621010

收稿日期:2021-10-08 修回日期:2022-01-08 出版日期:2022-03-20 发布日期:2022-02-24
通讯作者: 沈富军 E-mail:koujie@panda.org.cn;melady11@163.com;shenfj001@sina.com
作者简介:寇洁,博士,助理研究员,研究方向：保护遗传学。E-mail: koujie@panda.org.cn;|李严,博士,助理研究员,研究方向：保护遗传学。E-mail: melady11@163.com;
寇洁和李严并列第一作者。
基金资助:
成都大熊猫繁育研究基金项目编号(CPF-2014-08);成都大熊猫繁育研究基地自立课题编号(CPF-2014-08);四川省重点研发项目资助编号(2018JZ0074)

Optimization of microsatellite genotyping system used for genetic diversity evaluation of Ailuropoda melanoleuca

Jie Kou^1,²(), Yan Li^1,²(), Peng Wang³, Hong Liu^1,², Jiawen Liu^1,², Juan Wang^1,², Ye Wang^1,², Liang Zhang^1,², Fujun Shen^1,²()

1. Chengdu Research Base of Giant Panda Breeding, Chengdu 610081, China
2. Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Chengdu 610081, China
3. School of Life Sciences and Engineering, Southwest University of Science and Technology, Mianyang 621010, China

Received:2021-10-08 Revised:2022-01-08 Online:2022-03-20 Published:2022-02-24
Contact: Shen Fujun E-mail:koujie@panda.org.cn;melady11@163.com;shenfj001@sina.com
Supported by:
Supported by the Chengdu Giant Panda Breeding Research Foundation No(CPF-2014-08);Chengdu Giant Panda Breeding Research Base No(CPF-2014-08);the Key R&D Project of Sichuan Province No(2018JZ0074)

摘要/Abstract

摘要：

微卫星作为重要的分子标记之一,已被证明在大熊猫种群规模评估、亲子鉴定和遗传多样性分析方面是有效的。目前微卫星标记在大熊猫(Ailuropoda melanoleuca)染色体上物理定位方面的报道较少,而且缺乏微卫星基因分型系统的效能评估以及PCR扩增条件的优化。本研究基于大熊猫基因组参考序列(ASM200744v2),分析了34个大熊猫微卫星位点的染色体定位特征并评价了位点的应用价值。通过优化34个STR-PCR反应体系和扩增程序,结合微卫星的染色体定位数据确定了Ame-μ10标记的较低应用价值以及gpz-6重新筛选引物的必要性。本研究有助于提高基因分型结果的重复性和可靠性,对促进《大熊猫种群遗传档案建立技术规程》规范化应用和制定大熊猫保护策略具有重要意义。

关键词: 大熊猫, 遗传多样性, 微卫星标记, STR-PCR

Abstract:

Microsatellite DNA is one of the most widely used genetic markers of giant panda, especially in population size estimation, paternity testing, and genetic diversity analysis. However, there are few reports on the physical locations of microsatellite markers on the chromosomes of the giant panda (Ailuropoda melanoleuca) and research on the performance of microsatellite in genotyping system and the PCR amplification conditions. In this study, we analyzed the chromosomal locations and evaluated the application values of 34 microsatellite markers, based on the giant panda genome reference sequence (ASM200744v2). We optimized the PCR reaction systems and amplification procedures for these 34 microsatellite markers. We found the low value of the microsatellite marker of Ame-μ10 in genetic application, and the necessity in redesigning the primers for gpz-6. Our research helps to improve the reproducibility and reliability of genotyping results and is of great significance for promoting the establishment and standardized application of the “A Regulation for Giant Panda Population Genetic Archives” in giant panda conservation.

Key words: giant panda, genetic diversity, microsatellite, STR-PCR

寇洁, 李严, 王鹏, 刘红, 刘佳文, 王涓, 王也, 张亮, 沈富军. 大熊猫遗传多样性评估的微卫星分型体系优化[J]. 遗传, 2022, 44(3): 253-266.

Jie Kou, Yan Li, Peng Wang, Hong Liu, Jiawen Liu, Juan Wang, Ye Wang, Liang Zhang, Fujun Shen. Optimization of microsatellite genotyping system used for genetic diversity evaluation of Ailuropoda melanoleuca[J]. Hereditas(Beijing), 2022, 44(3): 253-266.

图/表 10

表1

大熊猫微卫星标记信息"

