ESTIMATION OF GENETIC VARIATIONS IN DIFFERENT TAXA IN BRASSICACEAE BY RAPD AND ISSR ANALYSIS

Twelve species from Brassicaceae family were studied using two different molecular techniques: RAPD and ISSR; both of these techniques were used to detect some molecular markers associated with the genotype identification. RAPD results, from using five random primers, revealed 241 amplified fragments, 62 of them were polymorphic (26%). ISSR results showed that out of seven primers, three (ISSR3, UBC807, UBC811) could not amplify the genomic DNA; other primers revealed 183 amplified fragments, 36 of them were polymorphic (20%). The Similarity evidence and dendrogram for the genetic distances of the incorporation between the two techniques showed that the highest similarity was 0.897 between the varieties red cabbage and red ornamental cabbage, meanwhile the lowest similarity index was between the varieties red radish and Green ornamental cabbage (0.169); thus these RAPD and ISSR markers have the possibility for the identification of species or varieties and the description of genetic variation within the varieties. Furthermore, it could be concluded that the Brassicaceae taxa have a suitable amount of genetic variance and a wide range in the genetic principle of the studied genotypes which can be used for output improvement.


INTRODUCTION
Although many of families in Flora of Iraq have been examined morphologically and phytochemically in some detail (Harborne et al., 1971), there are many open inquiries concerning the classification of particular genera within tribes and subfamilies.Molecular information enhances or even permits the illustration of phylogeny, and provides the fundamental information for comprehension taxonomy, breeding, and advancement of plants.Molecular techniques are being used increasingly in plant systematic (Soltis et al., 1992).In addition; the improvement of PCR-based molecular marker manner has prompted expanding the utilization of molecular marker technologies in many fields of science, including systematic studies.In this relation randomly amplified polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) techniques have drawn much attention in a wide variety of organisms, especially in plants.Therefore, this study chooses the Brassicaceae family because it is typical of families to compare it with other molecular and evolutionary studies (Franzke et al. 2011) for the following reasons.Firstly the consideration of Arabidopsis thaliana (L.) Heynh.which is a protrude model amongst the most vital plants in molecular studies (Meinke DOI: http://dx.doi.org/10.26842/binhm.7.2018.15.1.0001 et al. 1998) as well as many studies on Brassica spp.; secondly it investigated for different sides of botany, for example, biotic and abiotic stress tolerance, genome development (Vekemans et al. 2014), thirdly it encountered entire genome duplications and organismal radiation in its underlying formative history (Edger et al. 2015).
Mustard family (Brassicaceae or Cruciferae) is a fourth large and natural family which can be easily morphologically diagnosed by scrupulous flowering Actinomorphic form, Cruciform corolla and Silique fruits.It has a global apportionment predominately in the moderate area of the northern hemisphere (Al-Shehbaz, 1984).Most of the members of the understudied family are economically important and include vegetables such as cabbage, cauliflower and broccoli, oilseed from Brassica napus L., and B.rapa L., fodder, ornamental mainly in B.oleracea var.acephala (ornamental cabbage), and condiment plants like Brassica nigra (L) W.D.J.Koch.and B.juncea (L.) Czern, (Gomez-Campo and Prakash 1999).
Brassicaceae are divided into three to nineteen tribes and twenty to thirty subtribes.The phylogeny and ranking of the familial level like genera and tribe are still suspicious (Appel and Al-Shehbaz, 2003).This obstruction was performed in a lack of understanding of the number and limits of phylogenetically established tribes and genera and gave rise to various distinct classification systems which have been submitted previously (Warwick et al. 2010).Later, Al-Shehbaz (2012) estimated the number of tribes as approximately 51 which included 321 genera and 3660 species.Description of the plant with the utilization of molecular markers is a typical way to memorize plant genetic assets and molecular depiction helps to determine the breeding behavior of species (Prasad, 2014).In this way the objective of the present investigation is to estimate of degrees of relationship between different varieties and species of different taxa within the Brassicaceae by using random primers in RAPD and ISSR techniques a further important use of these markers are to distinguish between genotypes

