Molecular characterization of curry leaf genotypes using ISSR markers

Murraya koenigii (L.) Spreng is a perennial herbal spice, deciduous to semi evergreen aromatic tree that belongs to the family Rutaceae. India is the leading producer and exporter of curry leaves¹. Tamil Nadu is the leading state in curry leaf cultivation with an area of 2, 543. 00 ha and a production of 35,932.81 tonnes². It is mainly grown in Coimbatore, Theni and Salem districts and Coimbatore stands in the first position with an area of 1,215.00 ha and a production of 17,010.00².

Few varieties like DWD-1, DWD-2 and Senkaampu (a local type found in the Tamil Nadu region) are cultivated commercially in India¹. Information related to the genetic variability in curry leaf is very limited. In a previous study a total of 152 bands were amplified, out of which 136 were polymorphic corresponding to 89.47% polymorphism across the accessions³. Genetic diversity of 61 fenugreek accessions by using 18 random primers resulted in 66–100% polymorphism with genetic similarity of 0.66 to 0.90, indicating a moderate to high genetic variability⁴.Twenty eight ISSR primers amplified 6.89 average numbers of bands per primer and 5.07 average numbers of polymorphic bands per primer in Coriander⁵. An evaluation of fifteen cultivars of Betelvine (Piper betle) by using 18 ISSR primers resulted in average polymorphic information content (PIC) of 0.32 over all primers used. Jaccard’s similarity coefficient showed dendrogram grouped cultivars in 3 main clusters⁶.Thirty four ISSR markers were used for nutmeg diversity analysis. They were polymorphic with a band size of 300 to 1500 bp. Two major clusters were classified based on the Jaccard’s similarity coefficient at a coefficient value of 0.62⁷. Moringa accessions showed the average band size of 202–3010 bp. Three clusters were made based on the RAPD fingerprints with 88.5% similarity level⁸.

Phenotype of a crop is greatly influenced by genotype and environment. In perennial crops, a study of stability of a morphological character in varying environment and to develop varieties which show small genotype-environment interaction is time consuming⁹. Identification of suitable genotypes, therefore, becomes imperative for promoting its quality and production, productivity and quality. Hence the present study was formulated with the objective of evaluation and characterization of different Curry leaf accessions through molecular techniques.

DNA extraction

Fresh healthy and recently matured leaves were collected from each accession.The germplasm material used for the studyis given under Table 1. One hundred milligrams of fresh leaf tissues were ground using liquid nitrogen in a sterile pestle and mortar. The powdered leaf tissues were collected in a 2 ml microcentrifuge tube and 500 µl freshly prepared CTAB extraction buffer (2% CTAB, 100 mMTris-base, 20 mM EDTA and 1.4 M NaCl) with a pH of 8, 1 µl of mercaptoethanol and 1% PVP was added. The microcentrifuge tube was then incubated at 65^oC in the water bath for 1 h with intermittent shaking. The samples were then treated with an equal volume of 24:1 chloroform: iso-amyl alcohol and incubated for 15 min with intermittent shaking followed by centrifugation at 12,000 rpm for 10 min. The supernatant was collected in a fresh 1.5 µl micro centrifuge tube and an equal volume of ice cold isopropanol was added to the supernatant and incubated at -20 C over- night. The contents were centrifuged at 12,000 rpm and the supernatant was decanted. The collected pellet was washed with 100 µl of 70% ethanol (centrifuged at 10000 rpm for 10 min) twice, air dried immediately and dissolved in 50 µl of st erile water followed by storage at -20 °C until utilization¹⁰. The integrity and purity of genomic DNA was confirmed by gel electrophoresis in 0.8% agarose gel, carried out at approximately 70 V for 30 min in 10X TAE buffer and visualized with ethidium bromide staining under UV light. The DNA concentrations were determined at 260 nm and 280 nm using the Nanodrop^® ND-1000 spectrometer and where necessary the DNA solution was diluted to a working concentration of 50 ng µL-1 using sterile distilled water.