编号	标记名称	核心序列	GenBank号	引物序列(5′→3′)	目的片段长度(bp)	参考文献
1	gpz-6	(AAAG)₁₁	KF907161	F: CCTGGCAGGGCAAAGTATT	194~222	[9]
1	gpz-6	(AAAG)₁₁	KF907161	R: CCCCGTGAAAACATCAAGAC	194~222
2	gpz-47	(AATG)₂₀	KF907176	F: GACCTCAGTGTACGCCCAGT	174~210
2	gpz-47	(AATG)₂₀	KF907176	R: CTGGACAGGCAGGTAGAAGC	174~210
3	gpz-20	(AAAG)₁₀	KF907169	F: CCCTCTCGTTGTGTCTCTCTG	258~326
3	gpz-20	(AAAG)₁₀	KF907169	R: CACCTGGTAAATGGCACCTT	258~326
4	gpz-51	(AGAT)₁₁	KF907179	F: GGGGAGGATATGTGTTGTGG	163~179
4	gpz-51	(AGAT)₁₁	KF907179	R: TGCTTTGGATTTATTGGAGCA	163~179
5	GPL-29	(ATCC)₁₉	KF907139	F: TCCAAGGCTTCAAACAAGGT	155~179
5	GPL-29	(ATCC)₁₉	KF907139	R: CACCACAGGTGCCAATTATG	155~179
6	GPL-60	(TCTT)₁₂	KF907152	F: TGCCGGAAAGTTCTAAGCAT	218~238
6	GPL-60	(TCTT)₁₂	KF907152	R: TTTCTCTCCCTCTCCCCTTC	218~238
7	Ame-μ10	(CA)₁₆	未注册	F: ACCGTGCTCTTAATCCCCTT	138~160	[8]
7	Ame-μ10	(CA)₁₆	未注册	R: CCCATGCTTATGAGAAACAGG	138~160
8	Ame-μ11	(CA)₁₂	未注册	F: TATGCCACCTGCCCAGAC	228~236
8	Ame-μ11	(CA)₁₂	未注册	R: GATGGAAAGAGTAGAGCCAAGG	228~236
9	Ame-μ13	(CA)₁₈	未注册	F: GGAAGCATTAAGGAAAACATGC	142~171
9	Ame-μ13	(CA)₁₈	未注册	R: AATGATGACCATTTCAAACGC	142~171
10	Ame-μ15	(CA)₁₃	未注册	F: AAGCAGTTGTTTTTGCTTAGTG	122~130
10	Ame-μ15	(CA)₁₃	未注册	R: TGTCAAAGTATTTGCCTCACA	122~130
11	Ame-μ22	(CA)₁₁	未注册	F: AGGAAACATGTTGCCTTTTCA	127~129
11	Ame-μ22	(CA)₁₁	未注册	R: AGAGGGCAAATAGGAGGGAA	127~129
12	GPL-8	(ATCC)₁₁	KF907132	F: TGGTTTTGCAAGGATGACAG	224~236	[9]
12	GPL-8	(ATCC)₁₁	KF907132	R: TTGTGACAAGCAAGCTCCAC	224~236
13	GPL-28	(ATAA)₂₁	KF907138	F: GAAAGAAGGGCAGGGATAGG	186~194
13	GPL-28	(ATAA)₂₁	KF907138	R: TGACCAAGAACTCACGGTTG	186~194
14	gpy-5	(AACT)₁₆	KF907157	F: CTCGGGAGCTTTGTACCATC	200~212
14	gpy-5	(AACT)₁₆	KF907157	R: CAGAGAGCCCAAACCTCAAC	200~212
15	gpy-20	(TTTG)₁₆	KF907159	F: GCAGGCACTCAAGAGGTGTT	149~165
15	gpy-20	(TTTG)₁₆	KF907159	R: CCTTGTGCTAAACACAGGTGA	149~165
16	GPL-53	(ATTT)₂₁	KF907148	F: CCAGAAAATGGCTTTCATGC	150~174
16	GPL-53	(ATTT)₂₁	KF907148	R: TCTCTTTCTCTGCCCCACAC	150~174
17	GPL-44	(ATAA)₂₁	KF907146	F: TTCTCCCTCTGTCTGCCACT	172~184
17	GPL-44	(ATAA)₂₁	KF907146	R: ACCATTCTGGGTGCGATAAC	172~184
18	Panda-06	(TG)₂(GT)₂₀	AY161179	F: AGGCAGATTTCCCACAGAGAGC	159~163	[6]
18	Panda-06	(TG)₂(GT)₂₀	AY161179	R: GGTGGGCAGGAATGATTGGGC	159~163	[6]
19	Panda-22	(CAA)₁₂	AY161195	F: AGGGGAGAGAACATTGCTCG	177~186
19	Panda-22	(CAA)₁₂	AY161195	R: GAAGCCAGCCCAACTTTTCC	177~186
20	Panda-05	(GT)₂₀	AY161178	F: GCAAGGATTCATAACGGTAGGG	98~108
20	Panda-05	(GT)₂₀	AY161178	R: GTGTCTTGACCACCAGTGATGTAAGCCG	98~108
21	Panda-40	(TATC)₁₁	AY161213	F: CCTACCTATTTACCTACTTACCTACC	119~129
21	Panda-40	(TATC)₁₁	AY161213	R: GATGCTATTAAGCAACAGAC	119~129
22	Panda-44	(TATC)₁₀	AY161216	F: TATCAGGCTCTGTGCTCAGC	230~278
22	Panda-44	(TATC)₁₀	AY161216	R: GTTTGGTCTTCTCCAGTGGC	230~278
23	Panda-45	(TATC)₁₀… (TATC)…(TATC)₄	AY161217	F: AGGCAGATTTCCCACAGAGAGC	159~163
23	Panda-45	(TATC)₁₀… (TATC)…(TATC)₄	AY161217	R: GGTGGGCAGGAATGATTGGGC	159~163
24	Ame-μ24	(CA)₁₁	未注册	F: ATGCATGACATTTTGGGTAGC	248~258	[8]
24	Ame-μ24	(CA)₁₁	未注册	R: TGAAGACCCTAGATGAAGGCA	248~258
25	Ame-μ26	(CA)₁₁	未注册	F: TTTTCAGGCCTCCGAAAAC	114~120
25	Ame-μ26	(CA)₁₁	未注册	R: ATTCCCAATAAAGCAAATCAGA	114~120
26	GP001	(CA)₁₃A(CA)₂	未注册	F: ACGGGAAGCCTGCTTCTACACTC	144~152	[7]
26	GP001	(CA)₁₃A(CA)₂	未注册	R: AGACACCCAACCGACTAAACCAC	144~152
27	GP008	(AC)₁₆GCAC	未注册	F: AACATCCTGGGTATTCTCCATGC	154~160
27	GP008	(AC)₁₆GCAC	未注册	R: TGCAGAGTGAGGACCTAGGTGTC	154~160
28	GP901	(CA)₂₃	未注册	F: AGCTAATTTTCCAAGTTACCTTTCC	156~164
28	GP901	(CA)₂₃	未注册	R: GGATCTGGGTGTTATTTGCAATG	156~164
29	GPL-31	(ATCT)₂₁	KF907141	F: GCATCCTTGTCCTCTTGGAG	127~147	[9]
29	GPL-31	(ATCT)₂₁	KF907141	R: GCATTGTTTTCTACTCTACAAATATCC	127~147
30	GPL-47	(TCTA)₂₀	KF907147	F: TCCCCCTCTATGGTAAAAGG	140~172
30	GPL-47	(TCTA)₂₀	KF907147	R: CCATGTTGGGTGTAGGGATT	140~172
31	Ame-μ14	(CA)₁₇	未注册	F: CCACCCAGGCACATCTATCT	139~147	[8]
31	Ame-μ14	(CA)₁₇	未注册	R: TTTACTGTGGTGGAAGTTAGGG	139~147
32	Ame-μ19	(CA)₁₈	未注册	F: CAGGCAGCACAGCTATACCA	154~162
32	Ame-μ19	(CA)₁₈	未注册	R: CCACCTGATACCTATGCACAT	154~162
33	Ame-μ21	(CA)₁₈	未注册	F: TATGAAAAGAGCCCAAATGTCA	156~174
33	Ame-μ21	(CA)₁₈	未注册	R: TAGCTCCATCCACGTTGTTG	156~174
34	Ame-μ25	(CA)₂₂	未注册	F: CATAATTCCCTGGCAATGCT	219~237
34	Ame-μ25	(CA)₂₂	未注册	R: TAGCCCGCATTGAAAAATG	219~237

