c Indian Academy of Sciences

ONLINE RESOURCES

Development of twenty microsatellite loci from the Tibetan ground ( humilis)

PING FENG1, GUANG YANG2 and XIN LU1∗

1Department of Zoology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China 2Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, 1 Wenyuan Road, Nanjing 210046, People’s Republic of China

[Feng P., Yang G. and Lu X. 2013 Development of twenty microsatellite loci from the Tibetan ground tit (Parus humilis). J. Genet. 92, e68–e72. Online only: http://www.ias.ac.in/jgenet/OnlineResources/92/e68.pdf]

Introduction isolated and characterized 20 polymorphic microsatellite loci from the Tibetan ground-tit. These markers were success- The Tibetan ground tit Parus humilis is a sexually monomor- fully amplified in 30 individuals from four distinct popula- phic endemic to the Tibetan plateau. It inhab- tions. The number of alleles, expected heterozygosity and its the alpine meadows at elevations of 2500–5500 m across observed heterozygosity varied from 5 to 20, 0.612 to 0.934, the plateau. This is particularly interesting for its and 0.033 to 0.967, respectively. These markers will be use- phylogenetic position. It was initially assigned to the fam- ful in assessing genetic diversity, population structure, gene ily (crows and jays) based on lifestyle and mor- flow and paternity determination in the Tibetan ground tit. phology (Hume 1871), and recent evidence of nuclear and mitochondrial genes along with osteology suggested it to be a ground-adapted tit in the family Paridae (tits and chick- Materials and methods adees) (James et al. 2003). Moreover, the ground tits have also been explored with respect to phylogeography using Genomic DNA was extracted using the standard proteinase mtDNA sequences (Yang et al. 2006). In terms of social K/phenol extraction protocol (Sambrook and Russel 2001) behaviour, this species is a socially monogamous, faculta- from four individuals’ blood previously preserved in 100% tively cooperative breeder (Du and Lu 2009;Luet al. 2011; ethanol. Microsatellite loci were isolated from the genomic Oh 2011), which is uncommon in high-altitude because libraries enriched for AC/AG repeats following the FIASCO most cooperative species occur in the tropical or southern protocol (Zane et al. 2002). Later genomic DNA from the temperate regions (Jetz and Rubenstein 2011). It has been four individuals was purified and mixed. The digestion– shown that there is a difference in social system among ligation mixture included 24 ng mixed template DNA,  ) different ground tit populations (Johannessen et al. 2011). 10 pmol AFLP adaptor1 (5 -TAC TCA GGA CTC AT-3 ,  ) Ground tits are obligate primary cavity-nesters, excavating 10 pmol AFLP adaptor2 (5 -GAC GAT GAG TCC TGA G-3 , μ burrows under the earth for breeding and roosting (Ke and 5UMseI (New England Biolabs, Beverly, USA), 2.5 L × Lu 2009). Ground tit burrows may provide nest sites for sev- 10 NBE buffer (New England Biolabs), 2.5 ng Puri- μ eral secondary cavity-nesters (Zeng and Lu 2009a, b;Liand fied BSA (New England Biolabs), 5 mol ATP (Promega, Lu 2012). Therefore, the birds are a keystone species for Madison, USA), 10 U T4 DNA ligase (New England Bio- μ ◦ biodiversity in the species-poor, vulnerable alpine meadow labs), 16.75 L ddH2O, and was incubated at 37 Cfor ecosystems. 3 h. The digestion–ligation product was diluted 10-fold in Despite the significant implications of ground tits for ddH2O, and a polymerase chain reaction (PCR) was car- studies of systematics, evolutionary ecology and conserva- ried out on a DNA Engine Dyad Cycler (BioRad, Hemel tion biology, no species-specific microsatellite markers are Hempstead, UK) with the following conditions: initial denat- uration step of 5 min, 20 cycles of denaturation at 94◦Cfor available, which limits our ability to make further inves- ◦ ◦ tigations into these fields. In response to such a need, we 30 s, annealing at 53 C for 1 min, elongation at 72 Cfor 8 min. The PCR reaction contained 10 μL ddH2O, 4 pmol dNTP, 2 μL10× PCR buffer (Mg2+ including) (Takara, ∗ For correspondence. E-mail: [email protected]. Tokyo, Japan), 1 U rTaq DNA polymerase (Takara), 10 pmol Keywords. Tibetan ground tit; microsatellite loci; population genetics.

