Jia Tian1, Bin Zeng1, Mengwen Liu1, Shuping Luo2, Xiugen Li3, Jiang Li1*

Development of polymorphic microsatellite markers for the wild ‘Duli’ pear (Pyrus betulaefolia Bunge)

Jia Tian1, Bin Zeng1, Mengwen Liu1, Shuping Luo2, Xiugen Li3, Jiang Li1*

1Collage of Forestry and Horticultural, Xinjiang Agricultural University, Urumqi, 830052, P.R.China

2Collage of Agronomy, Xinjiang Agricultural University, Urumqi, 830052, P.R.China

3Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou, 450009, P.R.China

E-Mails:

(J. T), (B. Z), (M.-W. L), (S.-P. L), (X.-G. L), (J. L).

*Corresponding author: Jiang Li

Tel: +86-0991-8762363

Fax: +86-0991-8762363

Abstract

Wild Pyrus betulaefolia Bunge in China is threatened by excessive land reclamation and utilization. To facilitate studies of wild P. betulaefolia Bunge biodiversity, 150 microsatellite markers were developed by the whole-genome sequencing data of P. bretschneideri Rehd and tested in eight individual trees from three provinces. There were 25 polymorphic SSRs with four to eight alleles. The polymorphic information content (PIC) ranged from 0.371 to 0.864. The observed (Ho) and expected (He) heterozygosities ranged from 0-0.750 and 0.492-0.875, respectively. These microsatellite markers can improve strategies and conservation measures for wild P. betulaefolia Bunge diversity hotspot.

Keywords: Wild Pyrus betulaefolia Bunge; SSR; genetic biodiversity

Wild ‘Duli’ pear (Pyrus betulaefolia Bunge) with developed root system, belonging to the Rosaceae, Maloideae, is mainly distributed in northern China and has strong resistance. Moreover, it has well grafting affinity with Asian pear and European pear, and is the source of good characteristics of Pyrus plants as well as the important part of biodiversity. With the impacts of global warming, excessive land reclamation and utilization, the genetic diversity of the wild P. betulaefolia Bunge is suffering serious threat (Zong et al., 2013). To conserve this species, microsatellite or simple sequence repeat (SSR) markers are required to facilitate genetic characterization.

To develop and characterize markers, 8 individuals of wild P. betulaefolia Bunge were collected in Liaoning, Shandong and Gansu provinces, China. The genomic DNA were extracted from young leaves using the CTAB method described by Bokszczanin (Bokszczanin and Przybyla, 2008). 150 primers were designed based on the whole-genome sequencing data of Pyrus bretschneideri Rehd (Wu et al., 2013).

All the primer pairs were validated using the M13-tail technique by polymerase chain reactions (PCRs), which assessd the polymorphism of the amplified products. Three primers including 5' M13-tailed forward primer, reverse primer and fluorescently labeled M13 primer were synthesized in the genotyping experiments. Fluorescently labeled M13 primers carrying four fluorescent labels (FAM, HEX, TAMARA, and ROX) were used independently. PCR was carried out in a 10 μl volume containing 20 ng DNA, 0.4 pmol forward primer, 1.6 pmol reverse primer, 1.6 pmol fluorescently labelled M13 primers and 2×PCR Master Mix (New England Biolabs) under the following conditions: 94℃ for 5 min; 25 cycles at 94°C for 40 s, 55°C for 45 s, and 72°C for 40 s; 10 cycles at 94°C for 40 s, 53°C for 45 s, an 72°C for 40 s; and a final 7 min extension at 72°C. The products of PCR were analyzed by the ABI 3730 Prism Genetic Analyser (Applied Biosystems, Foster City, CA, USA).

144 of 150 loci showed the strong expected fragment size, while other loci failed to amplify any product. Through the test of capillary electrophoresis, of the 144 loci, 94 showed clear, single peaks for each allele, of which 25 were polymorphic and 69 were monomorphic.

The raw data was analyzed with GeneMarker ver. 2.6.3 (Soft Genetics, State College, PA, USA), and the number of alleles, observed heterozygosity (Ho) and expected heterozygosity (He) were calculated with GenAlEx ver. 6.5 (Peakall and Smouse, 2012). The number of alleles for these 25 SSRs per locus ranged from four to eight alleles. Ho and He ranged from 0-0.750 and 0.492-0.875, respectively. The polymorphism information content (PIC) of each locus was calculated with Cervus ver. 3.0 (Kalinowski et al., 2007) and ranged from 0.371 to 0.864 (Table 1). 25 microsatellite loci target sequences are showed as supplementary material (SM1)

The 25 polymorphic loci developed in this report can be used to facilitate estimation of genetic diversity and population genetic structure in wild Pyrus betulaefolia Bunge, as well as to develop a conservation strategy for this species.

Acknowledgments

We thank Dr. Quanxin Bi for assistance in experiment. We also thank Dr. Jianfeng Mao for analyzing Illumina sequence data, and his helpful discussions. This work was supported by the National High Technology Research and Development Program of China (No. 2011AA10020604), the Pomology Major Projects of Xinjiang Uygur Autonomous Region (No. 201130102–1) and the Special Research Projects of National Forestry Industry of Public Benefit (No. 201304701–1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

Zong Y, Sun P, Niu Q F et al (2013) Distribution situation and assessment of morphological diversity of wild Pyrus betulaefolia in Northern China. Journal of Fruit Science 30(6): 918-923.

Bokszczanin K, Przybyla, AA. (2008) New simple and efficient method of DNA isolation from pear leaves rich in polyphenolic compounds. International Journal of Horticultural Science 12(4): 21–24.

Wu J, Wang Z W, Shi Z B et al (2013) The genome of the pear (Pyrus bretschneideri Rehd.). Genome Research 23: 396-408.

Peakall R, Smouse PE (2012) GenAlEx 6.5:genetic analysis in Excel. Population genetic software for teaching and research-an update. Bioinformatics 28: 2537-2539.

Kalinowski ST, Taper ML, Marshall TC (2007) Revising how the computer program CERVUS accommodate genotyping error increases success in paternity assignment. Mol Ecol 16: 1099-1106.