C O N T E N T S JOURNAL OF HORTICULTURAL SCIENCES Volume 16 Issue 1 June 2021 In this Issue i-ii Review Moringa (Moringa oleifera L.): An underutilized and traditionally valued 1-13 tree holding remarkable potential Jattan M., Kumari N., Raj Kumar, Kumar A., Rani B., Phogat D.S., Kumar, S. and Kumar, P. Original Research in Papers Characterization and evaluation of mountain sweet thorn 14-25 (Flacourtia montana J. Grah) collections Tripathi P.C., Ganeshan S., Radhika V. and Shetti D.L. Optimization of methodology for the extraction of polyphenolic compounds 26-35 with antioxidant potential and á-glucosidase inhibitory activity from jamun (Syzygium cumini L.) seeds Arivalagan M., Priyanka D.R. and Rekha A. Genetic variability studies in amaranthus (Amaranthus spp.) 36-44 Agadi A.H., Kolakar S., Lakshmana D., Nadukeri S. and Hanumanthappa M. Morpho-physiological parameters associated with chlorosis resistance to 45-52 iron deficiency and their effect on yield and related attributes in potato (Solanum tuberosum L.) Challam C., Dutt S., Sharma J., Raveendran M. and Sudhakar D. Responses of different Okra (Abelmoschus esculentus) cultivars to water 53-63 deficit conditions Ayub Q., Khan S.M., Hussain I., Naveed K., Ali S., Mehmood A., Khan M.J., Haq N.U., Shehzad Q. Induced variability for yield and its attributing traits in cluster bean 64-68 [Cyamopsis tetragonoloba (L. ) Taub] through gamma irradiation Lavanya H.N., Mishra S., Sood M., Aghora T.S., Anjanappa M., Rao V.K. and Reddy A.B. In vitro multiplication protocol for Curcuma mangga : Studies on carbon, 69-76 cytokinin source and explant size Waman A.A., Bohra P., Karthika Devi R. and Pixy J. Effect of fungicide and essential oils amended wax coating on quality and shelf life 77-90 of sweet orange (Citrus sinensis Osbeck) Bhandari M., Bhandari N. and Dhital M. Post-harvest quality and quantification of betalains, phenolic compounds and 91-102 antioxidant activity in fruits of three cultivars of prickly pear (Opuntia ficus-indica L. Mill) Gonzalez F.P.H., Saucedo V.C., Guerra R.D., Suarez E.J., Soto H.R.M. Lopez J.A., Garcia C.E. and Hernandez R.G. Soil microbial community dynamics as influenced by integrated nutrient 103-113 management practices in sweet basil (Ocimum basilicum L.) cultivation Baraa AL-Mansour and D. Kalaivanan Effect of spectral manipulation and seasonal variations on cut foliage production 114-120 and quality of Philodendron (Philodendron ‘Xanadu’) Sujatha A. Nair, Laxman R.H. and Sangama Short Communications Studies on mutagenic sensitivity of seeds of pummelo (Citrus maxima Merr.) 121-124 Sankaran M., Kalaivanan D. and Sunil Gowda D.C. Isolation and characterization of microsatellite markers from 125-129 Garcinia indica and cross species amplification Ravishankar K.V., Vasudeva R., Hemanth B., Nischita P., Sthapit B.R., Parthasarathy V.A. and Rao V.R. 125 J. Hortl. Sci. Vol. 16(1) : 125-129, 2021 Short Communication Garcinia indica Choisy (Thouars; Family Clusia- ceae), is a perennial tree. G. indica is commonly known as a Br indonia Tallow tr ee or ‘Kokum Butter ’ tree in English. Kokum has many uses in cuisines a nd an important ingr edient in locally prepared medicines. The seeds are a rich source of Kokum butter, which is nutritive, demulcent, agent for smoothening, softening a nd used for cosmetic, confectionery, culinary purposes. Raw fruits, young lea ves 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. (Ba liga