Genotypes Using Inter-simple Sequence Repeat ...

Biotechnology Journal International 20(1): 1-12, 2017; Article no.BJI.37053 ISSN: 2456-7051 (Past name: British Biotechnology Journal, Past ISSN: 2231–2927, NLM ID: 101616695)

Molecular Differentiation of Five Quinoa (Chenopodium quinoa Willd.) Genotypes Using Inter-simple Sequence Repeat (ISSR) Markers A. M. M. Al-Naggar1*, R. M. Abd El-Salam1, A. E. E. Badran2 and Mai M. A. El-Moghazi2 1

Department of Agronomy, Faculty of Agriculture, Cairo University, Egypt. Plant Breeding Unit, Department of Genetic Resources, Desert Research Center, Cairo, Egypt.

2

Authors’ contributions This work was carried out in collaboration between all authors. Author AMMAN designed the study, wrote the protocol and wrote the first draft of the manuscript. Authors AMMAN, RMAES and AEEB supervised the study and managed the literature searches. Author MMAEM managed the experimental process and performed data analyses. All authors read and approved the final manuscript. Article Information DOI: 10.9734/BJI/2017/37053 Editor(s): (1) Rashid Ismael Hag Ibrahim, Department of Biological Sciences, College of Science, King Faisal University, Saudi Arabia. Reviewers: (1) Marcos Vinicius Bohrer Monteiro Siqueira, Universidade do Sagrado Coração, Brazil. (2) António Alberto Neves de Alcochete, Agostinho Neto University, Angola. (3) Davi Coe Torres, Universidade Federal de Alfenas (UNIFAL-MG), Brasil. Complete Peer review History: http://www.sciencedomain.org/review-history/21757

th

Original Research Article

Received 28 September 2017 st Accepted 31 October 2017 th Published 6 November 2017

ABSTRACT Knowledge of genetic diversity is one of the important tools used for genetic management of quinoa accessions for plant breeding. This research aimed to molecularly characterize five quinoa genotypes using ISSR markers to reveal genetic polymorphism and identify unique markers for each genotype. Analysis of inter-simple sequence repeats (ISSR) revealed that 10 ISSR primers produced 53 amplicons, out of them 33 were polymorphic and the average percentage of polymorphism was 61.83%. The number of amplicons per primer ranged from 3 (HB-13, HB-10, HB-8 and 17898A) to 10 (HB-15) with an average of 5.3 fragments/primer across the different quinoa genotypes. Data showed a total number of unique ISSR markers of 24; eleven of them were positive and 13 were negative. Using ISSR analysis, we were able to identify some unique bands _____________________________________________________________________________________________________ *Corresponding author: E-mail: [email protected], [email protected];

Al-Naggar et al.; BJI, 20(1): 1-12, 2017; Article no.BJI.37053

associated with quinoa genotypes. The genetic similarity ranged from 49% (between Ollague and each of QL-3 and Chipaya) to 76% (between CICA-17 and CO-407). The results indicated that all the five quinoa genotypes differ from each other at the DNA level where the average of genetic similarity (GS) between them was about 59%. The dendrogram separated the quinoa genotypes into two clusters; the first cluster included two genotypes (QL-3 and Chipaya). The second cluster was divided into two groups; the first group included two genotypes (CICA-17 and CO-407) and the second group included only one genotype (Ollague).

Keywords: Chenopodium quinoa; unique markers; cluster analysis; genetic similarity. institutions mainly from Bolivia, Perú, United States and India [14]. In Colombia, Corpoica Tibatitatá reports a germplasm bank with 28 accessions of quinoa [14], however, small collections are conserved in the main producer departments. In the country, the characterization of this plant genetic resources only morphoagronomic studies developed by Torres et al. [15] in the Savannah of Bogotá. Molecular markers are also employed for the genetic characterization of Chenopodium germplasm. They have been used to differentiate genotypes under environmental conditions that confounded their phenotypes [16]. Simple sequence repeats (SSR) are one of the frequently used molecular markers for genotyping crops [17-22]. A number of research studies have demonstrated the use of SSRs and ISSRs to detect polymorphism and diversity in quinoa [23,24] related species like amaranth [25,26] and others [27,28]. However, inter-simple sequence repeat (ISSR) markers are simpler to use than SSR technique [26,27]. The use of ISSR does not require prior knowledge of the target sequences flanking the repeat regions, is not expensive and is relatively easy to score manually compared to SSR.

