Development of an ISSR based SCAR marker to identify small cardamom Malabar (prostrate panicle) variety (Elettaria cardamomum Maton.)

Small cardamom (Elettaria cardamomum Maton) a perennial monocot, exhibits an array of variation in nature, mainly due to cross pollination. Based on the nature of the panicle orientation, cardamom is broadly grouped into three main ‘cultivated types’ – Malabar, Mysore and Vazhuka having prostrate, erect and semi-erect panicles respectively. These morphologically discriminative markers manifest itself during panicle emergence as is only possible. Among the three varieties Malabar variety is relatively superior with respect to different qualitative and quantitative characteristics. The objective of the present study was to develop and characterize molecular markers for enabling differentiation of Malabar variety at juvenile stage. One accession specic ISSR marker generated by UBC 866 was selected which consistently amplied an intact, distinct, (cid:0) 1500bp band specically in individuals of Malabar variety, which was therefore cloned, sequenced and characterized. Ten primers were designed from the sequences for converting them to SCAR markers. The developed SCAR markers were tested for variety specicity and one primer pair (SBBT4F/SBBT3R) was validated using small cardamom accessions belonging to Malabar variety from different geographic locations and varieties with erect panicles as well as hybrids. The ndings suggest that the SCAR marker is promising in identifying cardamom varieties having prostrate panicle (Malabar) and therefore is expected to make signicant contributions in selection of F1 hybrids during breeding programmes.


Introduction
Small cardamom (Elettaria cardamomum Maton) popularly known as 'Queen' among spices, is a rhizomatous perennial belonging to the family Zingiberaceae. It is considered as one of the most favoured spices in national and international cuisines by virtue of its versatility to impart the unique avour -the sweetness together with pungency and aroma, to sweets and savouries. Because of its richness in essential oil and therapeutic components, small cardamom becomes the raw material for various pharmaceutical formulations. India has a rich wealth of genetic resources of cardamom as the crop originated in the tropical forests of Western Ghats (Mayne 1951;Abraham and Tulasidas 1958) and thus has a long tradition since centuries in the country. The cardamom varieties are highly location speci c and therefore different varieties are adapted to different geographical regions along the Western Ghats (Anon 2009). Hence development of high yielding varieties speci c to particular eco-geographic situation is of paramount importance.
The three main cultivated types or natural varieties of cardamom viz. Malabar, Mysore and Vazhukka were grouped mainly based on the nature of the panicle -prostrate, erect and semi erect respectively and is regarded as the major discriminative morphological markers of cardamom (Madhusoodanan et al. 2002).
Among the three varieties of small cardamom, Malabar variety possesses several distinctive characters such as its relative tolerance to drought (Sudarsan et al. 1991), higher number of panicles per plant (Padmini et al. 1999), early maturing character (Madhusoodanan et al. 2002) which is suitable for high density planting (Madhusoodhanan et al. 2002) and the relatively higher avonoid content of 0.159% (Govindarajan et al., 1982). It was also reported that most of the resistant genotypes reported in cardamom are of Malabar type (Madhusoodhanan et al. 2002). However, all the three varieties and races of cardamom are inter-fertile and the observed variations are probably due to natural crossing (Korikanthimath 2003).
Cultivar selection during breeding programmes can be extremely di cult for a perennial like small cardamom as morphological discrimination of the three varieties is possible only after panicle emergence.
Molecular markers including SCAR markers are an effective tool that can be used for varietal authentication and species identi cation at a very early developmental stage itself when key morphological features are indistinguishable (Das et al. 2008, Ghosh et al. 2011. SCAR markers have emerged as tools to distinguish between species that have similar morphology and phenotype, in several plants such as in spruce (Nkongolo et al. 2003 Variety speci c ISSR markers were previously identi ed in cardamom by the present authors (Sherin et al. 2013) but were not developed and validated as SCAR marker. In the present study we describe the conversion of ISSR derived speci c marker to SCAR and its validation on small cardamom varieties. This is the rst report on generation of SCAR markers in small cardamom.