Inter simple sequence repeat (ISSR) analysis

Thirteen primer sets designed for curry leaf were used to amplify ISSRs in 30 curry leaf genotypes. The primers used in the study are listed below (Table 2).

Components of PCR reaction mixture for ISSR

The amplification was performed in a total reaction volume of 10 µL containing the following:

• DNA sample (50 ng): 1 µl.

• Primer (10 µM) : 1 µl.

• Master Mix : 4.0 µl.

• Sterile distilled water : 4 µl.

• Total volume : 10 µl.

The PCR amplification procedure is as follows:

1. 1.

   Initial denaturation at 94 ⁰ C for 4 min.

2. 2.

   Denaturation at 94 ⁰ C 60 s.

3. 3.

   Annealing (Depends upon the primer used) 60 s 35 cycles.

4. 4.

   Extension at 72 ^o C 2 min.

5. 5.

   Final extension at 72 ^o C for 7 min.

6. 6.

   Hold at 4 ^o C till storage.

Data analysis and data scoring

Polymorphic bands were scored visually for their presence or absence for each primer. The scores were obtained in the form of a matrix with ‘1’ and ‘0’, which indicate the presence and absence of bands in each genotype respectively.

Polymorphism information content (PIC) value

Polymorphism information content (PIC) values were calculated for ISSR markers, to characterize the capacity of each primer to reveal or detect polymorphic loci among the genotypes. PIC values may range from 0 (monomorphic) to 1 (very highly discriminative), with many alleles in equal frequencies.

PIC value was calculated using the formula¹¹. PICJ = 1- ∑ P2 I = 1 to L ij Where ij is the relative frequency of the lth allele for the locus j and is summed across all the alleles (L) overall lines.

Cluster analysis

Genetic dinversity estimate related analyses were done using NTSYSpc software, version- 2.02i¹². The binary data scoring was used to construct a dendrogram. The genetic associations between accessions were evaluated by calculating the Jaccard’s similarity coefficient for pairwise comparisons based on the proportions of shared bands produced by the primers¹³. The similarity matrix was generated using the SIMQUAL programme and the clustering of genotypes was done using an Unweighted Pair Group Method (UPGMA) with an arithmetic average algorithm following the SAHN module. The clustering result was used to construct a dendrogram following the TREE module¹⁴.

Polymorphic information content (PIC)

The Polymorphic Information Content is a measure of polymorphism. Among the 13 primers, only 12 produced the discrete bands. The PIC value of 12 ISSR primers ranged from 0.56 to 0.92 (Table 3). Twelve ISSR markers recorded PIC values greater than 0.5 and UBC-856 recorded the highest PIC value (0.92) followed by UBC-807, UBC-834 (0.91) and UBC-835 (0.88) indicating the markers are efficient in revealing diversity among the studied genotypes. The average PIC value was 0.83. The banding pattern of UBC_856 and UBC_834 were presented in Figs. 1 and 2 respectively.

ISSR banding pattern for UBC_834.

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ISSR banding pattern for UBC_856.

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The results revealed that ISSR markers were able to do estimate the available genetic diversity within and among different accessions. The polymorphism detected revealed higher genetic diversity as the curry leaf is an out breeding species. Our results are inline with the earlier studies in different species viz., M. koenigii¹⁵Gaultheria fragrantissimsa¹⁶, Lens culinaris¹⁷ and Punica¹⁸.

The higher genetic diversity is also may be maintained from many generations as the natural populations were able to combat with the heterogenetic variations in the ecosystem¹⁹.

The per cent of polymorphism was recorded maximum for the primer UBC_ 836 (62.5%), followed by UBC_881, UBC_856, UBC_857 (50.00%) and UBC_810 (42.86%).Similar results were reported in curry leaf^20,15 and related species M. paniculata²¹.

Cluster analysis using ISSR marker data

The banding pattern of ISSR markers were scored in the form of binary data.

The data was used to compute Jaccard’s similarity index. The similarity index obtained for each pair wise comparison of ISSR markers among 30 genotypes were used to construct dendrogram based on hierarchical clustering and the result is presented in Fig. 3.

Dendrogram of curry leaf accessions based on ISSR analysis using the UPGMA method.