1. INTRODUCTION Quinoa (Chenopodium quinoa Willd.) is a pseudo-cereal of the Amaranthace family which originated from the Andes of South America where it has been cultivated since more than 5,000 years [1]. Quinoa is an allotetraploid (2n=4x=36), thus exhibits disomic inheritance for most qualitative traits [2]; its seeds, and to some extent its leaves, are traditionally used for human and livestock consumption in the Andean region and have exceptional nutritional qualities [3,4]. Moreover, the species, being adapted to the harsh climatic conditions of the Andes [5], exhibits remarkable tolerance to several abiotic stresses such as frost [6], salinity [7] and drought [8]. Cultivated quinoa display a genetic diversity, mainly represented in an ample range of characters like plant coloration, flowers, protein content, seeds, saponin content and leaf calcium oxalates content, which allows obtaining a wide range of adaptability to agroecological conditions [9]. The adaptation capacities of quinoa are huge since we can find varieties developed from sea level up to 4,000 m above and from 40°S to 2°N of latitude [10]. The genetic bases of several quinoa traits was identified several decades ago [11], but the first true genetic descriptions more recently provided the starting point for improvement of quinoa. Several genetic tools have been developed, and today molecular markers are an effective way to enhance breeding efficiency [12].

Over the past few years, scientists have characterized the genetic diversity of quinoa to understand its biological diversity as a function of its eco-geographic distribution and to identify genetically distinct groups [29]. This knowledge is a prerequisite in quinoa conservation strategies and effective germplasm management and characterization [30]. The study of quinoa’s genetic diversity is also essential in plant breeding programs, especially in the identification of diverse parental combinations [31]. Yazici and Bilir [32] reported that genetic knowledge is one of the important tools used for different purposes such as gene conservation, managing of genetic resources, evolutional and genetic management of populations for plant breeding. The objectives of this research were to molecularly characterize five quinoa genotypes using ISSR markers, to

Quinoa is one of the Andean crops with little research in the area of genetics and plant breeding, although, it has a high variability in its characteristics [13]. Collecting, conservation and characterization studies are necessary for the development of strategies to improve of this species. At the international level, approximately 16,263 Chenopodium accessions are collected worldwide, which have been preserved and characterized in part by 2

Al-Naggar et al.; BJI, 20(1): 1-12, 2017; Article no.BJI.37053

2.2.1 Extraction buffer

reveal genetic polymorphism and identify unique markers for each genotype.

Tris-HCl (100 mM, pH 8.0), KCl (0.5 M) with MgCl2 (15 mM), NaCl (0.5 M), EDTA (20 mM), CTAB 2% (w/v), β-Mercapto ethanol (0.1%) was added to buffer just before use, 0.25 g Polyvinylpyrrolidone (PVP) per 1.0 g of tissue (added in the mortar while crushing the tissue), Phenol:chloroform: Isoamlyalcohol (25:24:1), Ribonuclease A (10 mg /ml).

2. MATERIALS AND METHODS 2.1 Plant Material Seeds of five quinoa (Chenopodium quinoa Willd.) genotypes obtained from Madison University, Wisconsin, USA were used in this study. The origin and some traits of these genotypes are presented in Table 1.

2.2.2 TE buffer Tris (10 Mm, PH 8.0), EDTA (1 Mm, PH 8.0) were the additional solutions prepared for experimentation.

The inter-simple sequence repeats (ISSR) analysis was used in the present study to investigate the genetic diversity among five quinoa genotypes. This experiment was carried out in the Molecular Genetics Laboratory of the Desert Research Center, Cairo, Egypt.

2.3 Quantity and purity of DNA For testing the purity, DNA concentration was measured by UV- spectrophotometer by estimating the ratio of absorbance at a wave length of 260 nm to 280 nm. To confirm the concentration and purity, the DNA was subjected to electrophoresis on 0.8% agarose gels prepared in 1X TAE with lambda DNA ladder.