Journal of Genetics Vol. 92, Online Resources e68 Ping Feng et al.

MseI-N primer (5-GAT GAG TCC TGA GTA AN-3), 5 μL of 72◦C for 8 min. The PCR products were genotyped on a diluted product. Biotinylated probes hybridization, magnetic LI-COR 4300 Automated DNA Sequencer using 6.5% dena- beads elution and strands recovery were performed accord- turing polyacrylamide gels, bands with a size of 50–350 bp ing to an earlier study (Chang et al. 2008). The PCR prod- were analysed using LI-COR SAGAGT software. Null allele ucts were ligated into PMD-18T (Takara), and transformed tests in each locus were performed using Micro-Checker into DH-5α competent cells. Subsequently, the recombinants 2.2.3 (Van Oosterhout et al. 2004). were screened by PCR using a probe primer and the M13 primers. Positive clones were selected and sequenced. A total of 76 positive clones were identified and sequenced, 52 of which were suitable for primer design. Primers were Results and discussion designed using Primer Premier 5 (Premier Biosoft Internatio- nal, Palo Alto, USA) and were commercially synthesized. Twenty loci were successfully genotyped. The variabil- After a series of PCR amplifications, we found 29 primer ity at each locus was measured by numbers of alleles, pairs could be successfully amplified. The 29 primer pairs observed heterozygosity and expected heterozygosity. The were further tested in 30 individuals, of which, seven were polymorphism of each locus was measured by polymor- from Tianjun population in Qinghai province, China; eight phism information content (PIC). Numbers of alleles, pair- from Gangcha population in Qinghai province, China; six wise linkage disequilibrium (LD) and Hardy–Weinberg equi- from Gahai population in Gansu province, China; and nine librium (HWE) were tested with GenePop 3.4 (Raymond from Dangxiong population in Tibet, China (figure 1). and Rousset 1995), and observed heterozygosity, expected Out of the 29 primer pairs, 22 produced a constant heterozygosity and PIC were calculated by Cervus 3.0 product in all or most of the 30 individuals. The 22 (Marshall et al. 1998). In total the 20 polymorphic loci primer pairs were in turn selected to add M13 (-29) (5- produced 209 alleles ranging from 5 to 20 with an aver- CAC GAC GTT GTA AAA CGA C-3) to 5 end of the age of 10.45 alleles per locus (table 1). The observed het- either one primer of a pair as a label. PCR amplification erozygosity values ranged from 0.033 to 0.967, and the was performed in a volume of 20 μL containing 2 μL expected heterozygosity ranged from 0.612 to 0.934. Analy- 10× PCR buffer (Takara), 4 pmol dNTP, 12 nmol Mg2+, ses using GenePop revealed that no significant linkage dise- 0.5 μL the M13(-29)-labelled forward primer, 5 pmol the quilibrium between all locus pairs, although cz29 and cz70b reverse primer and 5 pmol the fluorescent-labelled M13 were found to significantly deviate from HWE, with the esti- primer (either IRD700 or IRD800; LI-COR, Lincoln, USA), mation of exact P values by the Markov chain method (P < 1UrTaq DNA polymerase (Takara), 11.3 μL ddH2O, 30 ng 0.001) after sequential Bonferroni correction (Rice 1989)in DNA template. The PCR condition was as follow: 94◦Cfor Dangxiong or Tianjun populations (see details in table 2). 2min,35cyclesof94◦C for 15 s, primer-specific annealing These deviations from HWE may result from the occurrence temperature (table 1) for 20 s, 72◦C for 30 s, and a final step of null alleles (table 2) or insufficient sample size.

Figure 1. Sampling sites of the four populations of Tibetan ground tits (site 1, Tianjun; site 2, Gangcha; site 3, Gahai; site 4, Dangxiong).