表1

表2

大熊猫染色体中32个微卫星分布情况"

单位点微卫星	所在染色体	上游引物起始位置 (bp)	上游引物终止位置 (bp)	上游引物所在链	下游引物起始位置 (bp)	下游引物终止位置 (bp)	下游引物所在链	参考基因组预测区域长度 (bp)	上游引物完全匹配到基因组上的序列长度(bp)	下游引物完全匹配到基因组上的序列长度(bp)
Ame-μ11	10	39,305,873	39,305,852	-	39,305,639	39,305,656	+	234	21	20
Ame-μ13	8	87,108,620	87,108,599	-	87,108,459	87,108,479	+	161	20	19
Ame-μ14	2	61,503,868	61,503,849	-	61,503,723	61,503,744	+	145	20	19
Ame-μ15	12	36,518,091	36,518,112	+	36,518,211	36,518,191	-	120	22	22
Ame-μ19	9	7,900,919	7,900,900	-	7,900,765	7,900,785	+	154	20	19
Ame-μ21	5	21,973,805	21,973,826	+	21,973,974	21,973,955	-	169	20	19
Ame-μ22	X	170,977,937	170,977,957	+	170,978,065	170,978,046	-	128	19	19
Ame-μ24	5	115,728,956	115,728,970	+	115,729,181	115,729,195	-	253	14	14
Ame-μ25	3	66,347,364	66,347,383	+	66,347,593	66,347,575	-	229	19	21
Ame-μ26	7	12,967,654	12,967,636	-	12,967,537	12,967,558	+	117	19	26
GP-001	10	27,662,835	27,662,857	+	27,662,980	27,662,958	-	145	19	19
GP-008	14	3,182,498	3,182,476	-	3,182,341	3,182,363	+	157	19	19
GP-901	3	123,173,863	123,173,839	-	123,173,705	123,173,726	+	158	18	19
GPL-8	15	85,949,861	85,949,842	-	85,949,622	85,949,641	+	239	21	20
GPL-28	15	31,662,502	31,662,521	+	31,662,695	31,662,676	-	193	18	19
GPL-29	15	85,915,338	85,915,319	-	85,915,163	85,915,182	+	175	19	22
GPL-31	3	96,376,304	96,376,323	+	96,376,442	96,376,416	-	138	21	20
GPL-44	15	19,483,022	19,483,003	-	19,482,839	19,482,858	+	183	19	19
GPL-47	2	128,393,268	128,393,287	+	128,393,419	128,393,400	-	151	19	19
GPL-53	1	69,519,555	69,519,574	+	69,519,720	69,519,701	-	165	17	19
GPL-60	16	76,223,926	76,223,907	-	76,223,701	76,223,720	+	225	21	20
gpy-5	16	59,142,088	59,142,069	-	59,141,881	59,141,900	+	207	20	18
gpy-20	12	62,877,774	62,877,792	+	62,877,927	62,877,908	-	153	20	19
gpz-20	18	36,319,158	36,319,138	-	36,318,883	36,318,902	+	275	20	19
gpz-47	4	35,501,607	35,501,588	-	35,501,414	35,501,433	+	193	20	19
gpz-51	X	164,010,225	164,010,206	-	164,010,055	164,010,075	+	170	20	19
Panda-05	1	156,590,990	156,590,969	-	156,590,885	156,590,905	+	105	19	20
Panda-06	2	144,883,392	144,883,413	+	144,883,553	144,883,533	-	161	20	19
Panda-22	3	46,782,254	46,782,273	+	46,782,440	46,782,421	-	186	20	19
Panda-40	1	17,064,978	17,064,953	-	17,064,855	17,064,874	+	123	20	19
Panda-44	10	35,791,472	35,791,491	+	35,791,702	35,791,683	-	230	20	18
Panda-45	2	144,883,392	144,883,413	+	144,883,553	144,883,533	-	161	20	19

表2

表3

表4

表5

表6

图1

图2

图1

表1

附两对微卫星引物(Ame-µ10、gpz-6)的扩增情况"