MATERIALS AND MATHODS
Plant materials and extraction of genomic DNA: Twelve taxa which used in this study are listed in Table (1) and Plate (1).Fresh leaves, clean and free from dirt and bacterial and fungal infections were selected then treated with liquid nitrogen carefully in mortar and pestle until being a fine powder.Genomic DNA was isolated from leaves according to the method protocol of the Genomic DNA Mini Kit (Geneaid Biotech.Ltd; Taiwan Company).
Polymerase chain reaction: 25 µl a final volume of amplification reactions containing 5 µl of DNA, 1µl of primer (50 pmol), 12.5 µl of the master mix including (dNTPs, PCR buffer, Taq DNA polymerase (5U/µl), MgCl2) and 6.5µl deionized water.PCR reaction was carried out in Thermal cycler PCR System (Verity, Applied Biosystem).The subsequent different trials for upgrading the best fitting conditions, a program for PCR response was standardized with following settings according to Table (3).Then amplified DNA were separated by electrophoresis in 1.3 % agarose gels (stained with 0.3 µl of ethidium bromide at 3-4 hr on 70V).Data analysis: RAPD and ISSR markers produce DNA amplification signals that can be changed over into a measurement of similarity or dissimilarity DNA electrophoretic pattern containing visible bands at a specific position in the individual lane.The banding patterns were transformed into binary characters where the appearance of a band was given the number (1) while the absence of the band was given the number (0).
A square symmetric matrix of similarity was acquired using Jaccard's similarity coefficient to draw the dendrograms.The unweighted pair group method with arithmetic average (UPGMA) was applied for cluster analysis using past software ver.1.92 (Hammer et al., 2001), and calculated the percentage of polymorphism, efficiency and discriminating power of primer as the following equation: Polymorphism% = (No. the polymorphic bands of random primer / the total number of bands of the same primer) × 100.
Efficiency of primer =(No.the polymorphic bands to each primer / total number of bands to the same primer) × 100 Discriminating power of primer = No. the polymorphic band to each primer /total number of the polymorphic band to all primer X 100 %. according to (Grudman et al., 1995).

