GENETIC VARIABILITY OF CHOSEN BULGARIAN MENTHA SPECIES

Transcription

1 Доклади на Българската академия на науките Comptes rendus de l Académie bulgare des Sciences Tome 69, No 6, 2016 BIOLOGIE Génétique GENETIC VARIABILITY OF CHOSEN BULGARIAN MENTHA SPECIES Elena Apostolova, Georgi Anachkov, Krasimir Todorov, Ivanka Dimitrova-Dyulgerova, Rumen Mladenov, Plamen Stoyanov, Galina Yahubyan, Samir Naimov (Submitted by Academician A. Atanassov on December 16, 2015) Abstract The systematics of genus Mentha from the family Lamiaceae of the major group Angiosperms is well known as complicated. It was previously shown that using morphological characters alone is insufficient when differentiating the mint cultivars. It is important to identify and characterize mint species for medical and industrial applications. The present study investigated the efficiency of ISSR markers for assessing the genetic variability within this genus and to make an attempt to differentiate the genotypes within the group of species. Eighteen different ISSR primer sets were tested on five Bulgarian mint species. On the basis of molecular characterization, a clear allocation of the genotypes was found on dendrogram. Key words: ISSR markers, molecular differentiation, Mentha Introduction. Although many Mentha species were used as medicinal plants for more than 250 years, the genetic relations in the genus remain unclear [ 1 ]. Taxonomy difficulties are often due to high incidence of polyploidy, diverse morphology, variation in base chromosome number, and frequent interspecific hybridization [ 2 5 ]. Prior information about DNA sequences is not always required when using molecular markers. It is valid for the molecular technique ISSR which is based on the amplification of inter-repeat sequences [ 6 ]. ISSR are dominant markers, which are a preferred system for DNA fingerprinting [ 7 ]. This work was supported by grant NI 13 BF

2 Rodrigues et al. [ 8 ] used a combination between morphological traits, essential oils profile and ISSR markers to analyze the genetic diversity of Portuguese endangered medicinal plant Mentha cervina. This Mentha species from the Iberian Peninsula was studied with ISSR markers to assess genetic diversity and population structure. The conclusions were low genetic diversity and indispensability of conserving Mentha populations in that region [ 8 ]. The investigation on four Mentha piperita species from central Gujarat (India) based on different molecular techniques including ISSR gives useful information for future improvement and investigation programmes [ 9 ]. ISSR marker technique was also used successfully to investigate genetic diversity and natural hybridization in populations of clonal plants of Mentha aquatica [ 10 ]. Molecular techniques and morphometric measurements were successfully used to analyze M. piperita, M. aquatica, M. spicata and M. suaveolens for genetic and morphological variability [ 11 ]. The combination of morphological features and molecular data has been proven as a successful strategy for phylogenetic analyses of closely related species. In this study we have tested seven different ISSR primer sets on five Bulgarian mint species. In order to test the accuracy of the ISSR system implemented, two of the mint species (M. aquatica and M. longifolia) were tested in duplicates, originated from geographically isolated habitats. In order to assess genetic relations among studied Mentha species a phylogenetic tree based on the data obtained was constructed. Materials and methods. Plant material. The plant material used in this study was collected from natural habitats, during the period , from mint species found in Bulgaria (Table 1). The research material consisted of seven accessions of five species of genus Mentha: M. pulegium, M. piperita, M. spicata, M. longifolia, and M. aquatica. The plant tissues were stored on ice until arrival at laboratory facility and immediately frozen in liquid nitrogen and stored at 80 C. T a b l e 1 Collection locality and altitude of the studied Mentha species Species/Accessions Locality, Floristic region Altitude, m Mentha pulegium L. Peshtera, Rhodope Mts (western) 411 Mentha piperita L. Yagodovo, Thracian lowland 160 Mentha spicata L. Shiroka poliana Dam, Rhodope Mts (western) 1543 Mentha longifolia (L.) Hudson M. longifolia 1 Trigrad, Rhodope Mts (middle) 1247 M. longifolia 2 Smolyan, Rhodope Mts (middle) 1100 Mentha aquatica L. M. aquatica 1 Peshtera, Rhodope Mts (western) 411 M. aquatica 2 Veselinovo, Balkan Range (eastern) E. Apostolova, G. Anachkov, K. Todorov et al.