疑似多位点扩增的微卫星	引物3′端碱基完全匹配个数	所在染色体	上游引物起始位置 (bp)	上游引物终止位置 (bp)	上游引物所在链	下游引物起始位置 (bp)	下游引物终止位置 (bp)	下游引物所在链	参考基因组预测目的片段长度(bp)
Ame-μ10	14	4	131,489,485	131,489,499	+	131,489,603	131,489,617	-	145
	13	4	131,489,486	131,489,499	+	131,489,603	131,489,616	-	145
	12	16	36,764,856	36,764,868	+	36,764,988	36,765,000	-	161
		4	131,489,487	131,489,499	+	131,489,603	131,489,615	-	145
		5	16,148,517	16,148,529	-	16,148,310	16,148,322	+	236
	11	X	132,730,681	132,730,692	+	132,730,693	132,730,704	-	42
		16	36,764,857	36,764,868	+	36,764,988	36,764,999	-	161
		17	30,754,694	30,754,705	+	30,754,723	30,754,734	-	59
Ame-μ10	11	4	131,489,488	131,489,499	+	131,489,603	131,489,614	-	145
		5	28,604,614	28,604,625	+	28,604,738	28,604,749	-	154
		5	16,148,311	16,148,322	+	16,148,517	16,148,528	-	236
	10	X	132,730,682	132,730,692	+	132,730,693	132,730,703	-	42
		X	152,835,037	152,835,047	+	152,835,225	152,835,235	-	219
		X	20,778,068	20,778,078	-	20,778,020	20,778,030	+	79
		10	63,974,531	63,974,541	+	63,974,893	63,974,903	-	393
		10	69,613,011	69,613,021	+	69,613,141	69,613,151	-	161
		10	68,396,457	68,396,467	-	68,396,383	68,396,393	+	105
		1	6,262,822	6,262,832	+	6,262,890	6,262,900	-	99
		16	36,764,858	36,764,868	+	36,764,988	36,764,998	-	161
		17	30,754,695	30,754,705	+	30,754,723	30,754,733	-	59
		2	73,354,403	73,354,413	+	73,354,800	73,354,810	-	428
		3	6,719,946	6,719,956	-	6,719,934	6,719,944	+	43
		3	14,322,072	14,322,082	-	14,321,912	14,321,922	+	191
		4	131,489,489	131,489,499	+	131,489,603	131,489,613	-	145
		4	99,891,241	99,891,251	-	99,891,189	99,891,199	+	83
		5	28,604,615	28,604,625	+	28,604,738	28,604,748	-	154
		5	51,567,159	51,567,169	+	51,567,548	51,567,558	-	420
		5	16,148,517	16,148,527	-	16,148,312	16,148,322	+	236
		6	520,074	520,084	+	520,086	520,096	-	43
		6	540,466	540,476	-	540,454	540,464	+	43
		7	118,728,758	118,728,768	+	118,728,958	118,728,968	-	231
		8	109,034,735	109,034,745	-	109,034,723	109,034,733	+	43
	9	X	8,079,746	8,079,755	+	8,080,188	8,080,197	-	474
		X	8,618,319	8,618,328	+	8,618,700	8,618,709	-	413
		X	8,618,319	8,618,328	+	8,618,381	8,618,390	-	94
		X	10,518,262	10,518,271	+	10,518,643	10,518,652	-	413
		X	10,518,262	10,518,271	+	10,518,324	10,518,333	-	94
		X	10,745,431	10,745,440	+	10,745,812	10,745,821	-	413
		X	10,745,431	10,745,440	+	10,745,493	10,745,502	-	94
		X	13,457,006	13,457,015	+	13,457,105	13,457,114	-	131
		X	132,730,683	132,730,692	+	132,730,693	132,730,702	-	42
		X	152,835,038	152,835,047	+	152,835,225	152,835,234	-	219
		X	159,843,935	159,843,944	+	159,844,004	159,844,013	-	101
		X	167,892,730	167,892,739	+	167,892,809	167,892,818	-	111
		X	189,375,181	189,375,190	+	189,375,546	189,375,555	-	397
		X	827,818	827,827	-	827,437	827,446	+	413
		X	827,818	827,827	-	827,756	827,765	+	94
Ame-μ10	9	X	991,436	991,445	-	991,074	991,083	+	394
		X	991,436	991,445	-	991,380	991,389	+	88
		X	20,778,068	20,778,077	-	20,778,021	20,778,030	+	79
		X	33,458,994	33,459,003	-	33,458,597	33,458,606	+	429
		X	36,191,802	36,191,811	-	36,191,535	36,191,544	+	299
		X	63,142,984	63,142,993	-	63,142,784	63,142,793	+	232
		X	64,811,456	64,811,465	-	64,811,129	64,811,138	+	359
		X	86,881,785	86,881,794	-	86,881,705	86,881,714	+	112
		X	121,616,191	121,616,200	-	121,616,113	121,616,122	+	110
		X	187,855,421	187,855,430	-	187,854,953	187,854,962	+	500
		10	23,057,908	23,057,917	+	23,058,358	23,058,367	-	482
		10	27,106,136	27,106,145	+	27,106,313	27,106,322	-	209
		10	61,020,072	61,020,081	+	61,020,298	61,020,307	-	258
		10	63,974,532	63,974,541	+	63,974,893	63,974,902	-	393
		10	69,613,012	69,613,021	+	69,613,141	69,613,150	-	161
		10	76,655,075	76,655,084	+	76,655,482	76,655,491	-	439
		10	41,611,089	41,611,098	-	41,610,736	41,610,745	+	385
		10	68,396,457	68,396,466	-	68,396,384	68,396,393	+	105
		11	32,448,396	32,448,405	+	32,448,441	32,448,450	-	77
		11	65,258,278	65,258,287	+	65,258,749	65,258,758	-	503
		11	83,412,270	83,412,279	+	83,412,347	83,412,356	-	109
		11	84,696,636	84,696,645	+	84,696,690	84,696,699	-	86
		11	105,989,329	105,989,338	+	105,989,407	105,989,416	-	110
		11	41,752,459	41,752,468	-	41,752,087	41,752,096	+	404
		11	77,185,067	77,185,076	-	77,184,857	77,184,866	+	242
		11	83,242,277	83,242,286	-	83,241,825	83,241,834	+	484
		11	90,174,704	90,174,713	-	90,174,419	90,174,428	+	317
		11	101,501,815	101,501,824	-	101,501,605	101,501,614	+	242
		12	24,020,517	24,020,526	+	24,020,572	24,020,581	-	87
