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Biharean Biologist (2010) Vol. 4, No.2, Pp.: 145-151 P-ISSN: 1843-5637, E-ISSN: 2065-1155 Article No.: 041119

Assessment of genetic variation within international collection of rapa genotypes using inter simple sequence repeat DNA markers

Reza TALEBI1*, Ali HAGHNAZARI2 and Iman TABATABAEI3

1. Department of Agronomy and Plant Breeding, Islamic Azad University-Sanandaj Branch, Iran. 2. College of Agriculture, University of Zanjan, Zanjan, Iran. 3. Department of Plant breeding, Faculty of Agriculture University of Zabol * Corresponding author, R. Talebi, e-mail: [email protected]

Abstract. Information on genetic diversity and genetic relationships among taxa of (n=10, AA genome) is currently limited. The genetic diversity and the relationships among germplasm, including a collection of 47 genotypes from different countries were evaluated using inter simple sequence repeat DNA markers. A total of 99 different polymorphic amplification products were obtained using 12 selected primers. ISSRs revealed a significant level of polymorphism among the accessions. Cluster analysis based on unweighted pair group method comparison divided the studied accessions into three main clusters. Group 1 consisted mainly of Oil and types. Group 2 included a mixture of all three oil, turnip and types. Group 3 consisted mainly of cabbage and oil types. Also based on the origin, most of the Chinese and Korean Brasica rapa accessions were grouped in cluster 3. The cabbage accessions that tested in this study, were found to be extremely genetically unique and closer together in comparison with Oilseed and turnip types. The results indicate the occurrence of a considerable genetic variation between Chinese and European accessions.

Key words: Brassica rapa, Genetic Diversity, ISSR.

Introduction (subspecies, varieties and/or ) and varying taxonomic treatments. The genus Brassica includes a number of important crop A number of genetic or phylogenetic relationships species, which are used for various purposes, e.g., to have been proposed for taxa within B. rapa, based on produce oilseed, as , fodder or condiments. morphology, geographical distribution, isozymes and One of the species is B. rapa L. (2n = 20, genome AA) different molecular markers (Song et al. 1988, Specht & (synonym, Brassica campestris L.) which includes several Diederichsen 2001, Huh & Huh 2001, Guo et al. 2002, groups of different crops such as var. chinensis (L.) Zhao et al. 2005). Various data have indicated a division Kitam. and var. oleifera DC. This investigation deals with of B. rapa into ‘western’ and ‘eastern’ groups, perhaps B. rapa L. var. r a p - turnip. Brassica rapa L. (n ¼ 10, AA corresponding to two independent centres of origin genome) is grown for edible and industrial oil, as a (McNaughton 1976, Denford & Vaughan 1977, Prakash and for fodder. It has been domesticated over & Hinata 1980, Song et al. 1988, McGrath & Quiros a long period of time in both Europe and Asia. Key 1992). The western, or European, group includes crops include: vegetable or fodder turnip (subsp. rapa, vegetable turnip and oilseed turnip rape; India–Asian formerly subsp. rapifera); oilseed turnip rape (subsp. oilseed types are putatively derived from the latter. The oleifera); brown sarson or toria (subsp. dichotoma) and eastern, or Asian, group contains the various Asian yellow sarson (subsp. trilocularis); broccoletto (B. ruvo), a vegetables indicated above. Polymorphism between European vegetable with an enlarged, compact inflo- species can result from different events and is an rescence; Chinese cabbage (subsp. pekinensis), an Asian important aspect of plant breeding. For the assessment heading vegetable with winged petioles; pak choi of diversity in plant populations different marker (subsp. chinensis), a Chinese non-heading systems have been established on morphological, with fleshy but non-winged petioles; mizuna (subsp. physiological and DNA levels. Each of the various nipposinica), an Asian non-heading leafy vegetable with marker systems utilized for the characterization of many tillers and either pinnate or entire leaves similarities and differences between individuals has its (Warwick et al. 2008). Such varied use and correspond- specific strengths and constraints, e.g. regarding the ing morphological variability has led to the recognition number of available markers, the polymorphism per of a large number of both specific and infraspecific taxa marker, the mode of inheritance or the genomic location