3 DNA extraction and ISSR analysis. After grinding the young leaves in a mortar with a pestle in the presence of liquid nitrogen, total DNA extraction was performed with DNeasy Plant Mini Kit (Qiagen). The young leaves, used as a starting material were grinded in presence of liquid nitrogen, and processed according to the manufacturer s requirements. The DNA concentration and quality were assessed spectrophotometrically and by 1% agarose gel electrophoresis. ISSR primers were designed as 14 to 18 bp long oligonucleotides, shown in Table 1, and synthesized by Microsynth AG, Switzerland. ISSR-based amplification was performed using the Applied Biosystems 2720 Thermal Cycler, programmed for an initial step of 4 min at 94 C, followed by 40 cycles at 94 C for 30 s, 46 C for 45 s, 72 C for 2 min and finally a 7 min extension step at 72 C. The PCR products were separated by electrophoresis in 1.5% agarose gels and staining with ethidium bromide. All gels were visualized by and recorded with a UV image digitalizer (BIO-VISION+3026.WL system). The size of amplified DNA fragments was estimated by comparison with a standard 100-bp DNA ladder (Fermentas GeneRuler SM#0331). Statistical analysis. Band profiles generated by ISSR were completed onto a data matrix on the basis of the presence (1) or absence (0) of selected bands. Data were statistically analyzed by the software program FreeTree. The program computes the distance matrix, constructs the phylogenetic tree by using the unweighted pair group arithmetic average-linkage algorithm. Tree was visualized by FigTree v Results. ISSR amplifications were performed with 18 primers (Table 2) using seven samples from five different cultivars. All tested primers amplified informative and reliable PCR products and were selected for subsequent ISSR analysis. In general, the size of amplified DNA fragments ranged from 200 to 1350 bp (Fig. 1). One hundred seventy two polymorphic bands were observed and they ranged from 3 [(CA) 8 RG] to 21 [(AC) 8 YT] per primer. Each primer generated polymorphisms on average. Genetic relationships among the genotypes, based on ISSR genetic similarities, are shown on the dendrogram, which clustered the 7 genotypes into four groups (Fig. 2). The two M. aquatica probes are furthest from all other samples and are clustered in a separate group. The other five mints are clustered in one big group. In this group, three separate sub-clusters are formed one sub-cluster of the two M. longifolia samples, a second sub-cluster of M. piperita and M. spicata specimens, and a third sub-cluster represented by M. pulegium. Discussion. Morphological traits depend on a number of ecological conditions like mutation, genetic drift, gene flow and selection [ 12 ]. Unlike them, ISSRs are a neutral marker system that is PCR-based and is commonly used tool for assessment of variability in plants [ 13 ]. ISSRs are randomly distributed in the whole genome and are not always associated with a specific phenotypic trait. Compt. rend. Acad. bulg. Sci., 69, No 6,

4 T a b l e 2 Number of ISSR bands scored with the 18 primers used No Primers Sequence (5 3 ) Total number Number of bands of bands Polymorphic Monomorphic 1 ISSR 3 (ACTG) 3RG ISSR 4 (GACA) 3RT ISSR 5 (GACA) 3YR ISSR 6 (AG) 8YA ISSR 7 (GA) 8RG ISSR 9 (AC) 8YA ISSR 10 (AG) 8YT ISSR 11 (AG) 8YC ISSR 13 (CA) 8RG ISSR 15 (GT) 8YG ISSR 16 (AG) 8YG ISSR 17 (AC) 8YT ISSR L2 (GA) 8YC ISSR L3 BD(AC) ISSR L5 HV(GT) ISSR L6 (AG) 8C ISSR L7 (CT) 8RC ISSR L8 (AG) 8VC Total Average % Polymorphisms Note: R = A + G; Y = C + T ; V = G + A + C; D = A + G + T ; B = G + T + C; H = A + C + T Fig. 1. Examples of ISSR-generated profiles: (A) the molecular profile generated with ISSR 3 and (B) the molecular profile generated with ISSR 10 (M DNA ladder; 1 M. longifolia 1; 2 M. longifolia 2; 3 M. spicata; 4 M. pulegium; 5 M. aquatica 1; 6 M. aquatica 2; 7 M. piperita) 728 E. Apostolova, G. Anachkov, K. Todorov et al.

5 Fig. 2. Dendrogram of genetic relationships of the 7 genotypes based on unweighted pair group arithmetic average-linkage algorithm from 172 ISSR products M. piperita is a hybrid between M. spicata and M. aquatica. Based on the AFLP data Gobert et al. [ 14 ] determined genetic diversity of 62 Mentha accessions. They established that M. longifolia and M. spicata belonged in one of the two big formed clusters on the dendrogramme. M. piperita and M. aquatica got together in the second cluster. In our study, 7 genotypes of five Bulgarian mint species were clustered into four clear groups. The observed ISSR genetic similarities among the five Bulgarian mint species demonstrated that we can easily apply the ISSR technique carried out with the tested primers for evaluation of genetic relationships between genotypes within the Mentha genus. Conclusions. This study presents sufficient and reliable information for successful application of ISSR technique in molecular and genetic characterization of the Mentha genotypes, as well as for variety identification. The used genetic markers show high reproducibility and are informative for determining the pattern of genetic relationships. Our observations reveal certain potential of the used PCR-based fingerprinting technique in subsequent application for analysis of representatives of the genus Mentha. The analyzed Bulgarian Mentha genotypes are promising germplasme for inclusion in hybridization schemes and for application in biotechnological practices. Compt. rend. Acad. bulg. Sci., 69, No 6,

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