		12	27,427,810	27,427,819	+	27,428,111	27,428,120	-	333
		12	29,333,609	29,333,618	+	29,333,736	29,333,745	-	159
		12	30,798,124	30,798,133	+	30,798,201	30,798,210	-	109
		12	50,527,208	50,527,217	+	50,527,282	50,527,291	-	106
		12	99,347,706	99,347,715	+	99,347,890	99,347,899	-	216
		12	8,332,784	8,332,793	-	8,332,450	8,332,459	+	366
		12	21,145,729	21,145,738	-	21,145,509	21,145,518	+	252
		12	53,168,082	53,168,091	-	53,167,844	53,167,853	+	270
		12	94,476,227	94,476,236	-	94,476,123	94,476,132	+	136
		13	48,798,773	48,798,782	+	48,798,848	48,798,857	-	107
Ame-μ10	9	13	79,155,883	79,155,892	+	79,155,923	79,155,932	-	72
		13	406,697	406,706	-	406,375	406,384	+	354
		13	2,182,788	2,182,797	-	2,182,464	2,182,473	+	356
		13	9,223,601	9,223,610	-	9,223,267	9,223,276	+	366
		13	9,324,183	9,324,192	-	9,323,849	9,323,858	+	366
		13	42,317,421	42,317,430	-	42,317,341	42,317,350	+	112
		13	59,447,086	59,447,095	-	59,446,667	59,446,676	+	451
		13	59,820,893	59,820,902	-	59,820,570	59,820,579	+	355
		13	66,813,718	66,813,727	-	66,813,241	66,813,250	+	509
		13	71,797,402	71,797,411	-	71,797,138	71,797,147	+	296
		13	78,678,961	78,678,970	-	78,678,773	78,678,782	+	220
		13	100,911,641	100,911,650	-	100,911,327	100,911,336	+	346
		1	1,877,039	1,877,048	+	1,877,408	1,877,417	-	401
		1	6,262,823	6,262,832	+	6,262,890	6,262,899	-	99
		1	38,339,109	38,339,118	+	38,339,181	38,339,190	-	104
		1	70,235,646	70,235,655	+	70,236,032	70,236,041	-	418
		1	101,351,108	101,351,117	+	101,351,187	101,351,196	-	111
		1	129,126,490	129,126,499	+	129,126,961	129,126,970	-	503
		1	132,037,273	132,037,282	+	132,037,691	132,037,700	-	450
		1	132,621,702	132,621,711	+	132,621,932	132,621,941	-	262
		1	145,300,563	145,300,572	+	145,300,836	145,300,845	-	305
		1	148,159,039	148,159,048	+	148,159,109	148,159,118	-	102
		1	157,983,589	157,983,598	+	157,983,805	157,983,814	-	248
		1	174,114,473	174,114,482	+	174,114,966	174,114,975	-	525
		1	183,381,014	183,381,023	+	183,381,069	183,381,078	-	87
		1	28,486,552	28,486,561	-	28,486,070	28,486,079	+	514
		1	37,559,138	37,559,147	-	37,559,078	37,559,087	+	92
		1	61,356,988	61,356,997	-	61,356,657	61,356,666	+	363
		1	67,205,149	67,205,158	-	67,205,110	67,205,119	+	71
		1	79,189,857	79,189,866	-	79,189,455	79,189,464	+	434
		1	126,390,220	126,390,229	-	126,389,852	126,389,861	+	400
		1	181,500,359	181,500,368	-	181,499,979	181,499,988	+	412
		1	181,500,359	181,500,368	-	181,500,297	181,500,306	+	94
		1	181,665,561	181,665,570	-	181,665,181	181,665,190	+	412
		1	181,665,561	181,665,570	-	181,665,499	181,665,508	+	94
		14	27,465,397	27,465,406	+	27,465,795	27,465,804	-	430
		14	38,913,183	38,913,192	+	38,913,257	38,913,266	-	106
		14	44,296,741	44,296,750	+	44,296,805	44,296,814	-	96
		14	49,460,590	49,460,599	+	49,460,803	49,460,812	-	245
Ame-μ10	9	14	62,340,165	62,340,174	+	62,340,251	62,340,260	-	118
		14	74,381,704	74,381,713	+	74,381,782	74,381,791	-	110
		14	49,317,341	49,317,350	-	49,317,072	49,317,081	+	301
		14	70,371,230	70,371,239	-	70,370,977	70,370,986	+	285
		14	90,579,666	90,579,675	-	90,579,286	90,579,295	+	412
		15	13,077,676	13,077,685	-	13,077,409	13,077,418	+	299
		16	36,764,859	36,764,868	+	36,764,988	36,764,997	-	161
		16	37,942,682	37,942,691	+	37,942,741	37,942,750	-	91
		16	20,943,834	20,943,843	-	20,943,405	20,943,414	+	461
		16	46,029,028	46,029,037	-	46,028,706	46,028,715	+	354
		16	50,568,011	50,568,020	-	50,567,896	50,567,905	+	147
		16	52,065,817	52,065,826	-	52,065,322	52,065,331	+	527
		16	56,236,420	56,236,429	-	56,236,230	56,236,239	+	222
		17	7,483,154	7,483,163	+	7,483,461	7,483,470	-	339
		17	30,754,696	30,754,705	+	30,754,723	30,754,732	-	59
		17	2,739,713	2,739,722	-	2,739,432	2,739,441	+	313
		17	40,108,540	40,108,549	-	40,108,399	40,108,408	+	173
		18	15,248,735	15,248,744	-	15,248,310	15,248,319	+	457
		18	17,197,700	17,197,709	-	17,197,547	17,197,556	+	185
		19	12,905,731	12,905,740	+	12,906,203	12,906,212	-	504
		19	26,022,634	26,022,643	+	26,022,835	26,022,844	-	233
		19	35,188,942	35,188,951	+	35,189,269	35,189,278	-	359
		19	35,296,070	35,296,079	+	35,296,476	35,296,485	-	438
		19	34,448,049	34,448,058	-	34,447,838	34,447,847	+	243
		2	26,750,388	26,750,397	+	26,750,546	26,750,555	-	190
		2	58,276,002	58,276,011	+	58,276,237	58,276,246	-	267
		2	66,781,230	66,781,239	+	66,781,580	66,781,589	-	382
		2	73,354,404	73,354,413	+	73,354,800	73,354,809	-	428
		2	3,044,878	3,044,887	-	3,044,825	3,044,834	+	85
