American Journal of Botany: e96e99. 2011.

AJB Primer Notes & Protocols in the Plant Sciences

DEVELOPMENT OF NEW MICROSATELLITE MARKERS FROM MANGO (MANGIFERA INDICA) AND CROSS-SPECIES AMPLIFICATION1
Kundapura Venkataramana Ravishankar2,4, Bellam Hanumantha-Reddy Mani2, Lalitha Anand2, and Makki Ramachandra Dinesh3
2Division

of Biotechnology and 3Division of Fruit Crops, Indian Institute of Horticultural Research, Hessaraghatta Lake Post, Bangalore 560 089, India

Premise of the study: Microsatellite markers were developed and characterized to assess the genetic diversity among mango (Mangifera indica) cultivars and to test their amplication in closely related species. Methods and Results: Thirty-six microsatellite (simple sequence repeats; SSR) loci were isolated by a microsatellite-enriched partial genomic library method. Primers designed for these loci were characterized using 30 diverse mango cultivars. The number of alleles ranged from 3 to 19 with an average of 9.2 alleles per locus. Polymorphic information content values ranged from 0.185 to 0.920 with a mean of 0.687. The total value for the probability of identity was 2.42 1031. Conclusions: The newly identied SSRs would be useful in genetic diversity studies, nger-printing, and mapping. Loci from ve related species, M. odorata, M. anadamanica, M. zeylanica, M. camptosperma, and M. grifthii, were successfully amplied using these SSR primers, showing their potential utility across species. Key words: genetic diversity; Mangifera; mango; microsatellites; SSR markers.

India is the center of origin for cultivated mango (Mangifera indica L.). Mango belongs to the family Anacardiaceae and is distributed in tropical and subtropical regions. Mango has been cultivated in India for more than 400 yr (Mukherjee, 1972). Mangifera indica is native to India and occurs abundantly in forests and cultivated areas. Hence, it is difcult to differentiate true wild forms from cultivated ones. The cross-pollination nature and a wide range of prevailing agro-climatic conditions have contributed to its wide genetic diversity in India in mango (Mukherjee, 1972). Commercially grown cultivars have arisen through seedling selections made for different fruit characters like color, taste, avor, size, etc. These cultivars have been vegetatively propagated and cultivated over a wide area. Traditional mango cultivars from a particular geographical region are genetically very similar (Ravishankar et al., 2000). Generally, mango cultivars are classied into two groups: a monoembryonic type (or Indian type) and a polyembryonic type (or Indo-Chinese type). In India, the majority of the cultivated types are monoembryonic. Polyembryonic types are also grown on the southwest coasts of India. Despite intercrossability of mono- and polyembryonic types and their wild occurrence, these two types have diverse genetic bases (Ravishankar et al., 2004). In this study an attempt has been made to develop SSR markers that would basically help in characterization of Indian mango cultivars. Further, they would be useful in distinguishing cultivars from wild relatives and in managing mango germplasm collections.
1

METHODS AND RESULTS

Total genomic DNA was isolated from fresh leaf samples of mango cultivar Alphonso. DNA was restriction digested with RsaI, and a microsatellite-enriched library was constructed following Glenn and Schable (2005). After RsaI digestion, DNA fragments were ligated to linkers and preamplied using linker-specic primers. Biotinylated probes of two dinucleotide repeats, four trinucleotide repeats, and four tetranucleotide repeats were used to capture fragments containing microsatellites. Using linker-specic primers, these fragments were enriched by PCR. The enriched fragments were cloned using a T/A cloning kit (MBI Fermentas, Burlington, Canada). A total of 278 plasmids with inserts were sequenced, and sequences containing microsatellites were selected for primer design. Primers were designed using Primer3 software (Rozen and Skaletsky, 2000). Eighty-six primer pairs were designed for putative microsatellite loci. They were screened for amplication and polymorphism using DNA from ve diverse mango cultivars (cv. Alphonso, Banganapalli, Langra, Neelum, and Totapuri). PCR amplication was carried out in 20 l containing 16 mM (NH4)2SO4, 67 mM Tris-HCl pH 8.8, 0.01% Tween-20, 2mM MgCl2, 0.1mM each dNTP, 0.4M each primer, 40 ng genomic DNA, and 0.5 unit of Taq DNA polymerase (Bioron GmBH, Ludwigshafen, Germany). PCR was carried out on a Master Cycler Gradient (Eppendorf AG, Hamburg, Germany) thermal cycler with the following temperature prole: initial denaturation at 94C for 1 min, 35 cycles of 30 s at 94C, 30 s at 55C, and 1 min at 72C, and a nal extension at 72C for 5 min. Amplication products were separated using 8% PAGE. The primers that showed polymorphism, and clear and scorable patterns were selected for further analysis (Table 1). The microsatellite sequences have been deposited in NCBI GenBank (accession numbers EF592181 to EF592216). These sequences have 15 perfect SSR repeats (14 dinucleotide and one perfect trinucleotide repeat). These SSR primers referred to as MiIIHR01- MiIIHR36 were used for further analysis. They were modied with HEX, FAM, and TET. PCR amplication was done on genomic DNA from 30 diverse mango cultivars (from different regions of India, plus Mexico, Florida, Thailand, Australia, and some polyembryonic types). We also tested ve Mangifera species M. odorata Pierre, M. anadamanica King, M. zeylanica Hook.f., M. camptosperma Pierre, and M. grifthii Hook.f. The plant material was collected from the mango germplasm collection of the Indian Institute of Horticultural Research (IIHR), Bangalore, India (Appendix 1). PCR products were analyzed using an automated DNA sequencer. FAM- and HEX-modied primer products were analyzed on an ABI 3730

Manuscript received 21 July 2010; revision accepted 28 February 2011.

