An ISSR-based genetic diversity analysis of Malus sieversii in Tienshan Mountains in Xinjiang, China and Kyrgyzstan

Malus sieversii constitute a valuable genetic resource in wild apple ecosystems. The aim of this study was to use inter-simple sequence repeat (ISSR) primers as an accessible tool to investigate the genetic diversity in Malus sieversii species. The experimental materials include 34 samples from Kyrgyzstan and Xinjiang of China. A total of 125 bands and 98 polymorphic bands were amplified using 47 ISSR primers. The polymorphism rate was 78.4%. The genetic similarity coefficient of Kyrgyzstan and Xinjiang of China population was 0.68; the genetic similarity coefficient of various populations in Xinjiang was 0.72~0.94. The samples in same population got into a category, but some samples in faraway geographic locations have cross clustering. Geographical isolation hindered the gene exchange of Malus sieversii in different populations for a long time, and Malus sieversii developed along the natural selection environment direction and generate genetic differentiation after that.


Introduction
Malus sieversii (Ledeb) Roem. is also known as Tienshan or Xinjiang wide apple, which is not only precious tertiary relict species [1], but also an important part of the world apple gene pool. Page: 41 www.raftpubs.com classification and so on. Among genetic diversity research, polyphenols [3,4] and fruit volatile compounds [5,6] has been studied. However, information regarding their genetic diversity remains poor.

Materials and Methods
The experimental materials included 34 samples representing 6 populations that distributed in Kyrgyzstan and China, which were collected from May 2012 to July 2013, including 6 materials from Balykchy and Karakol of Kyrgyzstan and 28 materials from Xinjiang of China(the geographical location of the samples sites is shown in Figure 1 and Table 1). All populations are wild ancient wild M. sieversii distribution areas, and there is no distribution of cultivated species. All samples were taken from selected trees, which we survey and make a record every year. The distance of two populations was more than 60 kilometers. Choose appropriate amount of leaves without plant diseases and insect pests and make a record of the sampled tree with GPS system. The leaves were kept in Hermetic bag with silica gel, which is replaced every two days until completely dry. The samples were ground with PVP (Polyvinyl Pyrrolidone) and dissociated with 10%CTAB (Cetyltrimethyl Ammonium Bromide) and 0.7M NaCl in the supernatant, deposited with C2H3NaO2 (Sodium Acetate ) and precooling C3H8O (Isopropyl Alcohol). The concentration and purity of DNA were assessed with Nanodrop 2.1 and 0.8% agarose gel electrophoresis. A total of 47 ISSR primers sequences were selected on the basis of University of British Columbia Biotechnology and synthesized by BGI (Supplementary Table  1). The ISSR-PCR reaction system used in the present study followed the technique described by Weisheng [27] : Each 20μl reaction solution included 14.4μl double distilled water, 2.0μl 10×buffer (including Mg 2+ ), 1μl primer (10nM), 1μl DNA template (200ng/μl), 0.5μl dNTPs (2.5nM), 0.1μl TaqDNA polymerase (5 units/μl). Amplification procedure were as follows: Initial denaturation at 94℃ for 5min; followed by denaturation at 94℃ for 30s, annealing at 50℃-60℃ (depending on the primer) for 1 min, extension at 72℃ for 8 min, 35cycles; 72℃ extended 7 min; Save 4℃. Page: 43 www.raftpubs.com America). All of the above repeated three times. Clear bands were marked with "1", and others marked with "0". The data construct binary data matrix in Microsoft Excel, and did clustering analysis with UPGMA method of NTsys2.1 software. Genetic similarity coefficient (GS) were calculated according to the formula NXY/GS=2 (NX + NY), where NX and NY was amplified fragments number of strains X and Y,NXY is the common bands of two strains.

ISSR polymorphism analysis
We identified 7 efficient ISSR primers with polymorphic from 47 ISSR primers. The criteria for selection of markers as follows: clear bands were marked with "1", and others marked with "0". By statistics, every primer could amplificated 9-26 bands and the average number of bands is 14. Among them, the primer (TG)8AC26 amplificated the most loci for 26. The primer (AT)8TC amplificated the least loci for 9. All amplified fragment sizes were between 200 and 1500bp. The highest percentage of polymorphic is primer (CA)8AT and (CA)8GT followed 100%. The primer CATGGTGTTGGTCATTGTTCCA polymorphic percentage is 82.3%, and a minimum of polymorphic percentage is 44.4% of (AT)8TC (Table 2). 7 primers amplificated 125 loci in total. The polymorphism loci was 98, and polymorphism percentage is 78.4%. Figure 1 showed the ISSR-PCR result of 11 Malus sieversii electrophoresis with (CA)8GT and CATGGTGTTGGTCATTGTTCCA.