©Biharean Biologist, Oradea, Romania, 2010 Biharean Biol. 4, 2010 http://biologie-oradea.xhost.ro/BihBiol/index.html Oradea, Romania 146 Talebi, R. et al. of the markers. Microsatellites (SSR) represent the most units (AT, AC, GT, AG, GA, TTC, GCC, CAA, ATG, CCA, abundant source of polymorphism from repetitive CAC, GAAA and ATCC) were tested using a specific and sequences. SSR are often used as molecular markers optimal annealing temperature for each one. PCR reactions were composed as described previously by Bornet and even if this technology is time-consuming and Branchard (2001) and PCR reactions were performed in a expensive. Inter simple sequence repeat (ISSR) is an volume of 10 μl in Eppendorf thermocyclers. The reaction alternative technique to study polymorphism based on mixture contained 0.6 μl of each primer, 100 μM of each the presence of microsatellites throughout genomes deoxinucleotide, 0.5 units of Taq polymerase (Fermentas), 10x (Zietkiewicz et al. 1994). Inter-simple sequence repeat Taq buffer containing 1.5 mM MgCl2, and 10 ng of template (ISSR) markers have been proposed as a new source of DNA. Amplification regime was 94ºC/5 min, followed by 30 genetic markers that are inherited in Mendelian fashion cycles of 94ºC/1 min, 48-52 ºC (specific for each primer)/1 min º º and are scored as dominant markers. ISSR markers are and 72 C/2 min, and ending with an extension step of 72 C/10 min. PCR products were analyzed using agarose (2% w/v) DNA sequences delimited by two inverted SSR electrophoresis gels stained with ethidium bromide and only composed of the same units which are amplified by a bands with high intensity and well separated were selected. single PCR primer, composed of few SSR units with or For each accession, a binary matrix reflecting specific ISSR without anchored end. ISSR-PCR gives multilocus band presence (1) or absence (0) was generated. Pairwise patterns which are very reproducible, abundant and comparisons were calculated using the Jaccard’s coefficient polymorphic in plant genomes (Bornet & Branchard (Jaccard 1907). The similarity values found were used to 2001, Fernandez et al. 2002, Bornet et al. 2002a). generate a phenogram via the unweighted pair group method Comparison of ISSR and other PCR-based markers have analysis (UPGMA). The correlation between the similarity matrix and the cophenetic matrix for the clusters was shown their efficiency in plant breeding (Adams et al. computed. Polymorphic information content (PIC) values were 2003, Archak et al. 2003, Galvan et al. 2003). As a result calculated for each ISSR primers according to the formula: of these advantages and their universality and easiness PIC= 1- Σ(Pij)2, where Pij is the frequency of the ith pattern of development (no needs to sequence data), ISSR revealed by the jth primer summed across all patterns revealed markers are more and more requested. ISSR markers by the primers (Botstein et al. 1980). The averages genetic were successfully used for estimating of genetic distance (GD) between any two cultivars is represented by its diversity in main crops for instance maize (Kantety et al. Fst value, and refers to as inter distance. Fst value were 1995), wheat (Nagoaka & Ogihara 1997), rice (Blair et al. used as input data for two-dimensional principal coordinate analysis (2D PcoA) (Huff 1997). All these analyses were 1999) and barley (Brantestam et al. 2004, Hou et al. performed with NTSYSpc version 2.0 (Rohlf 1998). 2005). In the present study, the genetic diversity in 47 Brassica rapa accession from different types and Results countries was examined using ISSR. The major objectives were to quantify the amount and distribution Approximately 90% of the ISSR bands that were present of genetic variation within and among these genotypes in individual plants were present in bulked samples of using genetic diversity measures. Information on the same accession (data not shown). The use of bulked genetic diversity would provide a much improved basis leaf samples from individual plants for AFLP analysis for understanding of the genetic diversity of different and assessment of genetic diversity was documented for Brassica rapa genotypes from a broad spatial scale and B. carinata and B. rapa (Warwick et al. 2006, 2008). for the appropriate management of this species in Although a sample size of five individuals is adequate breeding programme. to represent the diversity in dihaploid breeding lines, greater sampling per accession would be required in order to estimate diversity within genebank accessions Material and Methods or breeding populations. Twenty eight ISSR primers