et a l. , 20 11) . Ga rc in ia s pecies a r e endemic and distributed in tropical rain forests of the Western Ghats. Perceiving the threat of over exploitation, FRLHT (Foundation for Revitalization of L oc a l H ea lt h Tr a dit ions ) a nd I UC N (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 r epr odu cib le micr os a tellite ma r ker s or S SR markers in this species. Keeping this in view, an attempt has been made to develop microsatellite or SSR markers using next generation sequencing This is an open access article d istributed under the terms of Creative Commons Attribution-NonCommer cial-ShareAl ike 4.0 International License, which permits unrestricted non-commercial use, d istribution, and reproduction in any med ium, provide d the original author and source are credited. Isolation and characterization of microsatellite markers from Garcinia indica and cross species amplification Ravishankar K.V.*1, Vasudeva R.2, , Hemanth B.1, Nischita P., Sthapit B.R.3 Parthasarathy V.A.4 and Rao V.R.5 1ICAR-Indian Institute of Horticultural Research, Bengaluru - 560 089 India 2Department of Forest Biology and Tree Improvement, College of Forestry Sirsi - 581 401 University of Agricultural Sciences (Dharwad), India, 3 Regional project coordinator (UNEP-GEF), Bioversity International, Pokhara, Nepal, 4 National project coordinator (UNEP-GEF), ICAR-Indian Institute of Horticultural Research , Bengaluru, India 5Bioversity International, Rome *Corresponding author e-mail : kv_ravishankar@yahoo.co.in, ravishankar.kv@icar.gov.in ABSTRACT Garcinia indica popularly known as ‘Kokum’ or Murugalu”, is a medium sized evergreen tree found in western-ghats of India. This tree species is highly exploited to produce anti-obesity drugs and culinary purposes. Its population is threatened by over exploitation and loss of habitat. Development of microsatellite markers would help in understanding genetic structure and further to develop appropriate conservation strategies. In this study, using next generation sequencing platform Illumina Hiseq 2000, we have sequenced partial genome of G. indica and identified 3725 microsatellites. Forty-eight microsatellite markers were analyzed using 30 accessions. Polymorphism information content (PIC) values ranged from 0.718 to 0.968 with a mean value of 0.922. Allele per locus ranged from 3 to 33 per locus. Probability of identity values ranged from 0.00329 to 0.30489. Cross species amplification SSR primers in the related species, showed a moderate transferability from 12.5 % (for G. morella) to 18.7%(for G. gummigutta) Key words : Cross-species amplification Garcinia indica; Microsatellite markers and Next-generation sequencing (NGS) 126 Ravishankar et al J. Hortl. Sci. Vol. 16(1) : 125-129, 2021 t ec hnology. T he develop ment of molec u la r mar kers would help in studying its diver sity, analyzing the genetics of traits, and further help in evolving conservation strategies and improvement. T he p la nt ma t er ia l wa s ob t a ine d f r om t he germplasm collection of the College of Forestry, S ir s i ( Univer s it y of Agr icu lt u r a l S c ienc es , Dharwad), Karnataka state, India. Total genomic DNA was isolated from the leaves of G. indica genot yp es u s ing modif ied C TA B met hod (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 per formed 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 u s ing M I S A s of t wa r e ( ht t p :/ / p gr c . ip k- 