Full size image

The 30 curry leaf genotypes were grouped into nine clusters (Table 4). Cluster- I was the largest comprising of 15 genotypes (Acc. no.1, 2, 27, 29, 26, 7, 3, 4, 22, 11, 6, 19, 24, 28 and Check) followed by Cluster-II (Acc. no. 12 and 14) Cluster- III (Acc. no. 5 and 8) Cluster- IV (Acc. no. 16 and 18) Cluster V (Acc. no. 9 and 10) Cluster VI (Acc. no. 23 and 25) Cluster VII (Acc. no. 15 and 17) and Cluster VIII (Acc. no. 20 and 21) with 2 genotypes each while Cluster IX (Acc. no. 13) had 1 genotype.

The banding pattern of ISSR markers were scored in the form of binary data. Similarity index for each pair wise comparison of ISSR markers among thirty genotypes were used to construct dendrogram based on hierarchical clustering and the result is presented in Fig. 3.

The data was used to compute Jaccard’s similarity index and it ranges from 0.08 to 0.44 (Fig. 3).

The thirty curry leaf genotypes were grouped into nine clusters.Cluster- I was the largest comprising of 15 genotypes (Acc. no.1, 2, 27, 29, 7, 3, 4, 22, 11, 6, 19, 24 ,28, 26 and Check) followed by Cluster-II (Acc. no. 12 and 14) Cluster- III (Acc. no. 5 and 8) Cluster IV(Acc. no. 16 and 18) Cluster V (Acc. no. 9 and 10) Cluster VI (Acc. no. 23 and 25) Cluster VII (Acc. no. 15 and 17) and Cluster VIII (Acc. no. 20 and 21) with two genotypes each while Cluster IX had one genotype (Acc.no.13).These groupings showed distinct genetic relationships influenced by geographic distribution patterns. ISSR markers were able to group the accessions into distinct clusters independent of locality of collection. The markers were able to distinguish them into distinct genotypes with some located in different clusters. The reason behind this is, during molecular characterization the traits at molecular level not influenced by the external source.

The grouping the different accessions according to UPGMA dendrogram showed that the accessions are mixed together irrespective of their geographical origin. The earlier studies in curry leaf also revealed that genetic variation found was not always same with their origin¹⁵.

The genetic nature of the different accessions depicts the combined effects of different elements along with the species pedigree history²². In the present study the variations showed by different accessions followed the same pattern as found in other out crossing plant species^19,23,24.

The genotypes from one region were grouped together in some cases, while they were placed in different clusters in certain cases. The wide genetic variability observed using ISSR markers would be of much valuable for efficient management of germplasm and for effective utilization of materials in breeding programmes to select the superior genotypes of desirable characteristics.

In conclusion present study gave the clear picture of existence of diversity in genotypes at genetic level as DNA markers are not influenced by environmental effects or plant growth stages whereas morphological markers does. ISSR markers proved best for assessing genetic diversity in curry leaf germplasm. This shows that the genotypes which were used in the present study can be utilized for further crop improvement programme.

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Authors and Affiliations

1. Department of Spices and Plantation Crops, Tamil Nadu Agricultural University, Coimbatore, 641003, India

   Dharini Chittaragi & K. Venkatesan

2. Open Distance Learning (ODL), Tamil Nadu Agricultural University, Coimbatore, 641003, India

   M. Ananthan

3. Department of Cotton, Tamil Nadu Agricultural University, Coimbatore, 641003, India

   L. Mahalingam

4. Department of Crop Physiology, Tamil Nadu Agricultural University, Coimbatore, 641003, India

   P. Jeyakumar

5. Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore, 641003, India

   N. Manikanda Boopathi

Contributions

D.C. involved in carrying out the research and article preparation.M.A. involved in formulating the project, article writing, editing and correction. K.V. involved in formulating the project, article writing, editing and correction. L.M. involved in formulating the project, article writing, editing and correction. P.J. involved in formulating the project, article writing, editing and correction. N.M.B. involved in conducting the research, article preparation and correction.

Corresponding author

Correspondence to Dharini Chittaragi.

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