Discussion
Kyrgyzstan is located in the northeast of central Asia, which east and southeast is China. Malus sieversii distributed in XinJiang of China along the Tienshan and Kyrgyzstan and Kazakhstan and so on. In this study, the results show that Malus sieversii of Kyrgyzstan and XinJiang together into their respective category, and both genetic similarity coefficient was 0.72. Genetic similarity coefficient of above is lower than that inside of population, which indicated Malus sieversii of Kyrgyzstan and XinJiang have relative relationships. These populations may be existed in a similar form before, but they produced geographical isolation after the change of geographical environment, such as the block of Tienshan. Durable geographical isolation cut off genetic exchange, which made the populations use different resources to grow and reproduce in their respective geographic areas, and occurred genetic differentiation. Therefore, to some extent, Tienshan plays a key Xinjiang is broad, and the distance between YiLi and TaCheng is more than 700 kilometers. In this study, the majority of samples within a region belong to a class, such as EY-2 and EY-3, TY-3 and TY-4, XY-5 and XY-6. HR from XinYuan with XY-5, XY-6 together into a category; W-1 (The oldest Malus sieversii) and W-2 (The second oldest Malus sieversii) from XinYuan respectively with GY-3 and XY-9 together into a category. Both of XinYuan and GongLiu belong to Yili region, which geographic distance is closer than that between TaCheng and Yili. So, they have more opportunities to exchange genes, and facilitated higher genetic similarity coefficient and closer relatives. TuoLi and NaLaTi respectively belong to TaCheng and YiLi prefecture and geographic distance is far, but TY-2 and NY-2 together into a category and the genetic similarity coefficient is 0.88. This result may be connected with complex environment conditions such as the spread of insects, the influence of climate and human factors. Genetic diversity reflected the difference of genetic basis and genetic improvement. High genetic diversity is the basis of maintaining the longterm survival of species [28]. The genetic similarity coefficient of all Malus sieversii resources in the study is 0.68 to 1.00, which provides a certain theoretical basis for the development and utilization of new varieties, and lay the foundation for the germplasm resources protection or utilization.

Conclusion
ISSR molecular marker showed a higher level of polymorphism genotype differences, which was suitable for genetic diversity analysis and genetic relationship of Malus sieversii. ISSR-PCR has cleared about genetic similarity coefficient of Malus sieversii in different geographical area, which was advantageous to Malus sieversii germplasm resources utilization and protection, genetic research and breeding. The clustering analysis of Malus sieversii showed that the geographical isolation block gene exchange, and made each isolated population have firmly genetic stability. So, they developed along the appropriate direction in the natural selection of environment and rich genetic diversity all the time (Supplementary  Table 1 54℃  ISSR5  UBC 801  AT AT AT AT AT AT AT ATT  36℃  ISSR6  UBC 802  AT AT AT AT AT AT AT ATG  38℃  ISSR7  UBC 803  AT AT AT AT AT AT AT ATC  38℃  ISSR8  UBC 804  TA TA TA TA TA TA TA TAA  36℃  ISSR9  UBC 805  TA TA TA TA TA TA TA TAC  36℃  ISSR10  UBC 806  TA TA TA TA TA TA TA TAG TG TG TG TG TG TG TG TGA  50℃  ISSR21  UBC 829  TG TG TG TG TG TG TG TGC  52℃  ISSR22  UBC 830  TG TG TG TG TG TG TG TGG  52℃  ISSR23 UBC 832 AT AT AT AT AT AT AT ATTC 38℃ ISSR24 UBC 833 AT AT AT AT AT AT AT ATTG 38℃ ISSR25 UBC 840 GA GA GA GA GA GA GA GATT 52℃  ISSR26  UBC 847  CA CA CA CA CA CA CA CAGC  56℃  ISSR27  UBC 851  GT GT GT GT GT GT GT GTCG  56℃  ISSR28  UBC 854  TC TC TC TC TC TC TC TCAG  54℃  ISSR29  UBC 855  AC AC AC AC AC AC AC ACCT  52℃  ISSR30  UBC 856  AC AC AC AC AC AC AC ACTA  52℃  ISSR31  UBC 857  AC AC AC AC AC AC AC ACCG  56℃  ISSR32  UBC 858  TG TG TG TG TG TG TG TGAG  54℃  ISSR33  UBC 859  TG TG TG TG TG TG TG TGAC  54℃  ISSR34  UBC 855  TG TG TG TG TG TG TG TGAA  52℃  ISSR35  UBC 861  ACC ACC ACC ACC ACC  50℃  ISSR36  UBC 862  AGC AGC AGC AGC AGC  50℃  ISSR37  UBC 863  AGT AGT AGT AGT AGT  40℃  ISSR38  UBC 864  ATG ATG ATG ATG ATG  40℃  ISSR39  UBC 865  CCG CCG CCG CCG CCG  60℃  ISSR40  UBC 866  CTC CTC CTC CTC CTC  50℃  ISSR41  UBC 872  GATA GATA GATA GATA  40℃  ISSR42  UBC 874  CCCT CCCT CCCT CCCT  56℃  ISSR43  UBC 875  CTAG CTAG CTAG CTAG  48℃  ISSR44  UBC 876  GATA GATA GATA GATA  40℃  ISSR45  UBC 892  TAGATCTGATATCTGAATTCC  50℃  ISSR46  UBC 899  CATGGTGTTGGTCATTGTTCCA  56℃  ISSR47  UBC900  ACTTCCACAGGTTAACACA  47℃