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) pr imers wer e designed using Pr imer3 softwa re (http://bioinfo.ut.ee/primer3-0.4.0/; Untergrasser et al., 2012). From these, randomly 50 loci were selected for initial scr eening. Fina lly, 48 SSR primers were selected for genetic analysis based on c lea r a mp lif ic a tion of P CR pr odu cts . We employed Thirty genotypes of Garcinia indica for assessing polymorphism at each locus. The fluorescence based M13 ta iled PCR method of Schuelke (2000) wa s 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 7 2 ° C f or 5 min. Amp lif ied p r o du c t s wer e s ep a r a t ed on 9 6 c a p illa r y Au t oma t ed D N A Sequencer (Applied Biosystems, ABI 3730 DNA Analyzer) at M/S Eurofin facility, Bengaluru. T he r a w da t a gener a t ed wa s a n a lyz ed a nd comp iled us ing Pea k Sc a nner V1. 0 soft wa r e (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 a l . 2 0 0 7 ) . We ha ve c a lc u la t ed ob s er ved het er oz ygo s it y, ex p ec t ed het er oz ygos it y, p olymor p hic inf or ma t i on c ont ent ( P I C ) . T he 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 mea n va lu es of ob s er ved a nd ex p ec t ed heter ozygosity 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 c omp a r ed t o wha t ha s been obs er ved in G. gummigutta species (Ravishankar et al., 2017). T his is t he f ir s t r ep or t of S S R ma r ker s f or Garcinia indica, where 3725 microsatellites were identified and pr imers 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 ma ny S S R loc i is u s ef u l f or molec u la r characterization. Finally, the SSR developed will be useful in studying genetic diversity, mapping and finger pr inting of Garcinia indica a nd r ela ted species. 127 Microsatellite markers from Garcinia indica L oc us F or w ar d Se qu en ce R ev er se S eq ue nc e R ep ea t N um be r A lle le s iz e O bs er ve d E xp ec te d P ol ym or ph ic P ro ba bi lit y 5I  3I 5I  3I Ty pe of A ll el e ra ng e H et er oz yg os it y H et er oz yg os it y In fo rm at io n of I de nt it y (k ) (b p) (H o) (H e) C on te nt ( P IC ) (P I) G I_ K V R a5 77 T T T G G C G A G G G T G T T G G T G A G T A C A C G T G TA G G C T G A C A C C A A C C (G T )6 20 14 0- 23 0 0. 34 5 0. 92 4 0. 90 2 0. 01 28 28 G I_ K V R a6 14 T G T G A G T T G T T T G G C AT G G G T G A G G A G G G T G A G C A A A T C A C A G C T C A (T G )2 2 26 19 7- 29 0 0. 18 5 0. 96 2 0. 94 1 0. 00 52 54 G I_ K V R a6 15 T G T G A G G G G T G A G G T T G A G G C T A C A A A C G C AT C C C C A C T C T C G G (A T )6 27 28 3- 37 9 0. 25 9 0. 95 3 0. 93 3 0. 00 68 29 G I_ K V R a6 51 T G G G T G G C A A A T T T G G G A G G A A A T G C C G C C C A A G G A G A G A G G A A A (A C )8 24 18 5- 27 7 0. 2 0. 97 1 0. 95 0. 00 66 22 G I_ K V R a7 23 T G C A C C A G G A G G G T C A C A G A C T A C A A C G A G G C C T T C C A A C A G G A (A C )1 0 21 41 2- 48 8 0. 14 3 0. 92 6 0. 90 4 0. 01 19 16 G I_ K V R a7 47 T G A C A G A T C G A C A G G C TA G A C T C G A A T C G C C C C C G TC TA T G TA TC A G T C (A T )6 25 43 2- 53 1 0. 19 2 0. 96 2 0. 94 1 0. 00 65 35 G I_ K V R a7 48 T G A A T G C C G A G A G C A A T T G T G C C T C A C A T C A C A A G G C T T G C T C A A A C A (T A )6 33 14 0- 21 4 0. 51 9 0. 97 9 0. 96 0. 00 32 90 G I_ K V R a8 34 G T G C A C A T G T C G C C A TA A A G AT G G A A C C TA C C C C TC C AT A A C AT G C C T T (A T )6 16 10 5- 18 0 0. 13 3 0. 85 3 0. 82 8 0. 