Authors acknowledge nancial support for this work from the Department of Biotechnology (GOI), New Delhi. 4 Author for correspondence: kv_ravishankar@yahoo.co.in doi:10.3732/ajb.1000263

American Journal of Botany: e96e99, 2011; http://www.amjbot.org/ 2011 Botanical Society of America

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Table 1.

AJB Primer Notes & ProtocolsMANGIFERA INDICA microsatellites markers

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Characteristics of the 36 microsatellite markers isolated from Mangifera indica.

GenBank Accession No. EF592181 EF592182 EF592183 EF592184 EF592185 EF592186 EF592187 EF592188 EF592189 EF592190 EF592191 EF592192 EF592193 EF592194 EF592195 EF592196 EF592197 EF592198 EF592199 EF592200 EF592201 EF592202 EF592203 EF592204 EF592205 EF592206 EF592207 EF592208 EF592209 EF592210 EF592211 EF592212 EF592213 EF592214 No. of alleles 7 13 6 19 13 11 6 7 5 7 3 9 9 6 12 8 12 9 13 6 4 7 13 9 5 16 5 18 9 11 8 15 4 8 Allele size range (bp) 237261 165221 227235 138192 185219 93122 159185 252267 273291 161184 203213 154188 169194 329347 140194 186209 236268 155174 177208 177186 231239 209234 132154 237260 143154 131167 187195 101124 144155 190209 211230 176196 161173 222244

Locus MiIIHR01a MiIIHR02c MiIIHR03a MiIIHR04c MiIIHR05c MiIIHR06b MiIIHR07a MiIIHR08b MiIIHR09c MiIIHR10c MiIIHR11a MiIIHR12a MiIIHR13b MiIIHR14b MiIIHR15b MiIIHR16a MiIIHR17b MiIIHR18b MiIIHR19a MiIIHR20a MiIIHR21b MiIIHR22a MiIIHR23a MiIIHR24b MiIIHR25a MiIIHR26a MiIIHR27c MiIIHR28c MiIIHR29a MiIIHR30a MiIIHR31b MiIIHR32a MiIIHR33a MiIIHR34b

Primers (53) F: GGATGCACAACAACAAGCAC R: TCAGCAAGCAATCCCTTCTT F: CCCCAACATTTCATAAACACA R: CCTCCTTACATGCCTCCTTG F: GTCGATGCCTGGAATGAAGT R: AAGCATCGAACAGCTCCAAT F: CGTTTTTGACCCTCTTGAGC R: CCGCATACTTCCCTTCACAT F: CTCTCCCTCACTTGCTCCAC R: AGACCACCGACAACGAAAAC F: CGCCGAGCCTATAACCTCTA R: ATCATGCCCTAAACGACGAC F: GCCACTCAGCTAAATAGCCTCT R: TGCAGTCGGTAAAGTGATGG F: TGCTCTCTACTGCCCCGTAT R: GTCACACCAATCGGGAATCT F: GTTGTGACCGAGGCCTTAAA R: CTTTGACATCGCTGATCTGG F: CGATTCAAGACGGAAAGGAA R: TTCAAGCACAGACGACCAAC F: CAGTGAAACCACCAGGTCAA R: TGGCCAGCTGATACCTTCTT F: GCCCCATCAATACGATTGTC R: ATTTCCCACCATTGTCGTTG F: CCCAGTTCCAACATCATCAG R: TTCCTCTGGAAGAGGGAAGA F: CCGAAACAACTCTTCCTCCA R: TGCTCTCTGGCCTCTTCTTC F: CTAACCATTCGGCATCCTCT R: TCTGTGATAGAATGGCAAAAGAA F: TTTCACTTGGTTCTGGATTGC R: ATTTCCCACCATTGTCGTTG F: GCTTGCTTCCAACTGAGACC R: GCAAAATGCTCGGAGAAGAC F: TCTGACGTCACCTCCTTTCA R: ATACTCGTGCCTCGTCCTGT F: TGATATTTTCAGGGCCCAAG R: AAATGGCACAAGTGGGAAAG F: CCTAACGCGCAAGAAACATA R: ACCCACCTTCCCAATCTTTT F: TTTGGCTGGGTGATTTTAGC R: TTAATTGCAGGACTGGAGCA F: TGGCCGAACTAGCAAACTCT R: CCCCATTTCGAGAAAATTCC F: TCTGACCCAACAAAGAACCA R: TCCTCCTCGTCCTCATCATC F: GCTCAACGAACCCAACTGAT R: TCCAGCATTCAATGAAGAAGTT F: TGTGAGTCTCCGTTTGTGCT R: CCCTCTCATTTTCCCAGTCA F: GCGAAAGAGGAGAGTGCAAG R: TCTATAAGTGCCCCCTCACG F: TGGGGATTCATCGGAGATAG R: TGGAAGACCCATTCTCATGC F: GCGGTCGCAGACAAATTCTATAT R:ACAACTCGAGATTGTCACATCTTT F: CGATGAGGATGGTTGGTTTT R: CATCAACAGTCGCCATCAAT F: AGCTATCGCCACAGCAAATC R: GTCTTCTTCTGGCTGCCAAC F: TTCTGTTAGTGGCGGTGTTG R: CACCTCCTCCTCCTCCTCTT F: TGGTGGTGTTTGTTTGCAGT R: ACCACCCGCAGTATTGAAAG F: GAAGCACTTGTCTCCCTTGC R: CCTCACACTCCTCCACCTGT F: CTGAGTTTGGCAAGGGAGAG R: TTGATCCTTCACCACCATCA

