Detection of genetic relationships among some species belongs to genus Malus from mid Iraqi regions

RAPD-PCR method was used for systematic study and revealing the genetic relationships in Malus by using 7 apple cultivars from some mid Iraqi regions, DNA of fresh leaves was extracted using modified protocol of CTAB, 22 prechosen random decamer primers were applied to detect Malus genotypes. four primers gave reproducible and appeared polymorphism in the RAPD profile, a total 48 bands were produced out of which 36 bands were polymorphic, the results arise two main clusters, the first one included Malus sylvestris has unique amplified and discriminated from other Malus taxa, thus it can be a good molecular tool for taxa identification which separated at the similarity value of 0.48, and the second cluster contained two groups, one included M. domestica , and M. domestica var. ralls janet which appeared as closely related species with a stronger correlation at similarity range of 0.09, furthermore, it considered that the present study identifies reservoir of alleles that useful for breeding programs in parental crosses.


Introduction
The genus Malus (apple) has been considered as a one of the important fruit cultivated within Rosaceae family, also it has been characterized by broad diversity, the interspecific hybridization and breeding gene pools among this fruit groups and their wild relatives have probably have main roles in the evolution of the Rosaceae 1 , 2.This has caused to produce individuals with intermediate phenotypes and genotypes characters 3, Hybridization activities has led to add or reduce taxa according to commercial roles, this might have caused to change the genetic identify and complicated taxonomic relationships within this family 4,5,6,7 Therefore, clearing relationships, taxonomy, and diversity is important for developing breeding strategies, conserving biodiversity, and improving breeding efficiency.Also understanding genetic variability in Malus is critical for characterizing germplasm, controlling genetic erosion and the registration of new cultivars 8 , 9.Many genetic studies at Pomoideae like 10, 11, 12, 13, 14, 15, 16, 17, 18 ,19 mention that the Malus has been economic significance and large geographical distribution but the origin of it unclear.References listed one species of Malus in flora of Syria, Palestine, Sinai, Iraq, Iran, and Turkey 20, 21, 22, 23, while24 were mentioned 12 species of Pyrus in New Delhi flora belonged to Pomoideae subfamily.Molecular evidence have become forceful tools to identify the hybridization and evolution processes, as well as provide a basic background knowledge to implement conservation genetics programmers, mutation rate or in dominance characteristics 3, 25 ,26 The random amplified polymorphic DNA (RAPD) have been vastly used in DNA fingerprinting gene mapping as a reliable, fast, and simplest technique and to isolate phylogenetic relationships of many organisms taxonomy 27 , 28, and within many plant families as a molecular technique for cultivar identification 29 and 30.Other molecular markers (amplified fragment length polymorphism (AFLP), simple sequence repeat (SSR), inter simple sequence repeat (ISSR) also have been used to analyzing the genetic diversity into Malus species such as 3, 11, 12, 13, 31, 32 , 33.On the other hand chloroplast and mitochondrial DNA and minisatellite (M13) probes have also been used for DNA fingerprinting in several Rosaceae family 34 , 35.The objectives of this study were investigated and identify the level of genetic diversity among apple cultivars, systematic analysis using RAPD technique as reliable molecular markers of these taxa.

Materials and Methods
1. Plants materials: Seven taxa of Malus genus were collected during April 2015 from different Iraqi regions included Salmanpak and Alsiafiyah south east Baghdad, (Baquba) Dyiala, and (AlHindia) Babel.All taxa present in table 1.

DNA extraction:
Approximately 50 to 100 mg of young fresh leaf tissue put in 1.5 ml tube, after homogenizing the tissue using liquid nitrogen with a conical hand tissue grinder.Total genomic DNA was isolated from fresh leaves using modified of cetylrimethyl ammonium bromide (CTAB) method of 36, the powder suspend in 2.5 ml of CTAB extraction buffer (1.4 M NaCl, 2% CTAB) and 5ml of B -mecaptoethanol.The suspension was mixed well, ad put incubated at 60ºC for 20 min to homogenate, followed by chloroform: isoamyl alcohol extraction (24:1), and precipitation with two volume of isopropanol at -20ºC, then bring down the sample formed after centrifugation for 5min, was washed with 1ml of 70% ethanol and 10m M of ammonium acetate, the DNA by TE buffer (20m M EDTA, 0.1 M Tris-HCL p H= 8), for detection of the DNA samples that were electrophoresed in 1% agarose gel and stained with 0.5 mg/ ml ethidium bromide, with 1500 bp ladder (Sib Enzyme Ltd.Russia) with 100 v for 45 min visualized and photographed under a UV transilluminator 37.