Plant material
A total of 50 small cardamom accessions from the germplasm repository of Indian Cardamom Research Institute (ICRI), Spices Board, Kerala, India and two wild varieties from Regional Research Stations of ICRI were used to screen for variety speci c markers (Table-1). Sources of genetic materials included 39 accessions and 11 released varieties. The accessions were selected based on the morphological analysis (IPGRI, 1994) and the panicle morphology was con rmed with the previous records available at ICRI. All the information concerning the collected samples was shown in Table 1. Secondly, the variety-speci c marker was validated on 24 small cardamom accessions, which included 21 Malabar, 2 Mysore and 1 Vazhukka accessions. Fresh, young leaves of the small cardamom varieties were collected for DNA extraction. The method of genomic DNA isolation followed the procedure developed by the authors in a previous study (Mathew et al. 2013). The integrity and quality of DNA were evaluated using 1.0% agarose gels electrophoresis, and the concentration of genomic DNA was determined using a UV spectrophotometer (Hitachi) by measuring absorbance at A260 and A280 nm and also using Nanodrop UV/Vis spectrophotometer. Based on the quanti cation data, DNA dilutions were made in TE buffer to a nal concentration of 25ng/µl and stored at -20°C for further use.
Ampli cation Of ISSR Markers PCR ampli cations were performed using UBC 866 in 25µl reaction mixtures containing 25ng genomic DNA, 2.5mM MgCl2, 1X PCR buffer, 10µM primer, 200µM of each dNTP (Genei, Bangalore) and 1 unit of Taq DNA polymerase (Genei, Bangalore). Ampli cations were carried out using a thermal cycler (Biorad) with an initial denaturation/activation step of 4min at 95°C followed by 40 cycles of 30s at 94°C, 60sec at annealing temperature and 2min extension at 72°C. A nal extension for 10min at 72°C was included.
Optimal conditions were determined based on the resolvable PCR products generated by each primer. A negative control which contained all the PCR components except DNA (replaced by water) was included in every experiment to test for DNA contamination of the reagents. PCR products were subjected to electrophoresis on 2% agarose gels, stained with ethidium bromide (0.5µg/ml) and documented in a gel documentation imaging (Biorad, USA). The fragment sizes were estimated by comparing to 250bp and 100bp DNA ladders (Genei, Bangalore).

ISSR Analysis And Marker Selection
Each of ISSR ampli cation was performed at least twice to verify the reproducibility of the results and only repeatable amplicons were selected. The ampli ed products were recorded as '1' for presence or '0' for absence of the bands. The presence of an ISSR band in a particular variety, and its absence in all the other varieties, was considered to be a variety-speci c marker.

Identi cation and gel elution of variety speci c -ISSR markers
Among the generated ISSR ngerprints, the ~ 1500 bp fragment that ampli ed speci cally in Malabar variety (prostrate panicle) was eluted using GeneJET Gel Extraction kit (Thermoscienti c) following manufacturer's instructions.

Cloning And Sequencing Of ISSR UBC 866 Fragment
The puri ed DNA fragment was ligated into pTZ57R (Fig-2) using InsTAclone PCR cloning kit (Thermoscienti c) according to the manufacturer's instructions and transformed into ultra-competent Escherichia coli strain JM109 and then plated onto LB/ampicillin/IPTG/X-Gal plates. Positive colonies were determined by blue/white screening. The presence of the insert and its orientation in the puri ed plasmids was con rmed by colony PCR using M13 Universal primers. The reactions were performed in an appropriate thermocycler (Biorad, USA) adopting the following programme, 94°C for 2 minutes for initial denaturation, followed by 30 cycles of 94°C for 30s, 45°C for 30s and 72° C for 1min. This was followed by 72° C for ve minutes for nal extension. PCR products were separated and the colonies with desired size of insert were sequenced, using M13 universal primers in both forward and reverse directions, by Scigenome (Kochi, Kerala) using an automated ABI 3100 DNA sequencer (Applied Biosystems, Foster City, CA) method.

