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Introduction

Microsatellite markers, or short tandem repeats (STR), are polymorphic DNA loci containing repeated nucleotide sequences, typically from 2 to 7 nucleotides per unit. The length of the repeated unit is the same for the majority of the repeats within an individual microsatellite locus. By contrast, when the number of repeats for a specific locus differs, it results in alleles of varying length (1). The vast amount of data emerging for thousands of microsatellite markers across organisms makes microsatellite analysis a widely accepted tool for population genetic diversity studies, as well as for identifying individual organisms, including human and tree shrew. Microsatellite analysis includes polymerase chain reaction (PCR) amplification of the microsatellite loci using fluorescently labeled primers. The labeled PCR products are then analyzed by capillary electrophoresis or electrophoresis to separate the alleles by size (2). In the present study, we used and developed this microsatellite analysis for identifying the individual Chinese tree shrew.

Tree shrew (Scandentia, Tupaiidae) is a small mammal with a close affinity to primates. It belongs to the Family Tupaiidae of Scandentia and is native to tropical forests, widely distributed across Southeast Asia (3). The Chinese tree shrew (Tupaia belangeri) is distributed across southwest China and classified as six subspecies (Tupaia belangeri chinensis, Tupaia belangeri gaoligongensis, Tupaia belangeri modesta, Tupaia belangeri tonquinia, Tupaia belangeri yunalis and Tupaia belangeri yaoshanensis) based on morphological characteristics and geographical distribution. Tupaia belangeri yaoshanensis is the largest in size. It inhabits the Dayaoshan Mountain of Guangxi, China. Owing to its resemblance of ancestral primates (4,5), as well as their behavior and social monogamy (6), the Chinese tree shrew has been used as an experimental animal models for the study of human diseases (7).

Although some mitochondrial (mtDNA) and nuclear (nDNA) sequences and karyotype maps have been published (6,8,9)correctly, there few species or population level studies on the Chinese tree shrew. Microsatellite markers are an extremely important tool for genetic studies and for the conservation and management of genetic resources (10,11). Previous studies identified microsatellite markers of tree shrew (2,12–14); however, microsatellite markers specific to Tupaia belangeri yaoshanensis remain to be determined. For the conservation of genetic resources or for selecting a proper founder to establish the inbred lines, it would be worthwhile to screen the genetic diversity of these different subspecies (12).

In this study, we analyzed 16 microsatellite loci of genomic DNAs from a population of 76 individuals of wild Tupaia belangeri yaoshanensis by using the microsatellites developed from other subspecies of tree shrews, and evaluated the genetic diversity of this population. Our study suggests that these 16 microsatellite markers can be used to analyze the population genetics of Tupaia belangeri yaoshanensis and to establish animal models. Moreover, the findings of this study can be used for the protection and effective utilization of the germplasm resource of this species.

Materials and methods

Sample collection

Seventy-six wild Tupaia belangeri yaoshanensis animals were collected from the Dayaoshan Mountain of Guangxi, China and were used in this study. All the animals were raised at the Experimental Animal Center of Guangxi University of Chinese Medicine under specific pathogen-free conditions and were cared for in an environmentally controlled room with a 12-h light/dark cycle and a constant temperature of 22°C. The animals were provided with a standard diet and water in accordance with the guidelines for the treatment of experimental animals published by the Ministry of Science and Technology of the People's Republic of China in 2006. Genomic DNA was extracted from the blood of tail tissues of each individual Tupaia belangeri yaoshanensis by using the genomic DNA extraction kit (Tiangen, Beijing, China). All sampling procedures related to animal subjects were reviewed and approved by the Review Committee of Guangxi University of Chinese Medicine, in accordance with the institutional animal care and use regulations and rules. The present study was carried out in strict accordance with the recommendations established in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health (eighth version, 2010). The animal use protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Guangxi University of Chinese Medicine (Nanning, China; approval nos. GXUCM2014NSF01125 and GXUCM2014NSF01136).

Microsatellite screening and simple sequence repeat (SSR) analysis

Sixteen previously described microsatellite loci of other species of tree shrews were used in this study (Table I). The composition of the PCR reaction mixture and procedures of PCR were referenced as per the published methods (2,12–14), with minor modifications. Briefly, the PCR amplification was performed in 25 µl volumes containing 1 µl diluted DNA template, 1 µl forward and reverse primers (the forward primer was fluorescently labeled with FAM, HEX or TAM), 2.5 µl 10X PCR buffer [without Mg2+: 100 mM Tris-HCl pH 8.8; 500 mM KCl, 0.8% (v/v) Nonidet], 0.5 µl 10 mM dNTPs, 2 µl 25 mM MgCl2, and 0.2 µl 5 units TaqDNA polymerase (Takara Bio Inc., Otsu, Japan), and 17.8 µl sterilized water. Amplification was performed on an Applied Biosystems (T100; Bio-Rad, Hercules, CA, USA). Thermal cycling conditions were implemented as follows: 94°C for 3 min; followed by 30 cycles of 95°C for 30 sec, optimal annealing temperature (Table I) for 30 sec, 72°C for 30 sec, a final extension step at 72°C for 5 min and then holding at 8°C. PCR product (1 µl) was mixed with 0.1 µl GeneScan™ 500 LIZ® and 9.9 µl Hi-Di™ Formamide and denatured at 95°C for 5 min, then immediately chilled on ice. After this, it was separated on an ABI PRISM 3730 XL genetic analyzer (Applied Biosystems, Foster City, CA, USA).

