Genetic analysis of three Amaranth species using ISSR markers

Abstract

Amaranthus is one of the underutilized species distributed and utilized worldwide. The study used a ISSR approach to analyse intra and inter-specific variability of 16 Amarathus caudatus, 19 A. cruentus and 21 A. hypochodriacus accessions. Using of 11 ISSR primers, fragments levels ranged from 15 up to the 24 were obtained. The dendrogram based on ISSR data set individual species separation is observed except of three accessions. Two A. caudatus genotypes originated from India clustered with A. hypochondriacus accessions and one A. hypochondriacus genotype originated from Nepal clustered with A. cruentus accessions. Average similarity index among all 55 analysed genotypes ranged from 0,154 to 1,000 with a mean of 0,522. Highest intaspecific variability is reported for A. hypochondriacus accessions when comparing to the A. caudatus or A. cruentus. This study has demonstared that a single primer marker system as ISSR is able to generate a sufficient level of informative characters for intra and inter-specific molecular analysis of Amaranthus genus.

Headnote

Abstract

Amaranthus is one of the underutilized species distributed and utilized worldwide. In the study was used ISSR approach to analyse intra and inter-specific variability of 16 Amarathus caudatus, 19 A. cruentus and 21 A. hypochodriacus accessions. Using of 11 ISSR primers, fragments levels ranged from 15 up to the 24 were obtained. In the dendrogram based on ISSR data set individual species separation is observed except of three accessions. Two A. caudatus genotypes originated from India clustered with A. hypochondriacus accessions and one A. hypochondriacus genotype originated from Nepal clustered with A. cruentus accessions. Average similarity index among all 55 analysed genotypes ranged from 0,154 to 1,000 with a mean of 0,522. Highest intaspecific variability is reported for A. hypochondriacus accessions when comparing to the A. caudatus or A. cruentus. This study has demonstared, that a single primer marker system as ISSR is able to generate a sufficient level of informative characters for intra and inter-specific molecular analysis of Amaranthus genus.

Key words: Amaranthus caudatus, Amaranthus cruentus, Amaranthus hypochondriacus, ISSR markers

Introduction

The genus Amaranthus L. (Caryophyllales: Amaranthaceae) consists of about 60-70 species. Several of which are cultivated as leafy vegetables or forage, others for grains production and some are planted as ornamental plants. Unfortunately, some are aggressive weeds that affect many agricultural areas of the world. Despite this, cultivated of amaranth plants have a lot of positive. Grain amaranth's balanced amino acid composition is close to the optimum protein reference pattern in the human diet according to FAO/WHO requirements (Mlakar et al., 2010). Presently, amaranth is cultivated in many parts of the world. Mainly, three species of Amaranthus L. are commonly cultivated for grain production: Amaranthus cruentus L., Amaranthus caudatus L. and Amaranthus hypochondriacus L. (Williams and Brenner, 1995; Lanoue et al., 1996). Amaranth has been also regarded as relatively drought tolerant, thus, suggesting that reasonable yield can be realized with limited irrigation. So, amaranth (Amaranthus spp.) is one of the most important leafy vegetables widely grown in the tropics with a potential to broaden man's food base in Africa (Neluheni et al., 2007). Improvement of amaranth in nowadays breeding programms is realized worldwide (Gajdosová et al., 2008; Hricová et al., 2011) what corresponds to the potential of this underutilized crop. Transgenic manipulation was also suggested as an alternative to irradiation, which can reduce fitness of the males and necessitates removal of females for optimum effect (Heinrich and Scott, 2000; Thomas et al., 2000).

Genus Amaranthus consists a large number of species with good facilities so, it has given a signal to increase interest of this plant, one signal to next research of this rediscovered plant. Nowadays, molecular analyses methods provide a base for a wide range of plant biological material assessment. They are involved in different methods from epidemiological changes evaluation (Pochop et al., 2012; Zele?áková et al., 2012), continuing in authentication of food (Zele?áková et al., 2008; Z idek et al., 2012) up to the analyses of biodiversity of wild and cultivated plants (Gregá?ová et al., 2005; Vivodík et al., 2011; Raz ná and Z iarovská, 2011).

Nowadays, two basic data sources exist for molecular base of the Amaranthus genus. The first genomics - based resources derived specifically from Amaranthus species are available from BAC library constructed from A. hypochondriacus (Maughan et al., 2008). The second Amaranthus genomics resource to become available is a set of microsatellite markers (Mallory et al., 2008). Nearly 400 unique microsatellite markers were obtained primarily from microsatellite - enriched libraries but also from BAC - end sequence data. About 180 of these proved to be polymorphic across three grain Amaranthus species.