03 68 97 G I_ K V R a8 61 G G C C C AT G G C C T C C T C T C AT A C A A T G G G G A A G G A C A A T TA A G T C G G G A (T A )6 15 10 3- 18 5 0. 13 8 0. 72 1 0. 69 5 0. 08 74 01 G I_ K V R a8 62 G G C A C A T G T G T C TA C A C C G C A C T G T G G A C A G G TA G G G T C A C A G G T (A T )7 9 23 3- 29 4 0. 14 3 0. 85 5 0. 81 9 0. 03 73 16 G I_ K V R a9 61 C C A C A C A C A A A AT G C C A C A A T T C C A T G T G C G T G T G T G G T T G A C A G G T (C A )6 14 99 -1 24 0. 28 6 0. 84 7 0. 81 6 0. 03 62 13 G I_ K V R b0 69 A G A C A T C C G T C A C C G G G C T C AT T G C C AT T TG TA T G TG T TG T TG G C G G (C A )7 10 99 -1 25 0. 21 4 0. 87 3 0. 84 1 0. 02 98 37 G I_ K V R b1 30 A C C C G C AT T C A C A AT G C A C AT A C A G T G G C G C TA T T G G G A A AT G A G TA C A (C A )7 8 23 3- 34 1 0. 00 0 0. 86 0. 82 3 0. 03 36 81 G I_ K V R b1 31 A C C C C TA A C G G T G G G T T C G T C A T C G A G G G T C C T T G A G T T C T C C C C T (A T )6 13 99 -1 90 0. 14 8 0. 90 5 0. 87 9 0. 01 76 89 G I_ K V R b1 32 A C C C C TA A C G G T G G G T T C G T C A T G G C C T TC G G TT G A G T T G TC C C (A T )6 10 11 7- 15 7 0. 42 9 0. 77 4 0. 73 3 0. 06 76 68 G I_ K V R b1 74 A C A C C G G TA A G G T G G T G A G A A G G A A C A C A C A G A G TA C C C C AT AT A C G C A C A (T G )7 12 10 1- 14 8 0. 25 0. 78 3 0. 74 9 0. 05 49 54 G I_ K V R b1 75 A C A C C G G TA A G G T G G T G A G A A G G A A C A C A G A G TA C C T C A C AT A C G C A C A (T G )7 18 10 0- 16 5 0. 51 7 0. 91 5 0. 89 1 0. 01 63 65 G I_ K V R b1 76 A C A C C C G AT C C C AT T C C G A C C T A C A C C A A C C A C G C T C C C T T C C T (T A )7 24 45 3- 52 4 0. 27 6 0. 94 5 0. 92 5 0. 00 82 23 G I_ K V R b2 00 A A C TA C C A T C A A A C A T C A C C A A C A C G A T G G A A G G T G T T G A G G T C G G C C A (C A )6 22 43 0- 51 4 0. 32 0. 95 7 0. 93 4 0. 00 90 77 G I_ K V R b2 01 A A C G G C TA G C T TT T C A A C T G A C T G T T G G TA A G T C G AT T G T T G G G C T T C G (T A )6 17 11 6- 17 9 0. 16 0. 91 3 0. 88 7 0. 01 78 50 G I_ K V R a9 75 C A C C C C AT A C A C A A C C A C AT T C C C G G T G TA T G T G C C T G G AT A A A T G A A G G T (C A )6 23 20 1- 28 5 0. 10 3 0. 93 8 0. 91 8 0. 00 92 12 G I_ K V R a9 76 C A C AT C C T TA C AT G TA C A C G G T C C A C C T G A C C G G C TA A A C A TA C A A G T T C C A (T A )7 20 31 6- 39 7 0. 08 3 0. 92 6 0. 90 1 0. 01 67 75 G I_ K V R a9 77 C A C A TA A G G A A C A A C A A C A A G G C C T C A G C C G G A G G C C G TA C A AT T G T G T T (A T )7 24 99 -1 71 0. 43 3 0. 85 6 0. 83 5 0. 03 19 96 G I_ K V R a9 78 C A AT C T C AT T C C TA G A C A A C C T G C A C A A G T T G A T C C A G G A T T T G G C G A G G G T (A C )6 20 99 -1 48 0. 41 4 0. 93 3 0. 91 2 0. 01 12 02 G I_ K V R a9 79 C A A G G C T G C T C G G A C G T C G A A T A T C C C A C C G G C T C G A G C A A G A A (C T )6 23 42 8- 58 2 0. 28 6 0. 90 5 0. 88 3 0. 01 55 18 G I_ K V R a9 80 C A A C AT G C T T C A A C C A A G C A C A TA C A A T G C TA C TA C C T TA G G A G A C A T G C AT C A (T G )1 1 21 11 2- 19 8 0. 44 4 0. 94 2 0. 92 0. 00 92 96 G I_ K V R a9 81 C A A C A A A G G G C A T T C A T G C A C A C A T T G G G G G A G G A A C C A A G C A A G T (A T )6 24 31 3- 39 9 0. 63 3 0. 95 5 0. 93 6 0. 00 68 17 G I_ K V R b0 47 A G C G A G G A C A A G G G A A A G G A C G T G G C G G AT AT G T G T G C T T G G C G (T A )7 19 32 3- 36 5 0. 36 0. 91 1 0. 88 5 0. 