Table I. PCR primers and their sequences for the 16 microsatellite loci used in this study.

Table I.

PCR primers and their sequences for the 16 microsatellite loci used in this study.

Locus	Primer sequences (5→3)	Tm (°C)	Repeat motif	Size range (bp)	Accession no./(Refs.)
JS188	F: ACACACACAAAACTCATTTTATCC	57	(CA)n	186–196	DQ-334279
	R: TCTACACGAATGTGCCAACC
JS196	F: ACCTCCTGGTGGCTTGC	57	(GT)n	304–344	DQ-334280
	R: TAATTGCAGGATGCTTCAGG
TB2	F: AATATGCTGGTAGGACCAGAAAC	60	(AAG)n(GAGAA)n	189–275	(11)
	R: ATTGGGTTGTTGGGAAAGTC
TB3	F: TCTGGACCTAGCAACTTGCTTTC	58	(TAAA)n	254–259	(11)
	R: TTCTTCACCAGTGTTGAGCATAA
TB8	F: CTCTGACTTCAAGACCTGGAAC	55	((A/G)TC(C/T))n	453–465	(11)
	R: CTTGAAAGCAAAGGCAGTACAA
TB10	F: ATTGGAGACCACTCTACCCTTTA	60.4	(ATTT)n	456–461	(11)
	R: TGGATTCCCACATAGTCTACCG
TB16	F: TCCTTAGTATGGACTTGTTTGC	58	((A/G)AAG)n	212–228	(11)
	R: AGGATCACAGGGCAGGGAGACT
TB20	F: TGATCTCCAAAGGCTTCTCCAT	60	(AAAAC)n	221–237	(11)
	R: GAACCTTCCCGTATGGGTCAGT
TBC1	F: TGGCAATTGGGAGAATTAGG	48	(TG)n	177	(12)
	R: GCTGCCTTCATTCACCTCTC
TBC3	F: TGGCAGGATTTCCTTCATTC	44	(GT)nGC (GT)n	160	(12)
	R: TCATTGCACGAGAATTTCCA
TBC5	F: GCTGCCTTCATTCACCTCTC	48	(AC)nGCAG(AC)n	173	(12)
	R: TGGCAATTGGGAGAATTAGG
TBC10	F: CCTTACTGACCAGTGCTGAGG	52	(TGG)nCTGTA(G)n	113–182	(12)
	R: GCCCAGACACAGAGAGGTTC		A(GCTGGG)n
TBC11	F: GACCTGGTGGCATGAAAACT	51.5	(GT)nGC(CT)n	157–167	(12)
	R: TCGGGGAAATATGGAGAGAA
TG1	F: TTCCTGTTACTCTGTTTTTTTCAGG	52	(CA)n	152–183	AY-064160
	R: ACATGGCTAACTGTGTGCTTTG
TG4	F: TGAAAACTGGCAATTCATATGC	52	(CA)nTA(CA)n	152	AY-064161
	R: CAATCCTTTTTCGTTAGTTTTGTG		TA(CA)n
TG19	F: ACCCCTCCCTAAAGGAACT	55	(CA)nTAAA(CA)n	171	AY-064163
	R: CGCCCTATAGAAACCTCTCC

[i] PCR, polymerase chain reaction; F, forward; R, reverse.

Statistical data analysis

For each locus in the population, the number of alleles (NA), effective number of alleles (NE), observed heterozygosity (HO), expected heterozygosity (HE), and fixation index (Fis), as well as tests for deviation from Hardy-Weinberg equilibrium (HWE), were analyzed using Popgene version 1.32 (15). Allele frequency data were used to calculate polymorphic information content (PIC) using the methods described by Botstein et al (16).

Results

The details of variability measurement across 76 individuals of the Tupaia belangeri yaoshanensis are summarized in Table II. Among the 16 microsatellites, seven were perfect (43.75%), four were imperfect (25%), and five turned out to be compound (31.25%).

Table II. Polymorphic microsatellite markers identified in Tupaia belangeri yaoshanensis in this study.

Table II.