The genus Amaranthus L. is known with high inter- and intraspecies variability (Mosyakin and Robertson, 1996; Marhold and Hindák, 1998). Inter alia, molecular tools serve to detect of this inter- and intraspecies variability of organisms, considering they provide valuable data on diversity through their ability to detect variation at the DNA level (Somasundaram and Kalaiselvam, 2011). DNA markers are more suitable for detection of intra- and interspecies variability when compared to the morphological ones, because they are not affected by environment (Gilbert et al., 1999; Duran et al., 2009).

Different techniques are reported as suitable for amaranth germplasm evaluation and screening of genetic diversity using DNA based markers. RAPD (Randomlly Amplified Polymorphic DNA) utilization in studies on genetic diversity in Amaranthus species is reported by Popa et al. (2010) or Tony-Odigie et al. (2012), ITS (Internal Transcribed Spacers), AFLP (Amplified Fragment Length Polymorphism) and double-primer fluorescent ISSR (Inter Single Sequence Repeats Polymorphism) approaches are reported by Xu and Sun (2001) and ISSR markers was also used by Ray and Roy (2007) and Nolah at al. (2010) for different amaranth species molecular studies.

Microsatellites are a common source of polymorphism used for molecular analysis of wide range of plant species. Two basic approached are based on microsattelites - SSR (Gregá?ová et al., 2005; Vivodík et al., 2011; Hulín et al., 2012) and ISSR. Conventional ISSR approach is used with oligonucleotides composed of defined short tandem repeat sequences that represented a variety of different microsatellite types. As a marker, in ISSR the oligonucleotide is used in a single primer amplification reaction. Using this approach, 11 of 30 tested ISSR markers were used for estimation of genetic relationships among and within amaranths and chenopods by Ray and Roy (2007). A total of 177 loci were obtained and 42.5% polymorphic bands were reported for leafy amaranth types and 71.6% for grain types.

The objective of presented study was to evaluate and screen genetic diversity of three amaranthaceae species - Amaranthus caudatus L., Amaranthus cruentus L. and Amaranthus hypochondriacus L. by ISSR markers. Obtained data were used for dendrogram constructing where the relationships based on the detected length polymorphism are visualized.

Materials and Methods

The seeds of 16 Amaranthus caudatus L, 18 Amaranthus cruentus L. and 21 Amaranthus hypochondriacus L. genotypes (table 1) with different origin were obtained from North Central Regional PI Station (NC 7), Iowa State University, Ames.

The amaranth seedlings were cultivated under in vitro conditions on Murashige and Skoog (1962) medium. DNA from fresh young leaves was isolated according to Rogers and Bendich et al. (1994) optimized protocol. Each genotype was represented by ten individuals. The amount of DNA in reaction (10-80 ng in 25 µl total volume), the primer concentration (0.2-1.5 µmol.dm-3), MgCl2 concentration (0.75-1.5 mmol.dm-3), deoxyribonucleotides concentration (0.1-0.2 mmol.dm-3) and the annealing temperature (45- 60)°C.

A total of 11 primers were used in analysis (table 2). All reactions were repeated three times. According to the optimization, PCR conditions were assigned 20 mmol.dm-3 Tris-HCl, pH 8,0 (Invitrogen(TM), Life Technologies), 50 mmol.dm-3 KCl (Invitrogen(TM), Life Technologies), 1 U Taq polymerase (Invitrogen(TM), Life Technologies), 3 mmol.dm-3 MgCl2 (Invitrogen(TM), Life Technologies), 0,1 mmol.dm-3 deoxy ribonucleotides (Promega), 0,2 µmol.dm-3 primer (Invitrogen(TM), Life Technologies, Table 2), 20 ng DNA in 25 µl total reaction volume.

The PCR cycling conditions were as follows: 94°C for 2 minutes (initial denaturation), then followed by 45 cycles at 94°C for 1 minute (denaturation), 50°C for 1 minute (annealing), 72°C for 2 minutes (polymerisation) with a final 7 min extension at 72°C and then cool down to 4°C. The PCR products were separated by electrophoresis on 2% agarose gel (3: 1, Amresco) containing 0.5 µg.ml-1 ethidium bromide in a 1 × TBE buffer and then photographed under UV light using KODAK EDAS 290. The ISSR bands were scored using the binary scoring system that recorded the presence and absence of bands as 1 and 0 using KODAK 1 D program. Genetic similarity was calculated on the basis of Nei Li (1979) coefficient. The resulting matrix of genetic similarity was used to construct the dendrogram through UPGMA with statistic program SYNTAX.

