ARTICLE; AGRICULTURE AND ENVIRONMENTAL BIOTECHNOLOGY SRAP analysis on genetic relationships of genotypes in the genusMalusMill

Thirteen sequence-related amplified polymorphism (SRAP) primers combined with polymerase chain reaction (PCR) were used to evaluate the genetic relationships among 24 genotypes of the genus Malus Mill. including Sect. Malus, Baccatus, Sorbomalus and Chloromeles. Out of 115 surveyed fragments, 107 (93.04%) were polymorphic. Coefficients of genetic similarity ranged from 0.538 to 0.868, with an average value of 0.720 between pairs of materials, which indicated the high degree of polymorphism within this species. Cluster analysis showed that all genotypes were divided into six groups. A dendrogram showed that some of the clustered genotypes were largely congruent for geographical distribution. The species in Sect. Malus, Sorbomalus and Chloromeles were separated to a great extent. However, the DNA patterns for some genotype groups did not demonstrate relative agreement in their pedigrees. In Sect. Sorbomalus, M. yunnanesis was independently separated, while M. prattii were clustered in group III with M. bhutanica, M. platycarpa and M. fusca classified into Ser. Kansuenses. Five species originated from the American region were dispersed into two groups within the dendrogram. The conflicts were reflecting their complex genetic backgrounds.


Introduction
The genus Malus Mill. includes about 43 species of plant germplasm in the world at present. The species' genetic diversity originates and is centred on China, and is distributed mainly at two centralized locations, Xinjiang and Sichuan-Yunnan-Guizhou regions. There were about 27 wild species of Malus including 20 endemic species in China, with distributions held widely in different geographic positions. The status of wild genotypes was reflected by environmental factors and significantly varied. At the same time, the effects resulting from human activities also have their impact. Therefore, the features of these resources had made the mixed distribution of various species possible and gene interpenetration inevitable. Abundant genetic diversity was frequently observed among species, subspecies or variants. Researches on phylogeny and evolution of the genus Malus are considered hotspots in the long term. Studies concerning the morphology, [1] cytology, [2] enzymatics, [3] phytochemical properties [4] and other characteristics are still in progress. Random amplified polymorphic DNA (RAPD), simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) molecular markers were applied for analysis of the genetic diversity and were used to construct and identify the phylogenetic relationships in the genus Malus.
[5À7] However, some disputes in the classification system and genetic relationships still exist.
Sequence-related amplified polymorphism (SRAP) developed by Li and Quiros [8] is a technique that uses a set of designed primers to amplify the region-specific open reading frames (ORFs) in a particular gene. These polymorphisms occur due to the different lengths of the introns, the promoter and the spacer region in different individuals and species. Currently, SRAP had been successfully applied to analyse the genetic diversity, to build genetic maps and for cloning of genes.[9À12] The results provided strong evidence for the evolution of different species. Budak et al. [13] concluded that through the SRAP methodology better distinction results and more polymorphic sites could be obtained in comparison to the use of RAPD, SSR and inter-simple sequence repeat (ISSR) molecular markers within Buchloe dactyloides. Ferriol et al. [14] addressed that the use of SRAP was more consistent than AFLP in terms of morphology and evolutionary history for Cucurbita pepo. Ruiz et al. [15] detected genetic variation sites among tomato varieties by applying the SRAP markers while by SSR such were not identified.
Malus species play an important role in breeding, cultivation and germplasm utilization of plant resources around the world and many countries are very concerned about the wild germplasm resources. In order to address the problems related to the scientific area of genetic relationships between plant species and their phylogeny, *Corresponding author. Email: hudch78@163.com molecular biology would provide systematic evidence for evolution. In this study the SRAP technique was used to survey the genetic diversity and explore the genetic relationships among 24 genotypes including Sect. Malus, Baccatus, Sorbomalus and Chloromeles. The results aimed to provide an important theoretical basis for breeding, protection and utilization of the germplasm resources of the genus Malus Mill.

Plant materials in the study
The current study collected plant species representing 24 genotypes of the genus Malus Mill. from Yantai Academy of Agricultural Sciences (Shandong, China) Apple Germplasm Repository (Table 1), including 20 species (or subspecies) and 3 varieties. M. sieversii had two different genotypes, named as 'Xinjiangyepingguo' and 'Saweishipingguo'.