Journal of Genetics Vol. 92, Online Resources e69 Microsatellite loci of Tibetan ground tit PIC Accession no. e H o H A N a T 199–211 66.3 5 0.733 0.767 0.709 JF946684 152–348181–231 66.3133–177 59.2300–322 8 61.4107–291 20 0.154 67227–267 8 0.967 0.806 66.3266–280 11 0.934 0.667 66.1 0.748246–266 8 0.933 0.914 0.765 67162–202 16 JF946675 0.333 0.806 62 0.723 JF946676 0.567 5 0.793 59.2 0.830290–306 JF946677 0.910 7 0.233 0.749261–281 17 JF946678 0.887 66.3 0.233 0.679221–239 0.700 JF946679 66.3 0.629255–341 JF946680 0.605 6 0.924 66.3154–334 10 0.578 0.733 0.903 JF946681 66.3256–264 9 0.276 0.612 JF946682 63.9214–244 15 JF946683 0.810 0.267 66 0.543324–348 15 0.867 0.767 0.795 61.4167–205 0.600 JF946685 0.835 5 69.2 0.752201–227 13 JF946686 0.858 0.802 68 0.533 7 0.933 JF946687 0.827 67 0.618 JF946688 12 0.884 0.828 JF946691 0.538 12 0.033 0.856 0.799 0.310 JF946689 0.827 0.751 JF946690 0.788 0.794 JF946693 0.751 JF946694 JF946692 , expected heterozygosity. e H 8 7 14 3 ) 2 GAAG(CA) T(AC) TG(CA) (TG) 5 13 26 12 16 9 17 13 24 13 11 27 11 10 13 16 13 18 19 14 Repeat sequence Size range (bp) , observed heterozygosity; o H )  -3  , number of alleles; A N CAATGCAGTCCTGCTGGTTTAT CCGTAGCTTTCCTGTGGTTTT GCGAGTAACCCTCTAAGCAATG CAATTGTGAGCCTATGGGAACT GGAACATGCACAGTGAGAAAGC TGCCAGGTCCTCACAATGTAAG CCCGTTCTATCTTGAAACCTCTC CAAAACTTGCCTGCATTCCT GGAGGCTTCCTGTTTTTGC CGCCCAGACCAGTGTGTAAGTT CCAAGCATCCTTCAGCCCT GCGTATTACTTCATTTGCTTGGC CGAGGTTACGATTTCCCCAG GAAAAGGTACCCTCATGAATGCT CGCCTTCACTAATAATCTGGGTTT CGCACTGCTCTCTGGAGTTGAAC GCAATCTTCCTTGCCTTGAGTTC TCTCACTGGGAGCTGCCTCAT CGTTGCGAGGGGGACCAC GCGAGGATGTTACCTTAGGCAAT Characterization of the 20 microsatellite markers. , optimal annealing temperature; a Table 1. Locuscz1 (tailed) CGTCTGCTGGCTGGATTATG Primer sequence (5 (TG) cz50 CCCCTAACCCCCAAATACACA(tailed) (GA) cz7cz18 CGGTATCTCTTTCTTTCAGCCA(tailed)cz19 GCAAGTCCACCTTCCATTTCAG(tailed)cz21 (CA) CAGTGTAGGATGGGGAGAACC(tailed)cz29 (CA) GCTGCTGCAGTGGAGAAAAAAT(tailed)cz32 (AC) GCGAGTAACCCTCTAAGCAATGA(tailed) (AC) cz34 CTGCCTCCATCCTCACCATC(tailed) (CA) cz40 CGGCTCGTATGTTGTGTGG(tailed)cz44 CGCGAGTAACCCTCTAAGCAAT(tailed) (CA) AATCTTCCTTGCCTTGAGTTCG(tailed)cz73 (CA) (CA) cz74 (AC) GCAGATTTCTTCACTTGGACCC(tailed)cz81 GCTTTGCCCTCTTTCCCACT(tailed)cz24a (GA) CTCCTATTATAAGCCCAAGCAGT(tailed)cz47a GATGGGCAGGATCAGGCAATC(tailed) ((TC) (AG) cz154a GGCGTGTATTTTGGTGTCTGTTTA(tailed)cz59b (GT) CGCATTGTGAAAGGGTTTACTGT(tailed) (CA) cz70b GCATATCCACCTGCCAATTTACT(tailed) (GT) cz74b GCGCGGCACCTTCAGTTGT(tailed) (CA) CGGGGAGCACTCTGTTCTTTTC(tailed) (CA) (AC) T