		2	16,274,073	16,274,082	-	16,273,941	16,273,950	+	164
		2	108,304,382	108,304,391	-	108,304,263	108,304,272	+	151
		2	123,557,884	123,557,893	-	123,557,573	123,557,582	+	343
		2	131,075,362	131,075,371	-	131,075,284	131,075,293	+	110
		2	156,338,316	156,338,325	-	156,338,159	156,338,168	+	189
		3	51,871,447	51,871,456	+	51,871,561	51,871,570	-	146
		3	56,443,736	56,443,745	+	56,443,812	56,443,821	-	108
		3	80,048,234	80,048,243	+	80,048,244	80,048,253	-	42
		3	84,603,822	84,603,831	+	84,604,022	84,604,031	-	232
		3	93,227,650	93,227,659	+	93,227,729	93,227,738	-	111
Ame-μ10	9	3	106,772,441	106,772,450	+	106,772,593	106,772,602	-	184
		3	146,736,890	146,736,899	+	146,737,218	146,737,227	-	360
		3	6,719,935	6,719,944	+	6,719,935	6,719,944	+	43
		3	14,321,913	14,321,922	+	14,321,913	14,321,922	+	191
		3	57,883,379	57,883,388	+	57,883,379	57,883,388	+	118
		3	98,791,352	98,791,361	+	98,791,352	98,791,361	+	255
		3	123,482,355	123,482,364	+	123,482,355	123,482,364	+	85
		4	2,550,277	2,550,286	+	2,550,751	2,550,760	-	506
		4	100,271,492	100,271,501	+	100,271,606	100,271,615	-	146
		4	115,282,118	115,282,127	+	115,282,442	115,282,451	-	356
		4	117,410,368	117,410,377	+	117,410,460	117,410,469	-	124
		4	121,363,376	121,363,385	+	121,363,483	121,363,492	-	139
		4	131,489,490	131,489,499	+	131,489,603	131,489,612	-	145
		4	57,678,463	57,678,472	-	57,677,975	57,677,984	+	520
		4	62,253,597	62,253,606	-	62,253,130	62,253,139	+	499
		4	77,028,111	77,028,120	-	77,027,655	77,027,664	+	488
		4	96,618,984	96,618,993	-	96,618,943	96,618,952	+	73
		4	99,891,241	99,891,250	-	99,891,190	99,891,199	+	83
		4	101,656,183	101,656,192	-	101,656,170	101,656,179	+	45
		5	21,551,923	21,551,932	+	21,551,975	21,551,984	-	84
		5	28,604,616	28,604,625	+	28,604,738	28,604,747	-	154
		5	46,104,340	46,104,349	+	46,104,424	46,104,433	-	116
		5	51,567,160	51,567,169	+	51,567,548	51,567,557	-	420
		5	99,521,693	99,521,702	+	99,522,124	99,522,133	-	463
		5	99,717,598	99,717,607	+	99,718,035	99,718,044	-	469
		5	134,087,212	134,087,221	+	134,087,555	134,087,564	-	375
		5	80,270	80,279	-	79,840	79,849	+	462
		5	16,148,517	16,148,526	-	16,148,313	16,148,322	+	236
		5	95,130,585	95,130,594	-	95,130,445	95,130,454	+	172
		5	117,971,512	117,971,521	-	117,971,481	117,971,490	+	63
		6	520,075	520,084	+	520,086	520,095	-	43
		6	2,623,853	2,623,862	+	2,624,080	2,624,089	-	259
		6	49,604,013	49,604,022	+	49,604,291	49,604,300	-	310
		6	65,637,321	65,637,330	+	65,637,378	65,637,387	-	89
		6	65,637,321	65,637,330	+	65,637,630	65,637,639	-	341
		6	85,154,395	85,154,404	+	85,154,526	85,154,535	-	163
		6	118,774,495	118,774,504	+	118,774,571	118,774,580	-	108
		6	118,774,495	118,774,504	+	118,774,799	118,774,808	-	336
		6	540,466	540,475	-	540,455	540,464	+	43
Ame-μ10	9	6	8,373,094	8,373,103	-	8,372,792	8,372,801	+	334
		6	31,269,130	31,269,139	-	31,268,640	31,268,649	+	522
		6	31,277,840	31,277,849	-	31,277,535	31,277,544	+	337
		6	45,597,232	45,597,241	-	45,596,966	45,596,975	+	298
		6	55,451,087	55,451,096	-	55,451,062	55,451,071	+	57
		6	66,130,370	66,130,379	-	66,130,270	66,130,279	+	132
		6	104,506,633	104,506,642	-	104,506,569	104,506,578	+	96
		6	110,065,154	110,065,163	-	110,065,076	110,065,085	+	110
		7	9,069,469	9,069,478	+	9,069,574	9,069,583	-	137
		7	10,527,332	10,527,341	+	10,527,354	10,527,363	-	54
		7	40,485,541	40,485,550	+	40,485,645	40,485,654	-	136
		7	41,703,556	41,703,565	+	41,703,635	41,703,644	-	111
		7	46,875,276	46,875,285	+	46,875,674	46,875,683	-	430
		7	57,177,754	57,177,763	+	57,178,087	57,178,096	-	365
		7	60,327,971	60,327,980	+	60,328,382	60,328,391	-	443
		7	64,163,698	64,163,707	+	64,164,114	64,164,123	-	448
		7	118,728,759	118,728,768	+	118,728,958	118,728,967	-	231
		7	129,239,902	129,239,911	+	129,239,970	129,239,979	-	100
		7	28,793,733	28,793,742	-	28,793,653	28,793,662	+	112
		7	34,938,193	34,938,202	-	34,938,118	34,938,127	+	107
		7	79,518,795	79,518,804	-	79,518,306	79,518,315	+	521
		7	86,659,696	86,659,705	-	86,659,361	86,659,370	+	367
		7	88,629,158	88,629,167	-	88,629,020	88,629,029	+	170
		7	115,600,867	115,600,876	-	115,600,539	115,600,548	+	360
		7	128,279,694	128,279,703	-	128,279,624	128,279,633	+	102
		8	13,153,118	13,153,127	+	13,153,475	13,153,484	-	389
		8	93,495,928	93,495,937	+	93,495,962	93,495,971	-	66
		8	101,175,655	101,175,664	+	101,175,916	101,175,925	-	293
		8	7,567,504	7,567,513	-	7,567,226	7,567,235	+	310