DNA extraction procedures
DNA was extracted using the cetyltrimethylammonium bromide (CTAB) method. The concentration and purity of DNA was determined by using an ultraviolet (UV) spectrophotometer and a 1.5% agarose gel electrophoresis. Total DNA was stored at À20 C.

SRAP-PCR
SRAP analysis was conducted according to previously established protocols. [8] In this assay, SRAP primers were synthesized by Shanghai Sangon Biological Engineering Technology and Service CO. LTD. The primer sequences are shown in Table 2. Seventy-seven pairs with primer combinations were obtained based on 7 forward and 11 reverse primers. The polymerase chain reaction (PCR) mixture in a total volume of 25 mL consisted of: 1X PCR buffer, 2.5 mM Mg 2C , 0.3 mM dNTPs, 0.3 mM primers, 1U Taq DNA polymerase (TaKaRa, Japan) and 30 ng of DNA. Amplifications were carried out in a MJ Researsh Thermocycler model PTC-100. The PCR profile was as follows: an initial denaturation at 94 C for 5 min; 5 cycles of 3 steps: 1 min for denaturation at 94 C, 1 min for annealing at 35 C and 2 min for elongation at 72 C; followed by 30 cycles with an annealing temperature increased to 50 C, and a final elongation step of 8 min at 72 C. PCR products were resolved by electrophoresis on 2% agarose gel in 0.5X TBE buffer stained with ethidium bromide. Bands were observed and recorded under UV light with the SYNGENE gel imaging system.

Data analysis
A binary matrix reflecting the presence (1) or absence (0) of each band was generated for each cultivar. After SM (simple matching coefficient) was calculated, the unweighted pair group method with arithmetic average (UPGMA) method was used with SAHN clustering program in NYSYS pc 2.10e to construct the dendrogram. The FIND module was used to identify all trees that could result from different choices of tied similarity or dissimilarity values.

Results and discussion PCR amplification
Out of 77 primer combinations, 13 primer pairs were best in terms of polymorphism level detection and were selected to survey the genetic diversity of the 24 genotypes of the genus Malus. The amplification results shown in Table 3

Cluster analysis
The similarity coefficients among the genotypes were computed by using NTSYS-pc2.10 (Table 4) Table 2. Primer sequences used for SRAP marker analysis.

No.
Sequence controversial. While some researchers insisted that M. bhutanica was part of the Sect. Sorbomalus based on its morphological characteristics, [16,17] Williams [4] considered it closer to the Ser. Baccatae according to the results from its flavonoid content, and an SSR analysis that supported the latter view. [18] In this assay, M. bhutanica and M. fusca were placed together the same group but divided in different smaller clusters. The obtained similarity coefficient was smaller with Ser. Baccatae; thus, it was considered by us that M. bhutanica should belong to Ser. Kansuensis in Sect. Sorbomalus, which was also consistent with the hypothesis of Shi et al.  Table 1.   are also in agreement with an isozyme analysis showing that the species of Ser. Yunnanensis in the Malus genus were more primitive. [19] Conclusions In this study, the genotypes with same parental blood were clustered together, and their similarity coefficients were higher, also reflecting the close relationship between them, such as with M. hupehensis var. mengshanensis and M. hupehensis, 'Shajinhaitang' and 'Duohuahaitang', etc.
The constructed dendrogram showed that some of the clustered biotypes were largely congruent in relation to their geographic distribution. The genotypes in Sect.
Malus, Sorbomalus and Chloromeles were separated to a great extent. However, the DNA patterns for some biotype groups did not demonstrate a relative agreement in their pedigrees. In Ser. Yunnanensis, M. yunnanesis was independently separated, while M. prattii was clustered in Group III with M. bhutanica, M. platycarpa and M. fusca classified into Ser. Kansuenses. Five biotypes that originated from the American Region were dispersed into two groups within the dendrogram. These conflicting observations reflect their complex genetic backgrounds. A follow-up research that includes combination with SSR, ISSR and other molecular markers can be carried out. Through a further increase in the number of markers and expansion in the scope of biotypes, a vaster theoretical basis addressing the Malus resources, genetic relationships and taxonomic status would be provided.