RESULTS AND DISSCUSION
Genetical variation indicates any alteration in nucleotides, gene, chromosomes or entire plant's genome (Kurane et al., 2009).This variation in DNA sequence (polymorphism) can result in different banding patterns which are assessed by agarose gel electrophoresis (Williams et al. ,1990).Thus, twelve primers were used to twelve individuals of Brassicaceae family for DNA amplification.The outcomes demonstrated various primers made distinctive fragment numbers and length of DNA amplification products as seen in Table ( 4) and ( 5).
An overall of 413 polymorphic amplified products was gained from five RAPD primers and seven ISSR primers distributed into 241 and 183 bands respectively.The total number of the amplified RAPDs produced by each primer varied from a minimum number of 21 amplified products by primer OPC14 to a maximum of 76 amplified products by primer OPC8.The polymorphic fragments number varied between 12 in OPC2, OPC8 and OPB18 to 13 in OPC14 and OPB11 from total 62 fragments were polymorphic thus generating 26% polymorphism and found the lower value of Primer efficiency is 9% in OPC14 and higher value is 32% in OPC8, also the results appeared the same value of primer discriminatory power in all RAPD primers approximately 19% except primer OPC14 is 21%.(Tab.4 and Pl. 2).
The ISSR profiles of the amplification products showed out of seven primers, three (ISSR3, UBC807, UBC811) could not amplify the genomic DNA, The primer ISSR1 accord the lowest number of fragments ( 11), while the largest number of fragments ( 67), was amplified with primer BH11.The minimum number of polymorphic bands was 5 with BH10, which appeared the polymorphism (8%) and 6 polymorphic bands in ISSR1 that represented (55%), BH11 and BH14 showed the largest number of polymorphic bands 12 and 13 in 18% and 33% respectively.And converged percentages of the efficiency of the primers BH10 and BH11 by 33% and 36%, and decreased ratio to 22% in BH14.On the contrary, the Primer discriminatory power in the last primer has a higher value to 36% but a lower value was 14% in BH10.It is remarkable that all used primers do not contain monomorphic bands except the primer BH10 which explained three bands have the same genotype (Tab.4 and Pl. 2).Also from Table (5) and Diagram (1) explained maximum number of polymorphism bands appeared in Green Cabbage and Green ornamental cabbage but minimum bands in Red and White radish, Some polymorphisms were easy to register whereas other bands appeared to produce nuclear fragments (Williams et al. ,1990).The best primers will output more than three clear bits.The number of fragments produced depends on the primer sequence rather than the nucleotide length.In general, show spacious whilst various studies have reported that of the vegetable Brassicas parental lines have emerged from a limit genetic base (Gray, 1993).Furthermore, Kurane et al. (2009) emphasis the ISSR markers proved to be very useful for accurate plant identification by recognized the Intra and Interspecies difference.ISSR strategies are almost indistinguishable to RAPD strategies exclude that sequences of ISSR primer are non-randomly outlined from microsatellite regions and the annealing temperatures applied are higher than utilized for RAPD markers.A difference of results was normal since just seven ISSR primers were utilized against five primers in the RAPD data analysis; notwithstanding, the average number of bands amplified per ISSR primer was higher.In this screening, RAPD markers were effectively exercised to distinguish 12 taxa of Brassicaceae from each to another and data from molecular markers display a good basis for better conservation approaches (Prasad, 2014).Thus, on the basis of RAPD, the discoveries of this investigation are closely resembling the notice of Hu and Quiros (1991) were demonstrated the use of RAPD-PCR in comparison of broccoli and cauliflower cultivar.An indistinguishable outcome was expressed in the dendrogram where two noticeable bunches were gotten The tree-cluster analysis explains the allocation of genotypes in two main clusters and genetic similarity among the 12 species ranged from 0.169 to 0.897 (Diag.2).Cluster I which was divided into group and subgroup.The first group included three subgroups, Red Cabbage and Red ornamental cabbage were represented as the first sub-group, The second sub-group consisted of genotypes labeled as Cauliflower and Broccoli, and Third sub-group included Green Cabbage and Green ornamental cabbage.Then Cress or RASHAD and Wild radish combined together with the first group, Then QUNAIBRA and Rocket Mustard attached with the previous group respectively.Cluster II comprised of two genotypes including White and Red radish.Thus the dendrogram showed the genetic relationships among species Brassica seem very closely related together which return to Brassiceae tribe.For the results of the current study agreed with the study Yang et al. (1999) that Confirmed the genus Brassica is a monophyletic group within the Brassicaceae very closely related to model crucifer plant Arabidopsis thaliana except their genomes are more complex because of nature of polyploidy.Also the combined RASHAD which belong to Lepidieae tribe with the Brassica group concurred with Zunk, et al. (1999) that indicated the current molecular systematic research reveals that exclusive Brassiceae and Lepidieae's tribes might to be counted a naturalistic composition.so the White and Red radish seem closely related together in the tree relationship because they have the same morphological and taxonomic feature and agreed with Cruz et al. (2014) when demonstrated that RAPD and ISSR biochemical and molecular markers are effective and promising when differentiating cultivars of the radish Details of RAPD and ISSR band sharing in twelve species and varieties in Brassicaceae.

Table ( 1
): Species and varieties included under study from Brassicaceae family with common names and tribes.

Table ( 2
): List of primers are used in RAPD and ISSR techniques and their sequences.

Table ( 4
): Details of RAPD and ISSR amplifications in twelve species and varieties in Brassicaceae.