Bull 1 Huda Jasim M. Al-Tameme Bull. Iraq nat. Hist. Mus. July, (2018) 15 (1): 1-13 ESTIMATION OF GENETIC VARIATIONS IN DIFFERENT TAXA IN BRASSICACEAE BY RAPD AND ISSR ANALYSIS Huda Jasim M. Al-Tameme Department of Biology, College of Science for Women, University of Babylon, Babylon, Iraq. Corresponding author: huda_jasim@yahoo.com Received Date: 13 December 2017 Accepted Date:09 January 2018 ABSTRACT 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. Keywords: Brassicaceae, Genetic similarity, ISSR, Polymorphism, RAPD. 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 2 Estimation of Genetic Variations in Different Taxa 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) and 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) has 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 the 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, were cleaned and triturated 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). Checking of the RAPD and ISSR primers: The amplification reactions were performed by five decamer oligonucleotide primers from (OPC2, OPC8, OPC14, OPB11 and OPB18 for RAPD analysis) and seven oligonucleotide primers (BH10, BH11, BH14, ISSR1, ISSR3, UBC807 and UBC811 for ISSR analysis ) (Tab. 2). 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). 3 Huda Jasim M. Al-Tameme 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). Table (1): Species and varieties included under study from Brassicaceae family with common names and tribes. No. Scientific name Common name Tribe 1. Raphanus sativus var. longipinnatus L. H. Bailey White radish Brassiceae 2. Raphanus sativus L. Red radish Brassiceae 3. Raphanus raphanistrum L. Wild radish or FIHAILA Brassiceae 4. Lepidium sativum L. Cress or RASHAD Lepidieae 5. Cardaria draba (L.)Desv. Hoary Cress or QUNAIBRA Lepidieae 6. Sisymbrium irio L. Rocket Mustard Sisymbrieae 7. Brassica oleracea var. botrytis L. Cauliflower or QARNABÎT Brassiceae 8. Brassica oleracea var. italic L. Broccoli Brassiceae 9. Brassica oleracea var. capitata L. Green cabbage-LAHANA Brassiceae 10. Brassica oleracea var. capitate L. Red cabbage Brassiceae 11. Brassica oleracea var. acephala DC. Green ornamental cabbage Brassiceae 12. Brassica oleracea var. acephala DC. Red ornamental cabbage Brassiceae 4 Estimation of Genetic Variations in Different Taxa Plate (1): Species and varieties included under study from Brassicaceae family. 5 Huda Jasim M. Al-Tameme Table (2): List of primers are used in RAPD and ISSR techniques and their sequences. Primer sequences 5' to 3' Primer Primer sequences 5' to 3' Primer GAGAGAGAGAGACC BH10 ISSR GTGAGGCGTC OPC2 RAPD GTGTGTGTGTGTCC BH11 TGGACCGGTG OPC8 CTCCTCCTCGC BH14 TGCGTGCTTG OPC14 TCTCTCTCTCTCTCTCC ISSR1 GTAGACCCGT OPB11 GGGTGGGGTGGGGTG ISSR3 CCACAGCAGT OPB18 AGA GAG AGA GAG AGA GT UBC 807 GAG AGA GAG AGA GAG AC UBC 811 Table (3): PCR amplification RAPD and ISSR Conditions. Name of Primer Initial denaturation Denaturation Annealing Extension Final extension Temp Cº Time (s) Temp Cº Time (s) Temp Cº Time (s) Temp Cº Time (s) Temp Cº Time (s) OPC2 OPC8 1 44 1 94 60 94 30 40 60 72 120 72 300 OPC14 1 40 1 94 