Screening of PCR :
Twenty two different 10mers RAPD primers were tested in this study (table 2) which supplied by Bioneer company were screened, four primers which had previously been shown indicated results of band patterns, multi master mix were used, the thermo profile for the PCR reaction was: 95°C for 5 minutes, then 35 cycles of 95°C for 30 sec, 37°C for 1 minute, and 72°C for 5 minute.Genotypes were visualized on 1% agarose gel, 1x TBE, 100 volts, for 55 minutes and scored as 1 or 0 based on presence or absence of a band, within the size range of 150-1800 base pairs (bp).

Results
48 RAPD bands were produced using the 4 primers, 36 (75 %) of which were polymorphic.The apple was shown to exhibit high interspecific polymorphism due the nature of cross-pollination in that culture genotypes.
The number of RAPD bands varied from 8 (the primer OPC-13) to 18 (the primers OPK-14) (table 3).Thirty six polymorphic bands were obtained, Some representative polymorphisms revealed by RAPD primers were presented in table 3, The dendrogram shown the genetic relationships among the 7 apple genotypes (Figure 2) showed that apple taxa were essentially divided into 2 main clusters, The two main clusters separated at the similarity value of 0.48, the 1st (cluster I) contained two groups with similarity value 0.40 which separated into 2 clades, (IA) containing M. pumila, and the second clade (IB) that separated to 2 subclades with value similarity of 0.23, (IB1) divided to 2 groups 2 taxa isolated from M. pumila var domestica with 0.21 similarity value, were M. domestica, and M. domestica var ralls janet with showed highest similarity range among taxa under study which was 0.09, while (IB2) including 2 taxa were M. sieversii and M. orientalis with 0.15 similarity, cluster (cluster II) consisted of Malus sylvestris according to the dendrogram linkage joining rule were more distantly related and separated from the (IA) group.

Discussion
The fingerprinting analysis with RAPDs showed a high genetic diversity among taxa under study, generally we can predict that a strong relationship within taxa characterized by high level of polymorphism due to vegetative propagation.One of the reasons that led to high level of polymorphism in fruit tree species they appear due to the high somatic mutation rates of different traits associated with sequences repetitive and constitutes an improvement over the basic genotype

Conclusion
The result indicate a high degree of correlation among studied taxa according reliable method of analysis that used in the present study provided an effective tool to understand evaluating germplasm material, or gene flow in order to identify the species that could be further evaluated.

Figure 1 .
Figure 1.Spectrum of DNA amplification products of 7 taxa of genus Malus (table 1), molecular weight marker 1500-bp DNA Ladder

Table 3 . Total number and size range of amplified bands obtained for each primer.
The outcome of RAPD-PCR analysis and index matrix based on all DNA bands that isolated by four primes observed the strongest homogeneity between M. domestica, and M. domestica var ralls janet, so it can be considered as a reservoir of alleles useful for breeding , due divergent genotypes may has a reliable breeding value 42, or has substitution rates and high levels of gene rearrangements, furthermore the origin of the cultivated species M. x domestica is an urgent taxonomic problem for apple breeding13.ccordingto US apple association the M. pumila var domestica progeny from M. domestica, and M. domestica var ralls janet.As noted by our results that species M. sieversii and M. orientalis were assumed to be ancestors of the domestic species, these apple are sweet and large 43.This assumption was supported by RFLP analysis 44 However, some authors assume M. sylvestris and M. pumila may also be ancestors of the domestic apple 31 , 45.Results showed that the M. sylvestris is genetically close to the M. orientalis and M. sieversii respectively, but there are genetic distance between in M. sylvestris and M. sieversii, this outcome against with 13 when he found the smallest genetic distance was between the M. sylvestris and M. sieversii.Moreover, our results deviate in part from those of another study 29 , 47 they regarding the identity of taxon but also by environmental differences such us geographic location, The Malus sylvestris indicated its unique banding form over the rest taxa, the rustles ensured it"s characterized by specific genotype, this lower genetic variability in this commercial hybrid compering with another taxa which is has narrower origin of hybrid and genetic erosion because intensive breeding 48.Actually hybridization, introgression, and migration with in species, notably out crossing and openpollinated these character genetically a great diversity of phenotypes, most of them being intermediate patterns 27.