Sequence Data Analysis, Scar Primer Design And Validation
With the obtained nucleotide sequence, the similarity of the sequence was determined using the BLAST program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Based on the sequences of the cloned ISSR product, ten pairs of SCAR primer was designed using the Primer3 software. Care was taken to avoid possible secondary structure or primer dimer generation, false priming to match melting temperatures and to achieve appropriate internal stability while generating SCAR primers.   The primer UBC866 produced a clear fragment (approximately 1500bp) unique to Malabar variety (prostrate panicle), but was absent in all DNA samples of Mysore and Vazhukka varieties (Fig. 2). This fragment produced by UBC-866 was selected as a putative variety-speci c marker.

Cloning of ISSR UBC 866 1500 fragment
The characteristic DNA fragment ampli ed by ISSR UBC 866 was eluted and ligated to pTZ57R vector by InsTAclone PCR cloning kit. Blue and white colonies were obtained and white colonies were cultured (Supplementary gure S1). The positive clones were then con rmed by PCR ampli cation using M13 universal primers (Supplementary gure S2). Blue colony (without insert) was used as a control for comparing the results. All clones from recombinant colonies showed fragments similar to the inserted fragment except the blue colony, which produced fragment of ~ 750bp in size. The recombinant clones were selected for sequencing.
The sequence of the prostrate panicle-speci c fragment of 1534 bp (Fig. 3) was deposited in GenBank (Accession number: MN276062). The sequence analysis of cloned product revealed that the 1534 bp ISSR fragment was bordered by the original bases of the microsatellite repeats and no repeats were found within the sequence. BLAST searches of the nucleotide sequences with the NCBI nucleotide database using BlastN tool did not reveal any signi cant match with any known nucleotide sequence, which con rmed the rst report of this SCAR marker.
To generate a stable Malabar-speci c diagnostic SCAR marker, ten pairs of primers were designed and synthesized based on cloned sequences and were named SBBT 1-10 (Supplementary table-1). The e cacy of each SCAR primer set for ampli cation of speci c PCR product size in particular variety was assessed using DNA from each of the three varieties. Of the 10 SCAR primers designed only a single primer pair SBBT4F and SBBT3R (Table 4) produced speci c fragment in Malabar variety and this was used for further validation. The SCAR marker primer pair (SBBT4F and SBBT3R) was used to amplify 24 samples to test varietyspeci city at an annealing temperature of 57°C (Fig. 4). The 24 tested accessions include 21 Malabar accessions (prostrate panicles), 2 erect panicles and 1 semi-erect panicle types. A clear speci c band was detected in 95% of prostrate panicle type but not in samples of erect and semi-erect accessions (Fig. 4). The result revealed that the SCAR primer SBBT 4 F and SBBT 3 R that produced speci c fragment (~ 1350 bp) in prostrate panicle is indicative of its Malabar-speci city. Moreover, the initial UBC866 primer was also analysed in the same gel in Malabar and Mysore accession to con rm the product size and speci city. This validated the relevance of designing Malabar-variety speci c SCAR marker.

Discussion
The present study was to identify suitable ISSR polymorphisms among cardamom varieties and to transform them into more-speci c SCAR markers in an e cient and reliable manner. ISSR reported rst by Zietkiewicz (1994) is a popular marker system, owing to their reproducibility, ability to detect polymorphisms, and cost-effectiveness. The reliability of the ISSR marker system has been supported by different investigators in a wide range of crop species including wheat (Nagaoka & Ogihara 1997) and rice (Joshi et al. 2000 Designing SCAR markers for varietal identi cation would be of immense use for crops like cardamom, where accurate identi cation of taxonomic status persists as a long-term problem. Due to many remarkable characteristics of Malabar variety, the present results with SCAR markers would be very useful for identifying Malabar varieties and thereby reducing the time and labour during breeding programmes.
This is the rst study on ISSR-SCAR in Elettaria for varietal identi cation.

Conclusion
The variety-speci c SCAR marker developed during the course of the present investigation proved to be reliable and e cient for discriminating small cardamom varieties based on panicle morphology. The

Ethics approval
This article does not contain any studies with human participants or animals performed by any of the authors.