were initially screened for their ability to produce Plant materials: Forty seven B. rapa (oilseed rape, cabbage and turnip) polymorphic patterns using 10 accessions. Twelve accessions were obtained from all canola and mustard- primers, which gave reproducible and distinct poly- producing countries of the world to form the germplasm morphic amplification products, were selected for eva- collection (Table 1). These accessions comprised 8 turnip, 18 luation of diversity across all the accessions. Each of the cabbage and 21 oilseed type from ten geographically diverse selected 12 primers varied greatly in the ability to countries. produce different polymorphic fragments ranging from 6 to 11 per reaction. A total of 99 polymorphic ISSR DNA extraction and ISSR analysis bands were scored using these primers. On average 7.6 Total genomic DNA was extracted from a pool of 10 plants of each accession following a CTAB extraction protocol (Lassner different polymorphic bands per reaction were recorded et al. 1989). Eighteen ISSR primers corresponding to 13 SSR (Table 2). The sizes of these bands were ranged from 190

Biharean Biol. 4, 2010 Genetic variation within international collection of Brassica rapa genotypes 147

Table 1. List of Brassica rapa accessions with their origin and growing type used for ISSR fingerprinting analysis.

No. Cultivar Country of origin Type 1 YS-180-5 India Oil 2 YS-180-1 India Oil 3 YS-179-1 Bangladesh Oil 4 YS-179-2 Bangladesh Oil 5 PC-169-1 Cabbage 6 PC169-2 China Oil 7 YS-184-3 India Oil 8 CGN06711(VT-008) India Turnip 9 SO-039 Bangladesh Oil 10 VT-009 India Turnip 11 BRO-123 Germany Turnip 12 CC-136 Cabbage 13 VT-113 Korea Cabbage 14 BRO-120 Germany Turnip 15 YS-143 India Oil 16 CC-192-1 China Cabbage 17 CC-191-1 China Cabbage 18 CC-190 China Cabbage 19 YS-189 India Oil 20 YS-188-1 India Oil 21 YS-871-1 India Oil 22 SO-032-1 Bangladesh Oil 23 SO-032-2 Bangladesh Oil 24 CC-48-2 China Cabbage 25 SO-32-7 India Oil 26 PC-078-8 China Cabbage 27 RO-18-8 Russia Oil 28 YS-143-7 India Oil 29 CWU-56-2 China Cabbage 30 PC-184-3 China Cabbage 31 CC-114-2 China Cabbage 32 CGN10999(VT-091) United Kingdom Turnip 33 CGN07223(FT-086) Pakistan Turnip 34 SO-035 India Oil 35 CGN07202 (CC-073) China Cabbage 36 CGN07199 (CC-070) Korea Cabbage 37 SO-034 Bangladesh Oil 38 CGN07188 (CC-061) Yugoslavia Cabbage 39 CC-062 Yugoslavia Cabbage 40 BRO-025 Germany Turnip 41 CC-072 Korea Cabbage 42 Ys-a95-1 Germany Turnip 43 CC-072 Korea Cabbage 44 YS-195-1 India Oil 45 CC-061-3 China Cabbage 46 CGN06832 (SO-031) USA Oil 47 -

to 2190 base pairs which was similar to the previously Branchard 2004). The average polymorphic information reported range in Brassica germplasm using ISSR and content (PIC) was 0.49, ranging from 0.33 to 0.77. The RAPD markers (Shengwu et al. 2003, Bornet & lowest and the highest PIC value were recorded for

Biharean Biol. 4, 2010 148 Talebi, R. et al.

Table 2. Single letter abbreviations for mixed base positions: N = (A, G, C, T); R = (A, G); Y = (C, T).