Polymorphic microsatellite markers identified in Tupaia belangeri yaoshanensis in this study.

Locus	NA	NE	HO	HE	Fis	PIC
JS188a	9.000	3.6098	0.6892	0.7279	0.0467	0.6884
JS196a	4.000	1.4777	0.1714	0.3256	0.4697	0.3052
TB2a	10.000	3.6554	0.7714	0.7317	−0.0619	0.6849
TB3	2.000	1.1573	0.1200	0.1368	0.1171	0.1266
TB8	2.000	1.2227	0.1486	0.1834	0.1840	0.1656
TB10a	6.000	2.3112	0.0870	0.5715	0.8467	0.4739
TB16	4.000	1.6284	0.3194	0.3886	0.1722	0.3500
TB20a	5.000	2.1457	0.4583	0.5277	0.1416	0.4417
TBC10a	6.000	1.2344	0.1781	0.1912	0.0623	0.1817
TBC11a	5.000	3.2996	0.5263	0.7016	0.2448	0.6468
TG1	8.000	4.6301	0.8947	0.7892	−0.1412	0.7505
Mean	5.5455	2.3975	0.3968	0.4796	0.1893	0.4378

{ label (or @symbol) needed for fn[@id='tfn2-br-0-0-969'] } N_A, observed number of alleles; N_E, effective number of alleles; H_O, observed heterozygosity; H_E, expected heterozygosity; Fis, fixation index; PIC, polymorphism information content.

a Significant deviations from Hardy-Weinberg equilibrium.

Overall, 11 loci (68.75%) were cleanly amplified and were shown to be polymorphic. We identified 2–10 alleles (with an average of 5.5455 alleles) per locus in these polymorphic markers. TB2 with 10 alleles was the most polymorphic microsatellite, while TB3 and TB8 were the least variable with only 2 alleles each. The effective number of alleles per locus ranged from 1.16 at TB3 to 4.63 at TG1, with an average of 2.3975. The observed and expected heterozygosities were 0.087 to 0.8947 and 0.1368 to 0.7892 (with an average of 0.3968 and 0.4796), respectively. The Fis pronounced the most loci with heterozygote deficiencies. Seven loci (JS188, JS196, TB2, TB10, TB20, TBC10 and TBC11) significantly deviated from the HWE in the sampled population after Bonferroni's correction. The average PIC per locus ranged from 0.1266 to 0.7505 (Table II), of which, three loci showed low polymorphism (PIC <0.25), four loci showed median polymorphism (0.25< PIC <0.5), while the other four loci were all high polymorphism (PIC >0.5).

In this study, we detected 16 population-loci out of the 35 population-loci examined, as we tested non-specific amplification in some loci in our samples. We showed that five (31.25%) (TBC1, TBC3, TBC5, TG4 and TG19) were monomorphic, and they failed to amplify in Tupaia belangeri yaoshanensis, and these population-loci were proved to be polymorphic microsatellite loci in Tupaia belangeri chinensis, Tupaiaglis and Tupaia minor (2,12–14).

Discussion

A high level of genetic diversity is essential for the long-term survival of populations, and the extent of variation determines their ability to adapt to changing environments. The provision of the primers for these microsatellite markers facilitates the studies of population structure, gene flow, and mapping of the tree shrew genome. In the present study, we analyzed 16 microsatellite loci in 76 animals of wild Tupaia belangeri yaoshanensis and showed that these Tupaia belangeri yaoshanensis had a considerably high heterozygosity, suggesting that the 16 markers may be useful for individual discrimination and parentage testing, as well as for the quantification of population heterogeneity.

We also compared our results with the same microsatellite markers reported in other subspecies including T. glias, T. minor and Tupaia belangeri chinensis, and found some genetic differences between Tupaia belangeri yaoshanensis and other subspecies (12,12–14). Since there are six subspecies in Chinese tree shrews, further study is necessary to determine the genetic diversity of other subspecies, as well as the genetic relationship between the subspecies.

Recent studies have shown that microsatellite markers are useful as a tool in the investigation of population genetics, ecology, and conservation of the Tupaia belangeri yaoshanensis. Our data reported here provide further scientific basis for the identification, breeding, and laboratory animalization of Tupaia belangeri yaoshanensis, and should pave the way for making use of the Tupaia belangeri yaoshanensis in Guangxi with both scientific and economic value.

Acknowledgements

We gratefully acknowledge the assistance from the Laboratory of Molecular Medicine Teaching (Guangxi Medical University), and thank Ms. Y.-F.G. (Guangxi University) for helping with the data analysis. This study was supported by grants from the Department of Science and Technology of Guangxi (nos. GKG1347003-6 and 14123006-1) and the Key Laboratory for Complementary and Alternative Medicine Experimental Animal Models of Guangxi, the Department of Education of Guangxi.

References