2.2 DNA Isolation The DNA was isolated from quinoa leaves collected from plants of age 40 days after emergence (leaf on the third node from the top of the main stem) according to the procedure of Junghans and Metzlatt [34]. From each sample, 0.5 g of leaf tissue was ground in a mortar and pestle in liquid nitrogen until a fine powder was obtained. After grinding, thawing of ground tissues was prevented and sample was transferred to 1.5 ml Eppendorf tubes. Then 700 µl of extraction buffer was added and mixed well. The tubes were incubated at 4ºC for 10 min. The tubes were centrifuged at 12000 rpm for 10 min. The supernatant were transferred to a new sterile Eppendorf tube. 500 µl of phenol: chloroform: isoamyl (25:24:1 v:v:v) was added to wash the supernatant. The upper phase was transferred to a new sterile 1.5 ml tube. Then, 500 µl of phenol: chloroform: isoamyl (25:24:1 v:v:v) was added and solution mixed. The tubes were centrifuged at 12000 rpm for 5 min. The aqueas phase was transferred to a new sterile tube. Cold isopropanol (750 µl) was added and solution mixed. The tubes were incubated at 4oC for 15 min. The tubes were centrifuged for 5 min to aggregate the DNA. Pellets were washed in 70% ethanol and left to dry for about 30 minutes. The pellets were re-dissolved in 100 µl TE buffer. Then, RNAase (5 units / µl for each sample) was added and solution incubated at 37ºC for 2 h and samples kept in refrigerator till use. DNA concentration was measured by UVspectrophotometer at a wave length of 260 -280 nm.

2.4 Polymerase Chain Reaction (PCR) ISSR-PCR amplification reactions were conducted using 10 specific primers, as presented in Table 2. The reaction conditions were optimized and mixtures consisted of the following: 25 µL reaction volume which contained varying concentration of template DNA (100 ng/ µl), dNTPs (0.1:0.2 mM), Taq DNA polymerase (1 unit), ISSR primers (0.1-0.3 µM) and 1X PCR buffer (10 mM TrisHCl pH 8.3, 50 mMKCl, 2 mM MgCl2).The PCR amplification was carried out in an Eppendorf Mastercycler-Gradient thermal Cycler as follows: One cycle (denaturation step) 94ºC for 4 min, followed by 34 cycles at 94ºC for 30 sec, 45 to 72ºC for 45 sec at the specific annealing temperature of each primer, 72ºC for 1 min and finally one cycle at 72ºC for 8 min, and hold temperature of 4ºC (infinitive). The PCR master mix for inter simple sequence repeat (ISSR) primers consisted of 2 µL of 20 ng / µL genomic DNA template, 0.40 µL of 10 µM a forward and reverse primer mixture, 0.18µL (0.9 U) of Taq Polymerase, 1.20 µL of 10X buffer (10 mM Tris-HCL, 50 mM KCl, 1.5 53 mM MgCl2, pH 8.3), 0.96 µL of a 100 µM mixture of dNTPs and 7.26 µL of water bringing the total reaction volume to 12 µL. Reaction conditions for ISSR markers were as follows: 8.33 µL ddH20, 2.4 µL 10X reaction buffer, 0.9 µL 50 mM MgCl2, 1.92 µL 2.5 mM dNTPs, 1.9 µL 1 pM of 19 bp M-13. 3

Al-Naggar et al.; BJI, 20(1): 1-12, 2017; Article no.BJI.37053

Table 1. Name, origin and seed color of quinoa genotypes under investigation Name Q-l3 Chipaya CICA-17 CO-407 Ollague

Origin Bolivia Altiplano Salares, Bolivia Peru Colorado State Univ, USA Altiplano Salares, Bolivia

Seed color* Light yellow Mixed (white & Peige) Yellow Mixed (yellow & white) Yellow

* Al-Naggar et al. [33]

Table 2. Description of the ISSR loci used in this study No. 1

Primer HB 15

2

HB 14

3

HB 13

4

HB 12

5

HB 11

6

HB 10

7

HB 9

8

HB 08

9

14A

10

17898A

Sequence F 5′-GTG GTG GTG GC-3′ R 3′- CAC CACCAC CG-5′ F 5′-CTC CTC CTC GC-3′ R 3′-GAG GAG GAG CG- 5′ F 5` GAGGAGGAGGC 3` R 3′-CTCCTCCTCCG- 5′ F 5` CAC CACCAC GC 3` R 3′-GTG GTG GTG CG-5′ F 5` GTG TGT GTG TGT TGT CC 3` R 3′-CAC ACA CAC ACA ACA GG -5′ F 5` GAG AGA GAG AGA CC 3` R 3′-CTC TCT CTC TCT GG5′ F 5` GTG TGT GTG TGT GC 3` R 3′-CAC ACA CAC ACA CG5′ F 5`GAG AGA GAG AGA GG 3` R 3′-CTC TCT CTC TCT CC5′ F 5′-CTC TCT CTC TCT CTC TTG-3′ R 3′-GAG AGA GAG AGA GAG AAC5′ F 5′-CA CA CACACACA AC -3′ R -3′ GT GTGTGTGTGT TG-5′ F = Forward, R = Reverse