Primer Sequence (5΄ to 3΄) No. of polymorphic PIC value bands UBC801 ATA TAT ATA TAT ATA TT 9 0.62 UBC807 AGA GAG AGA GAG AGA GT 8 0.44 UBC828 TGT GTG TGT GTG TGT GA 7 0.47 UBC878 GGA TGG ATG GAT GGA T 9 0.48 UBC899 CAT GGT GTT GGT CAT TGT TCC A 11 0.77 UBC822 TCT CTC TCT CTC TCT CA 8 0.49 UBC874 CCC TCC CTC CCT CCC T 6 0.41 UBC816 CAC ACA CAC ACA CAC AT 9 0.51 UBC814 CTC TCT CTC TCT CTC TA 9 0.57 UBC835 AGA GAG AGA GAG AGA GYC 7 0.39 UBC840 GAG AGA GAG AGA GAG AYT 8 0.33 UBC847 CAC ACA CAC ACA CAC ARC 7 0.41

primer UBC840 and UBC899, respectively. Reprodu- the different marker technologies employed in Brassica cibility is essential for a technique to be used in crops (Snowdon & Friedt 2004), ISSR markers have the fingerprinting. In the current experiment, we tested the advantage of being robust, cost effective and highly repeatability of ISSR markers by performing separate polymorphic (Bornet & Branchard 2004). The level of PCR runs. The amplification of these markers was polymorphic markers appeared to be adequate to consistent and no new differences were detected in clearly separate germplasm evaluated in this study. different experiments. Among the various similarity ISSR have the ability to generate a high degree of indices, those of Jaccard and Dice were chosen as the polymorphisms, and randomly selected unmapped most appropriate ones for dominant markers, like ISSR markers were used in this study. The usefulness of ISSR and RAPD, since they do not attribute any genetic for assessment of genetic diversity depends on their meaning to the coincidence of band absence. The high abundance and distribution across the genome (Bornet degree of genetic differentiation is confirmed by the & Branchard 2001). Previous studies have indicated that UPGMA dendrogram obtained using the 99 ISSR bands ISSR markers are usually randomly distributed over the scored in the 47 individuals showed well-defined genome, and therefore the results presented here should groups. Cluster analyses revealed three main groups give a good estimation of variation across the genome (designated I, II and III) (Fig. 2). Group 1 consisted (Bornet & Branchard 2004). The high level of genetic mainly of Oil and turnip types. Group 2 included a diversity between accessions showed that the genotypes mixture of all three oil, turnip and cabbage types. Group origin (country) had major impact on the outcome of 3 consisted mainly of cabbage and oil types. Also based our study. This is also indicated by the partly on the origin, most of the Chinese and Korean Brasica overlapping cultivar cluster derived from PCoA (Fig. 3) rapa accessions were grouped in cluster 3. Similar results and in the dendrogram (Fig. 2). The cabbage accessions were obtained using principal component analysis (Fig. we tested were found to be extremely genetically 3). PC1 and PC2 accounted for 29% and 11.7% of the unique and closer together in comparison with Oilseed total variation, respectively. Genotypes consisted in and turnip types. Genetically diverse Oilseed and turnip three main groups and 15 genotypes were not grouped rape genotypes represent a potentially valuable source closely with other genotypes. for improved pathogen and pest resistance (Lühs et al. 2003a, 2003b), and the same is true for the introduction of resistance genes from the B. rapa and B. oleracea gene Discussion pools via resynthesized B. napus (Rygulla et al. 2007a, 2007b, 2008). Furthermore, introduction of untapped In conservation programs for plant genetic resources, germplasm into breeding lines also has the potential to the availability of characterization data and information improve heterotic potential (Seyis et al. 2006, Basunanda on available genetic diversity can help germplasm users et al. 2007, Gehringer et al. 2007). ISSR is important to identify accessions of interest and also provide plant markers for assessing genetic relationships among breeders with initial data regarding materials for use in cultivars and for fingerprinting cultivars. For example, crop improvement programs (Cruz et al. 2007). Out of Bornet & Branchard (2004) showed that ISSR markers

Biharean Biol. 4, 2010 Genetic variation within international collection of Brassica rapa genotypes 149

Figure 1. ISSR fragments for 16 accessions using the primer UBC828. Arrows indicate the scored bands.

Figure 2. Dendrogram resulting from UPGMA cluster analysis of 47 B.rapa accession based on data derived from 12 ISSR markers.

Biharean Biol. 4, 2010 150 Talebi, R. et al.

Figure 3. The principal-coordinate analysis (PCO) of 47 accession of Brassica rapa. PCO axes 1, 2 accounted for 29% and 11.7% of the variation respectively.

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