01 81 87 T ab le 1 : G en et ic a na ly si s of m ic ro sa te lli te m ar ke rs d ev el op ed f or G ar ci ni a in di ca J. Hortl. Sci. Vol. 16(1) : 125-129, 2021 128 Ravishankar et al J. Hortl. Sci. Vol. 16(1) : 125-129, 2021 G I_ K V R b0 48 A G C G A A T G C A T G C G T G TA G C G A A C G A T C A C C T T G G G G A C G C T C A (A T )6 19 47 2- 52 7 0. 26 1 0. 87 1 0. 84 6 0. 03 17 85 G I_ K V R b2 04 A A C C C A G T G A G T G TA A T G C G A AT T G T T G T T G T T G G C T TA TA G C C G A AT G T G A (C A )7 21 10 2- 19 5 0. 10 7 0. 94 8 0. 92 7 0. 00 77 28 G I_ K V R b2 05 A A C C C A AT G A G T G TA AT G C C A G T T G T A C T G T G G T T G G C T TA T G G C C T G A (C A )6 21 10 3- 19 7 0. 5 0. 91 9 0. 89 8 0. 01 52 33 G I_ K V R b2 06 A A C A G G A C C G G T G T G C G G T T G A T C C G C A C AT G T G T C C A C A C C A A (T A )8 21 20 1- 34 1 0. 42 3 0. 90 9 0. 88 5 0. 01 63 89 G I_ K V R b2 07 A A C A C G T G G C A G A C G C T C A A G G T G G T G A G G T C G G T C C A A A C A G G A (A T )6 8 11 7- 17 8 0. 23 3 0. 79 3 0. 75 7 0. 07 08 82 G I_ K V R b2 08 A A C A C G C G C G A G G A C A TA C T G C C C A A G C C TC C TC TC C C AT TT G TG C (T A )6 7 15 4- 17 1 0. 67 9 0. 77 4 0. 72 0. 07 75 86 G I_ K V R b2 09 A A C A C C T G C A C G G G T T T C G T G G A C T T T C C AT C TC G A C C A C G C C G (T A )7 10 33 0- 41 3 0. 00 0 0. 89 0. 86 0. 02 37 26 G I_ K V R b2 13 A A A G G A C C G G C G A A G A A A G C G G C C C A G C T C A A A C C G A T G C C C A A (A G )6 10 13 4- 25 0 00 .0 00 0. 88 1 0. 85 0. 02 60 89 G I_ K V R b2 14 A A A G A G A G G T C A T C T TA G T G A G G G G G T G T T G G C T T G G T C G TA A C G G C T (G T )6 6 15 0- 25 1 0. 14 8 0. 79 2 0. 74 2 0. 06 27 89 G I_ K V R b2 19 T G T T G G G A A G TA A A A G G A G G G A G C A T G A C C TA G G C AT C C AT C T C C C C T (T G T) 5 7 11 3- 17 8 0. 5 0. 78 5 0. 73 3 0. 06 31 97 G I_ K V R b2 20 T G T G G G G A T G G C A A A T G A G G T G A T G C C AT T C G G T T G G G G C AT A C T (C A C )5 10 14 3- 17 3 0. 11 5 0. 82 9 0. 78 8 0. 04 43 38 G I_ K V R b2 34 T G G C G T G C A G TT C T TC C T C C C A G G G AT C G C AT C C A A C AT T C AT T T C C A (C A A )5 3 17 3- 21 5 0. 15 4 0. 33 5 0. 30 3 0. 30 48 96 G I_ K V R b2 42 T G C A A C A A C A G G C T C A G G C A C A T G G T G G A G G C A C G G G T T G A A C A (C C A )5 15 18 9- 21 5 0. 5 0. 90 7 0. 88 1 0. 01 80 89 G I_ K V R b2 43 T G A G C G A C C G T G C C T G AT G T T G A G G G C TC C C TC A C C C T C TA C C TT A (C A G )5 13 14 1- 17 1 0. 36 0. 86 4 0. 83 0. 03 20 98 G I_ K V R b3 41 A C A A G C A T G C C A A A C G TA G C C G A T G A A G A A G T G C C C A A C C C C A C T (T G G )5 12 13 6- 17 0 0. 51 7 0. 78 0. 74 1 0. 07 12 13 G I_ K V R b3 52 A A G A C G G G T G G C G G T G G A G A A A A G A A G C G A A C C C T C T C C T C C T G A (T CT )8 13 36 2- 40 3 0. 55 2 0. 86 6 0. 83 5 0. 03 36 09 G I_ K V R b3 57 T G A C A AT A C G T G G G G A G A T C C G T T G T T C A G G C TC A AT C C C TT C G TG C (A AT A )7 16 11 5- 19 1 0. 00 0 0. 88 6 0. 86 1 0. 02 13 33 G I_ K V R b3 68 T C C G T G C C A AT T C C C T G G C A A C T G A C C T G T C G C C TT A G C TA C C C T (A A A A T )5 17 24 9- 31 0 0. 19 2 0. 92 5 0. 9 0. 01 40 54 G I_ K V R b3 73 A G C TA G G G G G C A A C C T G TA C C A T G C TA T T G A AT T C G T G T T G G T G G T G A (C A AT A C )5 8 15 1- 16 8 0. 48 1 0. 81 8 0. 77 8 0. 04 80 49 G I_ K V R a0 11 TC C G TC C AT C C G T TC G TC C G TT A C C G G A T G G G A T C C A G C G A T G T (C G TC ) 12 10 0- 13 6 0. 17 2 0. 75 0. 72 2 0. 07 46 75 6c gt t (C G T C )7 T ab le 1 C on td ... . 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