		8	41,683,236	41,683,245	-	41,683,219	41,683,228	+	49
		8	91,767,600	91,767,609	-	91,767,209	91,767,218	+	423
		8	109,034,735	109,034,744	-	109,034,724	109,034,733	+	43
		8	119,385,527	119,385,536	-	119,385,417	119,385,426	+	142
		9	8,224,296	8,224,305	+	8,224,627	8,224,636	-	363
		9	20,730,280	20,730,289	+	20,730,747	20,730,756	-	499
		9	72,071,894	72,071,903	+	72,071,968	72,071,977	-	106
		9	97,192,300	97,192,309	+	97,192,378	97,192,387	-	110
		9	24,346,388	24,346,397	-	24,346,024	24,346,033	+	396
		9	55,810,907	55,810,916	-	55,810,638	55,810,647	+	301
Ame-μ10	9	9	68,353,870	68,353,879	-	68,353,778	68,353,787	+	124
		9	79,436,430	79,436,439	-	79,436,178	79,436,187	+	284
		9	104,272,086	104,272,095	-	104,271,606	104,271,615	+	512
gpz-6	14	3	82,719,983	82,719,997	+	82,720,168	82,720,182	-	210
	13	3	82,719,984	82,719,997	+	82,720,168	82,720,181	-	210
	12	3	82,719,985	82,719,997	+	82,720,168	82,720,180	-	210
	12	5	3,105,016	3,105,028	+	3,105,280	3,105,292	-	291
	11	3	82,719,986	82,719,997	+	82,720,168	82,720,179	-	210
	11	5	3,105,017	3,105,028	+	3,105,280	3,105,291	-	291
	10	3	82,719,987	82,719,997	+	82,720,168	82,720,178	-	210
		5	3,105,018	3,105,028	+	3,105,280	3,105,290	-	291
		5	118,940,700	118,940,710	+	118,941,099	118,941,109	-	428
		8	113,317,136	113,317,146	+	113,317,570	113,317,580	-	463
	9	X	9,605,646	9,605,655	+	9,606,042	9,606,051	-	426
		X	65,938,288	65,938,297	+	65,938,500	65,938,509	-	242
		X	91,365,658	91,365,667	+	91,365,672	91,365,681	-	44
		X	70,521,042	70,521,051	-	70,520,742	70,520,751	+	330
		X	158,752,051	158,752,060	-	158,751,663	158,751,672	+	418
		10	62,834,502	62,834,511	+	62,834,925	62,834,934	-	453
		11	76,640,765	76,640,774	+	76,641,095	76,641,104	-	360
		11	15,027,829	15,027,838	-	15,027,829	15,027,838	-	459
		11	33,527,982	33,527,991	-	33,527,982	33,527,991	-	500
		13	30,434,777	30,434,786	+	30,435,128	30,435,137	-	381
		13	38,865,914	38,865,923	+	38,866,291	38,866,300	-	407
		13	70,820,921	70,820,930	+	70,821,103	70,821,112	-	212
		1	13,401,614	13,401,623	+	13,401,768	13,401,777	-	184
		1	68,249,648	68,249,657	+	68,249,899	68,249,908	-	281
		1	21,445,703	21,445,712	-	21,445,334	21,445,343	+	399
		1	99,852,175	99,852,184	-	99,851,810	99,851,819	+	395
		1	117,654,292	117,654,301	-	117,653,826	117,653,835	+	496
		14	55,849,585	55,849,594	-	55,849,492	55,849,501	+	123
		15	41,580,119	41,580,128	+	41,580,351	41,580,360	-	262
		15	62,425,936	62,425,945	+	62,426,280	62,426,289	-	374
		15	79,866,861	79,866,870	+	79,867,156	79,867,165	-	325
		16	6,284,969	6,284,978	+	6,285,432	6,285,441	-	493
		16	81,120,429	81,120,438	+	81,120,896	81,120,905	-	497
		16	33,513,244	33,513,253	-	33,512,935	33,512,944	+	339
		17	17,788,713	17,788,722	-	17,788,505	17,788,514	+	238
		18	9,947,190	9,947,199	-	9,947,027	9,947,036	+	193
gpz-6	9	2	44,832,080	44,832,089	+	44,832,265	44,832,274	-	215
		2	144,076,375	144,076,384	+	144,076,512	144,076,521	-	167
		2	81,097,337	81,097,346	-	81,097,165	81,097,174	+	202
		2	126,424,262	126,424,271	-	126,423,908	126,423,917	+	384
		3	19,636,345	19,636,354	+	19,636,770	19,636,779	-	455
		3	82,719,988	82,719,997	+	82,720,168	82,720,177	-	210
		4	111,517,173	111,517,182	+	111,517,620	111,517,629	-	477
		4	127,405,094	127,405,103	+	127,405,298	127,405,307	-	234
		5	3,105,019	3,105,028	+	3,105,280	3,105,289	-	291
		5	18,840,252	18,840,261	+	18,840,331	18,840,340	-	109
		5	118,940,701	118,940,710	+	118,941,099	118,941,108	-	428
		5	6,982,351	6,982,360	-	6,981,916	6,981,925	+	465
		6	116,294,124	116,294,133	+	116,294,500	116,294,509	-	406
		6	82,123,233	82,123,242	-	82,122,738	82,122,747	+	525
		6	103,931,890	103,931,899	-	103,931,429	103,931,438	+	491
		6	125,162,004	125,162,013	-	125,161,788	125,161,797	+	246
		7	83,507,332	83,507,341	+	83,507,507	83,507,516	-	205
		7	4,724,187	4,724,196	-	4,724,149	4,724,158	+	68
		7	18,138,353	18,138,362	-	18,138,149	18,138,158	+	234
		7	116,373,027	116,373,036	-	116,372,679	116,372,688	+	378
		8	20,934,656	20,934,665	+	20,934,875	20,934,884	-	249
		8	21,197,576	21,197,585	+	21,197,769	21,197,778	-	223
		8	113,317,137	113,317,146	+	113,317,570	113,317,579	-	463
		8	6,148,525	6,148,534	-	6,148,131	6,148,140	+	424
		8	34,451,501	34,451,510	-	34,451,190	34,451,199	+	341
		9	12,598,302	12,598,311	+	12,598,594	12,598,603	-	322
		9	58,404,146	58,404,155	+	58,404,385	58,404,394	-	269
		9	87,055,111	87,055,120	+	87,055,534	87,055,543	-	453
		9	27,090,126	27,090,135	-	27,089,853	27,089,862	+	303