240 94 60 36 60 72 120 72 120 OPB11 OPB18 1 45 1 95 300 94 60 36 60 72 120 72 240 BH10 BH11 BH14 1 40 1 49 240 94 60 40 60 72 120 72 300 ISSR1 ISSR3 1 35 1 94 300 94 35 47 40 72 35 72 300 UBC 807 UBC 811 1 40 1 94 300 94 60 45 72 60 72 300 RESULTS AND DISSCUSION Genetical variation indicates any alteration in nucleotides, gene and 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 were 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% 6 Estimation of Genetic Variations in Different Taxa polymorphism and the lower value of primer efficiency is 9% in OPC14 and higher value is 32% in OPC8, also the results revealed the same value of primer discriminatory power in all RAPD primers approximately 19% except primer OPC14 is 21%. (Tab. 4, 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 other hand, 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) have 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. 7 Huda Jasim M. Al-Tameme Table (4): RAPD and ISSR amplifications in twelve species and varieties in Brassicaceae. P ri m e r d is c ri m in a to ry p o w e r (% ) P ri m e r e ff ic ie n c y (% ) P o ly m o rp h is m % N o . o f M o n o m o rp h ic b a n d s N o . o f p o ly m o rp h ic b a n d s N o . o f to ta l a m p li fi e d b a n d s P ri m e r 19 25 20 0 12 60 OPC2 RAPD 19 32 16 0 12 76 OPC8 21 9 62 0 13 21 OPC14 19 14 39 0 13 33 OPB11 19 21 24 0 12 51 OPB18 26 0 62 241 Total 14 36 8 3 5 65 BH10 ISSR 33 37 18 0 12 67 BH11 36 22 33 0 13 40 BH14 17 6 55 0 6 11 ISSR1 0 0 0 0 0 0 ISSR3 0 0 0 0 0 0 UBC 807 0 0 0 0 0 0 UBC 811 20 3 36 183 Total - - 3 98 424 Total RAPD+ISSR Table (5): RAPD and ISSR band sharing in twelve species and varieties in Brassicaceae. 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. The put markers of RAPD and ISSR together found a rising rank of distinction in the genetic relationship; the similarity coefficients extend from 0.169 (between varieties red radish and green ornamental cabbage) to 0.897 (between varieties red cabbage and red ornamental cabbage ) (Tab. 6). 8 Estimation of Genetic Variations in Different Taxa Plate (2): Banding patterns of RAPD and ISSR fragments of Brassicaceae individuals. (Lane L molecular size marker one step 100 bp ladder (Promega). Lane 1-12 species under study (1- White radish, 2- Red radish, 3- Wild radish or fihaila, 4- Cress or rashad, 5- Hoary Cress or qunaibra, 6- Rocket mustard, 7- Cauliflower, 8- Broccoli, 9- Green cabbage-lahana, 10- Red cabbage, 11- Green ornamental cabbage and 12- Red ornamental cabbage). Diagram (1): RAPD and ISSR band sharing in twelve species and varieties in Brassicaceae. 0% 20% 40% 60% 80% 100% OPC2 OPC8 OPC14 OPB11 OPB18 BH10 BH11 BH14 ISSR1 9 Huda Jasim M. Al-Tameme Table (6): Similarity matrix computed with Jaccard coefficient. 12 11 10 9 8 7 6 5 4 3 2 1 0.26 8 0.23 8 0.27 5 0.23 4 0.32 6 0.35 9 0.25 5 0.28 2 0.27 5 0.30 9 0.39 3 1 1 0.22 9 0.16 9 0.23 5 0.18 6 0.26 3 0.29 4 0.25 0 0.20 6 0.21 7 0.26 0 1 2 0.28 8 0.36 0 0.27 1 0.35 5 0.37 3 0.35 1 0.31 0 0.32 1 0.34 8 1 3 0.32 7 0.39 7 0.30 8 0.39 1 0.34 5 0.27 3 0.22 9 0.21 8 1 4 0.31 7 0.29 0 0.29 3 0.30 6 0.43 9 0.34 1 0.29 1 1 5 0.28 1 0.30 3 0.30 9 0.31 6 0.36 8 0.39 6 1 6 0.62 9 0.43 1 0.69 7 0.42 4 0.77 1 1 7 0.69 4 0.55 4 0.76 5 0.51 7 1 8 0.43 1 0.75 8 0.43 9 1 9 0.89 7 0.47 3 1 1 0 0.43 9 1 1 1 1 1 2 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 sub- groups, 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. 