The PCR master mix for sequence-tagged site (STS) was carried out in a volume of 20 µl and contained 200 ng of genomic DNA, 0.2 mM of dNTPs, 10 pmol of each primer, 2.0 mM of MgCl2, 50 mM of KCl, 10 mM of Tris-HCl (pH 9.0 at 25°C), 0.1% TritonX-100 and 0.5 U of Taq DNA Polymerase.

2.6 Gel Electrophoresis Gel electrophoresis was performed following the procedure of Sambrook et al. [35]. Agarose (1.2%) ultra pure (GIBCOBRL) was used for resolving the PCR products. One Kb plus DNA ladder (750 ng /3 µl) (GeNetBio) was used which was separated into fourteen bands with molecular weights of 100, 200, 300, 400, 500, 650, 850, 1000, 1650, 2000, 3000, 4000, 5000 and 12000 bp. The TAE buffer (50X) consisted of Tris 242 g, Glacial acetic acid 57.1 ml, EDTA 37.2 g, dd H2O up to 1L. The TBE buffer (10X) consisted of Tris 108.0 g, Boric acrid 55.0 g, EDTA 7.44 G, d H2O up to 1 L.

2.5 Sample Preparation For sample preparation PCR- product (20 µl) and Loading buffer (6X) 5 µl were used. The run was performed for 1 h at 100 V using Biometra gel electrophoresis submarine (20 cm x 10 cm) and the gel was stained with ethidium bromide. Bands were detected on UV- transilluminator and photographed by Gel documentation system (Biometra Bio Doc Analyze 2000). Loading buffer (6X) consisted of Bromophenol blue 0.25 g, Xylene cyanol 0,25 g, Glycerol (30%) 100 ml. For gel preparation, Agarose 1.2 g, TAE buffer (1X) 100 ml, Ethidium bromide (10 µg /µl) 1.5 µl were used.

2.7 Analysis of Gel Images Gel with amplification fragments were detected on UV- transilluminator and photographed using Gel Documentation System (Biometra Bio Doc Analyze 2000). Molecular ladder was used as molecular marker to know the size of the 4

Al-Naggar et al.; BJI, 20(1): 1-12, 2017; Article no.BJI.37053

fragments. 100 bp DNA ladder was used for determining the molecular weight.

2.8 Genetic Analysis

Similarity

and

to characterize the genetic markers and differences on a molecular level among the five quinoa genotypes and assay the genetic relationships among them.

Cluster

3.1 Genetic Diversity Genotypes

The banding patterns generated by ISSR-PCR marker analysis were compared to determine the genetic relatedness of the genotypes. Clear and distinct amplification products were scored as ‘1’ for presence and ‘0’ for absence of bands. Bands of the same mobility were scored as identical. The genetic similarity coefficient (GS) between two genotypes was estimated according to Dice coefficient [36].

among

Quinoa

Ten ISSR primers revealed discernible amplification profiles, therefore were employed to investigate the genetic polymorphism among the five quinoa genotypes (Tables 3 and 4 and Figs. 1 and 2). The 10 ISSR primers produced 53 amplicons, out of which 33 were polymorphic and the average percentage polymorphism was 61.83% (Table 3). The number of amplicons per primer ranged from 3 (HB-13, HB-10, HB-8 and 17898A) to 10 (HB-15) with an average of 5.3 fragments/primer across the different genotypes. However, the number of polymorphic amplicons varied from 1 (HB-13 and HB-8) to 6 (HB-15 and HB-12) with an average number of polymorphic amplicons of 3.3 fragments/primer. One out of the 10 primers exhibited 100% polymorphism (17898A), while two primers (HB-13, HB-8) showed low polymorphism (33.3%). The size of amplified fragment varied with the different primers, ranging from 130 to 1456 bp (Fig. 1).

The similarity matrix was used in the cluster analysis. The cluster analysis was employed to organize the observed data into meaningful structures to develop taxonomies. At the first step, when each accession represents its own cluster, the distances between these accessions are defined by the chosen distance measure (Dice coefficient). However, once several accessions have been linked together, the distance between two clusters is calculated as the average distance between all pairs of accessions in the two different clusters. This method is called unweighted pair group method using arithmetic average (UPGMA) according to Sneath and Sokal [36].