表1

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位点名称	20 µL反应体系								最适退火温度范围 (Tm, ℃)
位点名称	Taq DNA 聚合酶 (5 U/µL)	10×Taq Buffer with KCl	10×Taq Buffer with (NH₄)₂SO₄	dNTP (10 mmol/L each)	MgCl₂(25 mmol/L)	引物 (10 µmol/L)	DNA (20 ng/µL)	BSA (1 mg/mL)	最适退火温度范围 (Tm, ℃)
Ame-μ10	0.4 µL	2 µL,二者择一		0.6 µL	1 µL	0.6 µL×2	0.15~2 µL*	0.8 µL	55~61
Ame-μ11	0.4 µL	2 µL	?			0.4 µL×2			55~61
Ame-μ13	0.4 µL	2 µL	?						55~59
Ame-μ14	1.6 µL	?	2 µL						55~61
Ame-μ15	0.4 µL	2 µL	?						55~57
Ame-μ19	0.4 µL	2 µL,二者择一							55~61
Ame-μ21	0.4 µL								55~59
Ame-μ22	0.4 µL								55~57
Ame-μ24	0.4 µL	?	2 µL						55~57
Ame-μ25	0.4 µL	2 µL	?						55
Ame-μ26	0.4 µL	2 µL,二者择一							55~57
GP001	0.4 µL								55~65
GP008	0.4 µL								55~65
GP901	0.4 µL	2 µL	?						55~61
GPL-8	0.4 µL	2 µL,二者择一							55~59
GPL-28	0.4 µL	2 µL,二者择一				0.8 µL×2			55~61
GPL-29	1.2 µL	?	2 µL			0.8 µL×2			55~57
GPL-31	0.4 µL	2 µL,二者择一				0.4 µL×2			55~61
GPL-44	0.4 µL	?	2 µL						55~59
GPL-47	0.4 µL	?	2 µL						55~59
GPL-53	0.4 µL	2 µL,二者择一							55~59
GPL-60	0.4 µL								55~59
gpy-5	0.4 µL								55~61
gpy-20	0.4 µL	?	2 µL						55~61
gpz-6	0.4 µL	2 µL	?						55~59
gpz-20	0.4 µL	?	2 µL						55~61
gpz-47	0.4 µL	?	2 µL						55~63
gpz-51	0.4 µL	2 µL	?						55~59
Panda-05	0.4 µL	2 µL,二者择一							55~63
Panda-06	0.4 µL								55~65
Panda-22	0.4 µL								55~63
Panda-40	0.4 µL								55~57
Panda-44	0.8 µL	?	2 µL			0.6 µL×2			55~59
Panda-45	0.4 µL	2 µL,二者择一				0.4 µL×2			55~65

步骤	温度	时间	循环数
1	94℃	5 min	1
2	94℃	15 s	35
3	Tm	15 s
4	72℃	30 s
5	72℃	10 min	1

谱系号	疑似连锁等位基因大小(bp)		对照等位基因大小(bp)		疑似多位点扩增等位基因大小(bp)
谱系号	Panda-44	Ame-μ11	GPL-29	Ame-μ19	Ame-μ10	gpz-6
401	234/242	230/236	163	158/162	138/144	206/210
480	242	236	171/175	158/160	142	206
522	234/238	230/234	163/175	160	138/144	206/218
532	238/242*	232*	175	158	142/146	210/214
537	234/238	230/236	175	160/164	144	206/210
574	238/242*	234/236*	167/171	160	138	206/222
584	242	232/236	167/175	160	138/142	206/218
598	234/238	230/236	175	158/160	146	202/206
635	238/242*	228/236*	163	158/160	138/144	206
678	234	232/234	163/175	160	138/146	210/218
681	234	230/232	163/171	158/160	138/144	206/218
703	238/242*	232*	163/171	160	138	210
717	238/242*	228/236*	163/171	158	142/154	206
765	234/238	230/236	171/175	158/160	138	202/206
801	234	230/232	163/175	158/160	146	206/210
857	242	232/234	175	160	138/142	206
858	238/242	230/232	175	160	144/146	206/210
870	234/238	230/236	163/175	158/160	144	210
871	234	230/236	163/175	158/160	146	206/210
919	234/242	232/234	163/175	160	138/144	206/210
963	234/238	230/236	175	160	138/146	206
985	238/242*	236*	167/175	158	142/144	202/206
986	234/242	236	175	160	138/144	206/210
990	238	230/232	167/175	160	144/146	202/210
998	238/242*	232/236*	167	165	146	210/218
1016	234/242	232/236	175	160	138/144	210/218
1017	238/242*	234/236*	163/167	160	138/144	206
1077	234/242	230/232	163/175	158	146	206/210
1119	230/242	228	167/175	160	142/144	206
1160	234/238	232/236	171/175	156	142/146	206/210

分组	亲子关系	谱系号	疑似连锁等位基因大小(bp)		对照等位基因大小(bp)		疑似多位点扩增等位基因大小(bp)
分组	亲子关系	谱系号	Panda-44	Ame-μ11	GPL-29	Ame-μ19	Ame-μ10	gpz-6
1	母	401	234/242	230/236	163	158/162	138/144	206/210
1	女	635	238/242	228/236	163	158/160	138/144	206
2	母	401	234/242	230/236	163	158/162	138/144	206/210
2	女	681	234	230/232	163/171	158/160	138/144	206/218
3	母	480	242	236	171/175	158/160	142	206
3	子	717	238/242	228/236	163/171	158	142/154	206
4	母	480	242	236	171/175	158/160	142	206
4	子	584	242	232/236	167/175	160	138/142	206/218
5	父	584	242	232/236	167/175	160*	138/142	206/218
	母	537	234/238	230/236	175	160/164*	144	206/210
	子	985	238/242	236	167/175	158*	142/144	202^#/206
6	父	584	242	232/236	167/175	160	138/142	206/218
	母	537	234/238	230/236	175	160/164	144	206/210
	子	986	234/242	236	175	160	138/144	206/210
7	父	584	242	232/236	167/175	160	138/142	206/218
7	子	1016	234/242	232/236	175	160	138/144	210/218
8	父	532	238/242	232	175	158*	142/146	210/214*
8	子	857	242	232/234	175	160*	138/142	206*
9	父	532	238/242	232	175	158*	142/146	210/214
9	子	858	238/242	230/232	175	160*	144/146	206/210
10	母	598	234/238	230/236	175	158/160	146	202/206
10	子	963	234/238	230/236	175	160	138/146	206
11	母	598	234/238	230/236	175	158/160	146	202/206
11	子	1077	234/242	230/232	163/175	158	146	206/210

大熊猫遗传多样性评估的微卫星分型体系优化

Optimization of microsatellite genotyping system used for genetic diversity evaluation of Ailuropoda melanoleuca

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[2]	朱艳, 魏明, 周晓, 邓林华, 仇剑, 李果, 周世强, 谢浩, 李德生, 王承东. 大熊猫(Ailuropoda melanoleuca) miRNA研究进展[J]. 遗传, 2021, 43(9): 849-857.
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[6]	赵永欣, 李孟华, 赵要风. 中国绵羊起源、进化和遗传多样性研究进展[J]. 遗传, 2017, 39(11): 958-973.
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