Results and Discussion

Many different methods of identification have been used for evaluation of amaranth diversity. RAPD analysis was successful in the investigation of the relationships of four A. hypochondriacus varieties (Barba de la Rosa et al., 2009). AFLP markers were successfully used to determine species what demonstrated taxonomic ambiguity at the basic morphologic level (Costea et al., 2006). Other methods such as ITS, ISSR and isozyme profile were used to get exhaustive view of interrelationship and relative closeness among amaranth species (Das, 2011; Xu and Sun, 2001).

As AFLP results in amaranth germplasm analysis are reported as sharing many features in common with the ISSR dentrograms by Xu and Sun (2001), ISSR was chosen for sixteen A. caudatus L., eighteen A. cruentus L. genotypes and twenty one A. hypochondriacus L. genotypes. ISSR markers are more comfortable and lab effective and are able to produce uniquely fingerprints as well as RFLPs, AFLPs, RAPDs and SSRs as reported for Amaranthus accessions in Xu and Sun (2001). Not only leafy and grain species of Amaranthus are in the center of nowadays molecular markers research. Nolah et al. (2010) reported ISSR as amployed to measure both genetic diversity and the phylogenetic position of A. pumilus. Using ISSR markers, genetic variation was detected among and within A. pumilus populations, though variability was low. In the study, authors also compared genetic variability of A. pumilus to the variation of A. hypochondriacus and A. cruentus, because of the desirable characteristics of A. pumilus in plant breeding trials. A. pumilus is reported here as having lower genetic diversity as analysed grain species (Nolah et al., 2010).

Primers used in the study ranged 50-72% GC content. Seven from eleven primers generated consistent profiles. Three of analyzed primers (CA)6AG, (CT)8AC and (GA)6CC were used to assess inter and intra-specific diversity. The number of band levels ranged from 15 [primer (CT)8AC] to 24 [primer (GA)6CC]. An average of 6, 11 loci per primer was produced, ranging from a minimum of 4,8 loci using (CA)6AG to a maximum of 7.9 loci using (GA)6CC.

Cluster analyses performed with the ISSR data matrix generated by all primers could group genotypes at the inter-specific level. A. caudatus L. genotypes were clustered with the value of Euclidian Distance Averages of Clusters (EDAC) 0.414; A. cruentus L. genotypes clustered at EDAC 0.450 and A. hypochondriacus L. at EDAC 0.459. A. caudatus L. and A. cruentus L. genotypes clustered together at EDAC 0,533 that indicates more genetic relationships at the inter-specific level. A. hypochondriacus L. genotypes clustered with genotypes of two grain amaranths at EDAC 0,621 (Figure 1). A. caudatus L. PI 480816 IC-38286 and PI 480854 IC-38313 genotypes grouped with A. hypochondriacus L. genotypes at EDAC 0.459. Based on ISSR data two of A. caudatus L. genotypes originated from India are probably more related to A. hypochondriacus L. A. hypochondriacus L. Ames 2086 RRC 149 originated from Nepal grouped with A. cruentus L. at EDAC 0.471, which is probably more related to A. cruentus.

The average similarity index among 55 genotypes ranged from 0.154 to 1.000 with a mean of 0.522 indicating high variation among amaranth genotypes. The cophenetic correlation coefficient was 0.9317. These results are in concordance with finding of Ray and Roy (2007) who reported genetic similarities among Amaranthaceae individuals ranged from 0.06 to 0.85. In dendrogram the same authors have reported, A. caudatus and A. cruentus are grouped into the one cluster at the same level and A. hypochondriacus accessions are connected to them at the following closest level. This grouping pattern is repeated fully in the dendrogram obtained in this study (Figure 1).