The number of bands and the percentages of polymorphism found in this study are suitable for estimating genetic diversity when compared with others species that used ISSR markers [16,37,38]. ISSRs are polymorphic markers that are useful for the discrimination of closely related quinoa individuals [24,26,38].

3. RESULTS AND DISCUSSION In the present investigation, analysis of intersimple sequence repeats (ISSR) was conducted

Table 3. Number of monomorphic and polymorphic amplicons and percentage of polymorphism, as revealed by ISSR primers for five quinoa genotypes Primer HB-15 HB-14 HB-13 HB-12 HB-11 HB-10 HB-09 HB-08 14A 17898A Total Average

Ms (bp) range 1450-180 810-300 680-380 410-150 800-240 300-230 700-180 610-400 700-410 480-280

Polymorphic amplicons 6 5 1 6 3 2 3 1 3 3 33 3.3

Monomorphic amplicons 4 2 2 1 3 1 4 2 1 20 2.0

5

Total

Polymorphism %

10 7 3 7 6 3 7 3 4 3 53 5.3

60.0 71..4 33.3 85.7 50.0 66.7 42.9 33.3 75.0 100.0 61.83

Al-Naggar et al.; BJI, 20(1): 1-12, 2017; Article no.BJI.37053

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Fig. 1. Banding patterns of five quinoa genotypes amplified with the ISSR primers HB-15, HB14, HB-13, HB-12, HB-11, HB-10, HB-09, HB-08, 17898A and 14A M: 100bp DNA ladder, Lane 1: QL-3, Lane 2: Chipaya, Lane 3: CICA-17, Lane 4: C0-407, Lane 5: Ollague The genotype CICA-17 was characterized by two unique positive markers amplified by one unique positive marker amplified by the primer 17898A (410 bp) and one negative unique marker amplified by the primer HB-9(600 bp). The genotype CO-407 was characterized by one unique positive marker amplified by the primer HB-14 (810 bp) and three negative unique markers amplified by the primers HB-15 (180 bp), HB-14 (700 bp), HB-11 (450 bp). The genotype Chipaya was characterized by two positive unique markers amplified by the primer HB-15 (350 bp) and the primer HB-14 (480 bp) and three negative unique markers amplified by the primer HB-12 (300, 170, 150 bp). The quinoa genotype QL3 exhibited six positive unique markers HB-12 (410 bp), HB-11 (800, 700 bp), HB-10 (300 bp), 17898A (500 bp) and 14A (480 bp) and two negative unique markers amplified by the primers HB-9 (700 bp) and 14A (300 bp).

3.2 Genotype Identification by Unique ISSR Markers Unique markers are defined as bands that specifically identify an accession from the other by their presence or absence. The bands that are present in one accession but not found in the others are termed positive unique markers (PUM), in contrast with the negative unique markers (NUM), which are absent in a specific genotype. These bands could be used for genotype identification [20,21]. As shown in Table 4, the ISSR assay permitted the identification of all five quinoa genotypes by unique positive and/or negative markers. Data showed a total number of unique ISSR markers of 24; eleven of them were positive and 13 were negative. 7

Al-Naggar et al.; BJI, 20(1): 1-12, 2017; Article no.BJI.37053

Table 4. Unique positive and negative ISSR markers generated for five quinoa genotypes, marker size (bp) and total number of markers identifying each genotype Negative unique markers Primer Total (band size/bp) no. HB-9(700) 2 14A (300)

CICA-17

Positive unique markers Primer Total (band size/bp) no. HB-12(410) 6 HB-11 (800, 700) HB-10 (300) 17898A (500) 14A (480) HB-15 (350) 2 HB-14 (480) 17898A (410) 1

CO-407

HB-14 (810)

Ollague

HB-15 (910) -

Quinoa genotype

QL-3

Chipaya

Total

Grand total 8

HB-12 (300, 170, 150)

3

5

HB-9(600)

1

2

1

HB-15 (180) HB-14 (700) HB-11 (450)

3

4

1

HB-15 (800,450) 17898A (700) 14-A (400)

4

5

13

24

11

that ISSR analysis can provide a fast detection of ISSR markers linked to quinoa genotypes. These markers would help in breeding programs of quinoa.