10 Estimation of Genetic Variations in Different Taxa Diagram (2): UPGMA dendrogram indicating the genetic relationships among Brassicaceae taxa, based on RAPD and ISSR markers. 1-12 species under study: 1- White radish, 2- Red radish, 3- Wild radish or fihaila, 4- Cress or rashad, 5- Hoary cress or qunaibra, 6- Rocket mustard, 7- Cauliflower, 8- Broccoli, 9- Green cabbage- lahana, 10- Red cabbage, 11- Green ornamental cabbage and 12- Red ornamental cabbage. CONCLUSIONS Our investigation revealed significant variation in terms of RAPD and ISSR fingerprinting among the closely related species thought to be devoid of molecular variation and there by successfully drawing the interspecific phylogenetic relationships. LITERATURE CITED Al-Shehbaz, I. A. 1984. The tribes of Cruciferae (Brassicaceae) in the Southeastern United States. Journal of the Arnold Arboretum, 65: 343–373. Al-Shehbaz, I. A. 2012. A generic and tribal synopsis of the Brassicaceae (Cruciferae). Taxon, 61( 5):931-954. Appel, O. and Al-Shehbaz, I. A. 2003. Cruciferae. In: Kubitzki, K. and Bayer, C. (eds.), families and genera of vascular plants. 5: 75-174. Springer-Verlag, Berlin, Heidelberg. Cruz, S. M., Nery, M. C., Pinho, É. D. V. D. R. and Laia, M. L. 2014. Molecular characterization of radish cultivars. Revista Ciência Agronômica, 45(4): 815-822. http://www.ingentaconnect.com/content/iapt/tax;jsessionid=dop8aop6o84ha.alice 11 Huda Jasim M. Al-Tameme Edger, P. P., Heidel-Fischer, H. M., Bekaert, M., Rota, J., Glöckner, G., Platts, A. E., Heckel, D. G., Der, J. P., Wafula, E. K. and Tang, M. 2015. The butterfly plant arms-race escalated by gene and genome duplications. Proceeding of the National Academy of Sciences of the United State of America, 112: 8362–8366. Franzke, A., Lysak, M. A., Al-Shehbaz, I.A, Koch, M. A. and Mummenhoff, K. 2011. Cabbage family affairs: the evolutionary history of Brassicaceae. Trends Plant Sciences, 16: 108–116. Gray, A. R. 1993. Broccoli: Brassica oleracea (Italica Group). Genetic improvement of vegetable crops. In Kalloo, G. and Bergh, B. O.(eds.), pp 61-86. Gomez-Campo, C. and Prakash, S. 1999. Origin and domestication. In: Gomez-Campo C. (eds.), Biology of Brassica coenospecies, Elsevier, Amsterdam, pp 33-38. Grudman, H., Schneider, C., Hartung, D., Daschner, F. and Pith, T. 1995. Discriminatory power of three DNA typing techniques for P. aeruginosa. Clinical Microbiology, 3:528-532. Hammer, Ø., Harper, D. A. T, and Ryan, P .D. 2001. PAST: a palaeontological statistic software package for education and data analysis. Paleontologia Electronica, 4(1): 1-9. Harborne, J. B., Boulter, D. and Turner, B. L. 1971. Chemotaxonomy of the Leguminosae. Academic Press, London, New York, 612pp. Hu, J. and Quiros, C. F. 1991. Identification of broccoli and cauliflower cultivars with RAPD markers. Plant Cell