Garcinia indica Choisy (Thouars; Family Clusia- ceae), is a perennial tree. G. indica is commonly known as a Brindonia Tallow tree or ‘Kokum Butter’ tree in English. Kokum has many uses in cuisines and an important ingredient in locally prepared medicines. The seeds are a rich source of Kokum butter, which is nutritive, demulcent, agent for smoothening, softening and used for cosmetic, confectionery, culinary purposes. Raw fruits, young leaves and bark are also used as medications against several disorders. The fruit rind is a rich source of Hydroxy Citric Acid (HCA) that prevents accumulation of fat in the human body cells. Therefore, G. indica has become the natural source for production of anti-obesity drugs. (Baliga et al., 2011). Garcinia species are endemic and distributed in tropical rain forests of the Western Ghats. Perceiving the threat of over exploitation, FRLHT (Foundation for Revitalization of Local Health Traditions) and IUCN (International Union for Conservation of Nature) have recognized this species as ‘Vulnerable’ and ‘Threatened’ category respectively (Hareesh and Vasudeva, 2010). A few studies examined diversity in this species using general DNA markers like RAPD and ISSR markers (Thatte et al. 2012; Palkar and Sellappan, 2019). However, so far there are no efforts to develop species specific, highly reproducible microsatellite markers or SSR markers in this species. Keeping this in view, an attempt has been made to develop microsatellite or SSR markers using next generation sequencing technology. The development of molecular markers would help in studying its diversity, analyzing the genetics of traits, and further help in evolving conservation strategies and improvement.

The plant material was obtained from the germplasm collection of the College of Forestry, Sirsi (University of Agricultural Sciences, Dharwad), Karnataka state, India. Total genomic DNA was isolated from the leaves of G. indica genotypes using modified CTAB method (Ravishankar et al., 2000). Genomic DNA was sequenced using Illumina HiSeq2000 platform at M/ s Genotypic Pvt. Ltd, Bengaluru facility following manufactures instructions. High quality sequence data was used for assembly into contigs. De novo assembly of reads into contigs was performed using SOAPdenovo2-src-r240 software (Luo et al., 2012). This has resulted in 92125 contigs. The total assembled size of the contigs is approximately 25.6 Mbp. An SSR survey of genomic sequences using MISA software (http:// pgrc. ipk- gatersleban.de/misa), showed that 3590 contigs contained at least one microsatellite (Ravishankar et al. 2015). A total of 3725 microsatellite was identified. A total of 1374 microsatellites (ESM1) primers were designed using Primer3 software (http://bioinfo.ut.ee/primer3-0.4.0/; Untergrasser et al., 2012). From these, randomly 50 loci were selected for initial screening. Finally, 48 SSR primers were selected for genetic analysis based on clear amplification of PCR products. We employed Thirty genotypes of Garcinia indica for assessing polymorphism at each locus. The fluorescence based M13 tailed PCR method of Schuelke (2000) was followed to amplify the microsatellites in a quick, accurate and efficient manner. PCR was carried out in the 20µl reaction volume containing 2µl of 10X reaction buffer, 2.0µl of 1 mM dNTPs, 0.9µl (5 pmol) of forward, 0.9µl reverse primers (5 pmol), labeled M13 probe 1.2µl (5 pmol), 5.0 µl (50-75 ng) of template genomic DNA, 0.8 µl (2 U) of Taq DNA polymerase and 7.2 µl of nuclease free water. The PCR cycling profile was: initial denaturation at 94°C for 2 min, followed by 35 cycles of 94°C for 30sec., 55°C for 30 Sec., 72°C for 1 min and a final extension at 72°C for 5 min. Amplified products were separated on 96 capillary Automated DNA Sequencer (Applied Biosystems, ABI 3730 DNA Analyzer) at M/S Eurofin facility, Bengaluru.

The raw data generated was analyzed and compiled using Peak Scanner V1. 0 software (Applied Biosystems, USA) for estimating the allele size in bp. The allele size data was used for genetic analysis using Cervus 3.0 software (Kalinowski et al . 2007). We have calculated observed heterozygosit y, expected heterozygosit y, polymorphic information content(PIC). The probability of identity (PI) was calculated using IDENTITY1.0 software (http://www.uni-graz.at/~sefck/: Wagner and Sefc, 1999). Genetic analysis of 48 SSR loci, showed PIC values ranging from 0.718 to 0.968 with a mean value of 0.922. The mean values of observed and expected heterozygosity are 0.2813 (Table 1) and 0.933 respectively (Table 1 and 2). The allele per locus ranged from 13 to 41 with a mean of 16.395. The probability of identity (PI) values ranged from 0.00329 to 0.304896 with a mean of 0.03506. The total probability of identity is 8.132729x 10-80. In cross species amplification, out of 48 SSR primers, 6 amplified in G. morella , accounting 12.5 per cent transferability and 9 amplified in G. gummigutta accounting 18.8 percent transferability (ESM2). This relatively low cross-species transferability compared to what has been observed in G. gummigutta species (Ravishankar et al., 2017).

This is the first report of SSR markers for Garcinia indica, where 3725 microsatellites were identified and primers were designed for 1374 microsatellites. The genetic analysis showed that the majority of the SSR primers developed have high PIC values indicating high heterozygosity in the species. The low probability of identity values of many SSR loci is useful for molecular characterization. Finally, the SSR developed will be useful in studying genetic diversity, mapping and fingerprinting of Garcinia indica and related species.

Acknowledgments

Authors acknowledge financial support from UNEP/GEF regional project “Conservation and Sustainable Use of Cultivated and Wild Tropical Fruit Diversity: Promoting Sustainable Livelihoods, Food Security and Ecosystem Services”

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