Journal of Genetics Vol. 92, Online Resources e70 Ping Feng et al. 0.05 0.05 0.01 0.05 0.025 0.05 0.05 0.05 0.01 0.05 0.025 0.05 0.05 0.025 0.001 0.05 > > < < < < > > < > < > > < < > (Null alleles) 4) or Markov P ≤ A N values for all classes of alleles; P (HW) Null present P PIC (null alleles), combined P E H O H A N values for Hardy–Weinberg equilibrium were calculated by exact tests ( P 0.001) after Bonferroni correction; 0.01 6 0.571 0.791 0.701 0.0369 No 0.05 2 0.714 0.538 0.375 0.5105 No – 0.05 4 1.000 0.659 0.541 0.0909 No – 0.010.050.05 30.001 3 0.286 0.143 4 0.659 5 0.385 0.857 0.530 0.325 0.143 0.780 0.0270 0.0769 0.736 0.674 0.647 0.4139 Yes 0.0026 No No Yes – 0.001 8 0.857 0.923 0.841 0.5691 No 0.001 6 0.429 0.747 0.664 0.1165 Yes 0.0010.05 5 9 0.714 1.000 0.802 0.934 0.700 0.853 0.0290 1 Yes No 0.05 5 0.571 0.802 0.704 0.4598 No 0.05 6 0.857 0.835 0.744 0.6587 No 0.010.05 4 8 0.250 1.000 0.750 0.901 0.605 0.817 0.0286 1 Yes No 0.001 8 0.667 0.924 0.830 0.0905 No 0.001 6 0.000 0.879 0.791 0.0000* Yes < (HW), < > > < < > < < < < > > > < > < < (Null Alleles) P P , expected heterozygosity; E (HW) Null present H P PIC E H , observed heterozygosity; O H O 4); *, significant deviation from Hardy–Weinberg equilibrium ( H > A N A N Characterization of the 20 microsatellite markers for Tibetan ground tit from Dangxiong and Tianjun populations. , number of alleles; A cz21cz29 6 8 0.333 0.333 0.863 0.889 0.788 0.820 0.0028 0.0000* Yes Yes cz47a 5 0.556 0.739 0.650 0.1161 No cz154a 9 0.889 0.908 0.842 0.0256 No cz81 8 1.000 0.830 0.756 0.1320 No cz24a 3 1.000 0.582 0.448 0.0134 No – 10 0.571 0.923 0.844 0.0028 Yes cz32cz34cz44 4cz50 5cz73 0.222cz74 0.333 5 0.706 6 0.719 0.444 5 0.602 0.556 3 0.640 0.752 0.125 0.0038 0.686 0.000 0.0085 0.668 0.775 0.620 0.601 0.1002 0.679 Yes 0.0137 0.489 Yes 0.0029 0.0033 No No Yes Yes – – 4 6 0.714 0.429 0.626 0.802 0.520 0.715 0.1049 0.0359 No Yes – cz40 9 0.333 0.915 0.850 0.0031 Yes cz18cz19 4 7 0.222 0.778 0.725 0.824 0.624 0.747 0.0036 0.3155 Yes No cz70b 7 0.000 0.889 0.819 0.0000* Yes cz59b 4 0.875 0.758 0.658 0.2754 No cz7 11 0.889 0.915 0.852 0.2556 No Table 2. Locuscz1 5 0.200 0.822 0.701 Population (Dangxiong) 0.0042 Yes Population (Tianjun) cz74b 4 0.111 0.660 0.557 0.0033 Yes – indicates that the combined probability test could not be performed. chain method ( N

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Received 28 December 2012; in final revised form 25 February 2013; accepted 7 March 2013 Published on the Web: 29 July 2013

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