Report, 10: 505-511. Kurane, J., Shinde, V. and Harsulkar, A. 2009. Application of ISSR marker in pharmacognosy: a current update. Pharmacognosy Reviews, 3(6): 216-228. Meinke, D. W., Cherry, J. M., Dean, C., Rounsley, S. D. and Koornneef, M. 1998. Arabidopsis thaliana: a model plant for genome analysis. Science, 282: 662–682. Prasad, M. P. 2014. Determination of molecular characterization of Brassicaceae family using RAPD molecular markers. International Journal Advance Research Biology Sciences, 1(2): 56-61. Soltis, P. S., Soltis D. E. and Doyle, J. J. 1992. Molecular systematics of plants Chapman and Hall, New York, 11pp. Vekemans, X., Poux, C., Goubet, P. M. and Castric, V. 2014. The evolution of selfing from outcrossing ancestors in Brassicaceae: what have we learned from variation at the S-locus?. Journal Evolutionary Biology, 27: 1372–1385. Warwick, S. I., Mummenhoff, K., Sauder, C., Koch, M. A. and Al-Shehbaz, I. A. 2010. Closing the gaps: phylogenetic relationships in the Brassicaceae based on DNA sequence data of nuclear ribosomal ITS region. Plant System Evolutionary, 285: 209–232. 12 Estimation of Genetic Variations in Different Taxa Williams, J. G. K., Kubelik, A. R., Livak, K. J., Rafalski, J. A. and Tingey, S. V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research, 18: 6231-6235. Yang, Y. W., Lai, K. N., Tai, P. Y. and Li, W. H. 1999. Rates of nucleotide substitution in angiosperm mitochondrial DNA sequences and dates of diversification between Brassica and other angiosperm lineages. Journal of Molecular Evolution, 48: 597–604. Zunk, K., Mummenhoff, K. and Hurka, H. 1999. Phylogenetic relationships in tribe Lepidieae (Brassicaceae) based on chloroplast DNA restriction site variation. Canadian Journal of Botany, 77: 1504–1512. 13 Huda Jasim M. Al-Tameme Bull. Iraq nat. Hist. Mus. (2018) 15 (1): 1-13 تقدير التغايرات الوراثية لمراتب تصنيفية مختلفة من العائلة الصليبية باستخدام ISSRو RAPDتحليل هدى جاسم محمد التميمي العراق ،بابل ،جامعة بابل ،العلوم للبناتكلية ،قسم علوم الحياة 00/03/1032 :تاريخ القبول 31/31/1037 :تاريخ االستالم الخالصة من العائلة الصليبية باستخدام اثنين من التقنيات درست اثنا عشر نوعا استخدمت كل من هذه التقنيات للكشف إذ ؛ ISSRو RAPDالجزيئية المختلفة عن بعض الواسمات الجزيئية المرتبطة مع تحديد النمط الجيني اذ اظهرت ، من استخدام RAPDنتائج تضاعف العشوائي المتعدد االشكال لسلسلة الدنا متعددة منها كانت 21حزمة مضخمة، 192ات عشوائية، تمييزخمسة بادئ (. ٪12)األشكال من أصل سبعة بادئات، ISSRتكرار التسلسالت البسيطة أظهرت النتائج لم يظهر اي تضخيم للحامض ( ISSR3 ،UBC807 ،UBC811)ثالثة منها كانت 12 حزمة مضخمة، 281النووي الجيني، وكشفت البادئات األخرى وتبين ان مؤشرات التشابه والمخطط التشجيري (٪12)متعددة األشكال بين 29840للمسافات الوراثية عند الجمع بين الطريقتين أن أعلى التشابه كان أصناف الملفوف األحمر والملفوف الزينة األحمر، بينما ظهر أدنى مؤشر اي ان ؛(29224)ينة األخضر للتشابه بين أنواع الفجل األحمر والملفوف الز األصناف / لديها القدرة على تحديد األنواع ISSRو RAPD عالمات يمكن أن نستنتج أن انواع أيضا وتوصيف االختالف الجيني داخل األصناف العائلة الصليبية لها كمية كافية من التنوع الوراثي ومجموعة واسعة في القاعدة الدراسة والتي يمكن استخدامها لتحسين الوراثية للتراكيب المستخدمة في .المحاصيل