At the interspecific level, all individual primers with all genotypes used in the study were tested. Genetic diversity of Amaranthus caudatus L. was analyzed by eight ISSR primers. The average similarity index was 0.72 and the average resolving power (Rp) value was 6.30. The value of average similarity index in Amaranthus cruentus L. was 0.74 and Rp value 5.28. The Rp value in the analysis of Amaranthus hypochondriacus L. genotypes was lower than in other amaranth species (4.24) and the average similarity index was 0.73. More than 75% of Amaranthus caudatus L. genotypes were differentiated using four ISSR primers (CA)6GT, (CA)6AG, (CA)6GG and (GT)6CC. Primer (GT)6CC distinguished 88% genotypes of Amaranhus caudatus L. In Amaranthus cruentus L., only three primers differentiated more that 75% genotypes and in Amaranthus hypochondriacus L. only one primer (GTG)3GC distinguished 62% genotypes, that indicates less polymorphism at the interspecific level in comparison to Amaranthus caudatus L. and Amaranthus cruentus L.

Ray and Roy (2007) reported ISSR primers as producing varying numbers of DNA fragments among amaranthaceae, depending on their SSR motif. They reported, first - dinucleotide repeats (CA)8 and (AC)8 as having good fingerprint patterns and second - (CA)8T did not show any amplification by Amaranthaceae indicating that CA repeats flanked by either G or A and not by T. Very similar, in this study, primers with the core sequence of CA repeats give the highest polymorphism among all tested species - 88.24% for A. caudatus and 100% for A. cruentus and A. hypochondriacus. Wang et al. (1994) reported that dinucleotide microsatellites are prevalent in plants while mono-, tri- and tetranucleotide repeats are less common. This is confirmed again the results presented here. Dinucleotide cored primers (except of CA repeats) give the polymorphism ranged from 83.33% to 100% while trinucleotite cored primers polymorphism ranged from 61.11% to the 72%.

According to ISSR data, two Amaranthus caudatus L. genotypes PI 568147 from Bolivia and PI 175039 RRC from India clustered together using (GTG)3GC, (GAG)3GC, (GA)6CC and (CT)6CC, indicating higher level of genetic similarity between them. High level of genetic similarity revealed (CA)6GG, (GTG)3GC, (GAG)3GC, (CT)8AC and (GA)6CC primers in Amaranthus caudatus L. PI 480816 IC-38286 and PI 480854 IC- 38313. Two genotypes originated from India clusterd together with 0,000E+00 EDAC value.

On the basis of ISSR data, genotype Amaranthus cruentus L. PI 511876 Huatle originated from Mexico clustered as a single cluster using three primers - (CA)6GG, (CT)8AC, (GA)6CC in PCR, which indicates less genetic relationships between the PI 511876 Huatle genotype and other genotypes. Two primers (CA)6AG and (GTG)3GC were able to distinguish four genotypes of Amaranthus hypochondriacus L. originated from Mexico from other genotypes used in the study. Genotypes PI 477916 RRC 1023 with Ames 5209 RRC 457 and Ames 5132 RRC 363 with Ames 5321 RRC 539 were clustered with the 0,000E+00 EDAC value using ISLA-(GA)6CC in PCR (Figure 2). Amaranthus hypochodriacus Ames 2086 RRC 149 originated from Nepal clustered as a single cluster with different EDAC values ranging from 0,159E+0,2 to 0,377E+02 with three ISSR primers (GTG)3GC, (CT)8AC, (GA)6CC indicating less genetic similarity in comparison to other genotypes.

Amaranthus, as well as other crops from central and south America became rediscovered and are studied intensively from different points of view from antioxidant activity through sustainable management up to the molecular based polymorphism (Giuffré et al., 2011; Celis et al., 2011; Milella et al., 2011; Hernández et al., 2012; Martínez at al., 2012). Amaranth, as a promising crop will need a good knowledge about germplasm collections for genetic improvement. However, still only limited information is available on intra- and inter-specific genetic diversity and relationships within Amaranthus germplasm collections exist and results of this study report a new insight into it.

Conclusion

ISSR based evaluation of 55 accessions were done. The accessions belong to the three amaranthaceae species - Amaranthus caudatus L., Amaranthus cruentus L. and Amaranthus hypochondriacus L. As different origins and breeding stages of the tested species were involved, the ability of inter-species grouping of ISSR markers was proved in the study.

Acknowledgments

We thank to D. Brenner of the USDA North Central Regional Plant Introduction Station at Ames, Iowa, USA, for the supply of seed material. This work was supported by ECPUA - Excellent Center of Protection and Use of Agrobiodiversity (0.5) and KEGA - 001SPU-4/2012 - Plant Genetic Technologies (0.5).

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