However, the genotype Ollague exhibited one positive unique marker amplified by the primer HB-15 (910 bp) and three negative unique markers amplified by the primer HB-15 (800, 450 bp), 17898A (700). bp) and 14-A (400 bp). The size of these unique markers ranged from 150 to 910 bp.

3.3 Genetic Relationships among the Five Quinoa Genotypes

The number of bands and the percentages of polymorphism found in this study are suitable for estimating genetic diversity when compared with others species that used ISSR markers [16,37,38]. Using ISSR analysis, we were able to identify five unique bands associated with quinoa genotypes.

The scored data from the ISSR analysis in this study were used to compute the similarity matrices according to Dice coefficient [35]. As shown in Table 5 the genetic similarity ranged from 49% (between Ollague and each of QL-3 and Chipaya) to 76% (between CICA-17 and CO-407). The results of this investigation indicated that all the five quinoa genotypes differ from each other at the DNA level where the average of genetic similarity (GS) between them was about 59%.

Further experiments need to be achieved to determine the linkage between the ISSR markers used in the present study and gene(s) of quinoa genotypes. The present results support the idea

Table 5. Genetic similarity (GS) matrices among the five quinoa genotypes Genotype QL-3 Chipaya CICA-17 CO-407 Ollague

QL-3 1 0.64 0.51 0.52 0.49

Chipaya

CICA-17

CO-407

1 0.69 0.59 0.49

1 0.76 0.62

1 0.60

8

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Also, this technique could detect enough polymorphism in the studied quinoa genotypes to distinguish each genotype from the others by at least one specific fragment. Furthermore, the use of these results in the future is important for quinoa germplasm management and improvement as well as for the selection strategies of parental lines that facilitate the prediction of crosses in order to produce hybrids with higher performance as was indicated by several investigators [41-44].

In this context, some investigators reported wide genetic dissimilarity among quinoa accessions [23,24,26,27,39,40]. Genetic dissimilarity could be utilized by plant breeders in hybridization programs for using heterosis phenomenon or for initiating genetic variability amenable for efficient selection.

3.4 Cluster Analysis as Revealed by ISSR The Dice ISSR-based coefficients of genetic similarity among the five quinoa genotypes were employed to develop a dendrogram using the UPGMA method (Fig. 2). The dendrogram separated the quinoa genotypes from each other. Genotypes were separated into two clusters, the first cluster included two genotypes (QL-3 and Chipaya).

In general the overall results indicated the possible use of ISSR analyses to detect some species-specific markers for the five quinoa genotypes that can be used to discriminate among these quinoa genotypes and also, to detect genetic relationships among these genotypes which can be used in breeding programs. The molecular genetic results of these five quinoa genotypes are efficient tools for the characterization of these genotypes.

The second cluster was sub-divided into two groups; the first group included two genotypes (CICA-17 and CO-407) and the second group included only one genotype (Ollague).

Similar results have been reported in other studies of genetic diversity in the genus Chenopodium using microsatellite markers [26,38,39]. Considering both the conditions under which quinoa is cultivated and its genetic variability, the plant has a remarkable adaptability to different agro-ecological zones. This adaptability is of great importance for the diversification of future agricultural systems; however, there is an urgent need to strengthen the breeding programs in quinoa (conventional as well as biotechnological) for its genetic improvement and conservation.

In conclusion, the use of molecular markers can increase the efficiency of conventional plant breeding by identifying markers associated with the quantitatively inherited traits controlled by several genetic loci and their genetic components are difficult to measure. Our results indicated that ISSR technique is useful in the establishment of the genetic fingerprinting and estimation of genetic relationships among quinoa genotypes.

Fig. 2. A dendrogram illustrates the genetic distance among quinoa genotypes based on ISSR data

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4. CONCLUSION Our results indicated that ISSR technique is useful in the establishment of the genetic fingerprinting and estimation of genetic relationships among quinoa genotypes. Also, this technique could detect enough polymorphism in the studied quinoa genotypes to distinguish each genotype from the others. Furthermore, the use of these results in the future is important for quinoa germplasm management and improvement as well as for the selection strategies of parental lines that facilitate the prediction of crosses in order to produce hybrids with higher performance. Using ISSR analysis, we were able to identify unique bands associated with quinoa genotypes. These bands might also be used in breeding programs for differentiating among Chinopodium quinoa varieties.

6.

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9.

COMPETING INTERESTS Authors have interests exist.

declared

that

no

competing

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