Repeat motif (GAA)4CAG(CAA) 2(TA)2 (CA)2A(CA)7AG(CA)5 (CTT)6(CA)2 (CA)11 (CT)8C(CT)2TTTT(CT)4 (CA)7CG(CA)5 (GA)11 (GAA)2GTA(GAA)4 (CT)3TTGC(CT)2GT(CT)4TC(GT)2(CT)2 (GTT)6 (CT)2TT(CTT)5 (GA)11 (CCCTTT)3(CTCTTT)6 (GAA)3(AG)2A(AAG)3AG (GAA)2GGA(GAAA)2AA(GAA)3 (CTT)11CTA(CTT)5 (CTGCTT)2CTA(CTT)6 (GA)10 (GT)13GAGT(GA)10 (GT)12 (AC)11 (AT)2(GT)8 (GTTT)3(GT)2TTTTGTC(TG)4(AATGA)2 (GTCTC)2(TGTCTC)3T(CTC)2 (GA)17GG(GA)6 (CA)9TACC(CATA)6 (GTTT)3ATTTG(ATT)2 (GA)14GGA(GAA)2 (GT)8AT(AG)2 (GA)12 (GT)10 (CT)13 (GAC)6 (GA)12 (GA)12 (GGT)9(GAT)5

He 0.763 0.867 0.732 0.941 0.847 0.856 0.588 0.718 0.460 0.498 0.598 0.704 0.767 0.604 0.762 0.724 0.875 0.752 0.875 0.485 0.195 0.759 0.904 0.769 0.579 0.914 0.247 0.887 0.757 0.878 0.792 0.905 0.676 0.847

Ho 0.567 0.500 0.267 0.467 0.520 0.633 0.400 0.167 0.182 0.067 0.586 0.667 0.500 0.400 0.429 0.552 0.586 0.276 0.621 0.300 0.172 0.276 0.444 0.690 0.483 0.519 0.067 0.467 0.414 0.724 0.724 0.533 0.500 0.714

PIC 0.713 0.836 0.672 0.920 0.812 0.824 0.498 0.659 0.426 0.470 0.512 0.641 0.723 0.559 0.726 0.666 0.846 0.718 0.846 0.445 0.185 0.712 0.876 0.731 0.507 0.889 0.234 0.864 0.715 0.848 0.753 0.879 0.597 0.809

PI 0.17 0.07 0.22 0.02 0.08 0.08 0.41 0.22 0.37 0.32 0.39 0.24 0.15 0.27 0.13 0.22 0.06 0.13 0.06 0.37 0.67 0.16 0.04 0.13 0.37 0.03 0.60 0.04 0.15 0.06 0.13 0.04 0.31 0.09

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Table 1.

American Journal of Botany

Continued.
GenBank Accession No. EF592215 EF592216 F: R: F: R: No. of alleles 7 12 Allele size range (bp) 185192 214247

[Vol. 0

Locus MiIIHR35a MiIIHR36b

a

Primers (53) TGGTGAAGCTTGTTGTCTGC TGGCTTGACTGTTTTTCAGC TCTATAAGTGCCCCCTCACG ACTGCCACCGTGGAAAGTAG (GA)7 (TC)17

Repeat motif

He 0.811 0.869

Ho 0.345 0.759

PIC 0.767 0.838

PI 0.13 0.07

FAM; bHEX; cTET modied forward primers. He, expected heterozygosity; Ho, observed heterozygosity; PIC, Polymorphic Information Content (calculated using CERVUS); PI, Probability of Identity (calculated using IDENTITY).

instrument, and TET-modied products were analyzed on an ABI 377 system (Applied Biosystems, Foster City, California, USA). These SSR primer pairs for 36 loci generated one or two products per cultivar (Table 1). Genetic analysis was done for mango cultivars using Cervus 3.0 (Kalinowski et al., 2007). The expected heterozygosity (He) values ranged from 0.195 to 0.941 with a mean of 0.728. Polymorphic information content (PIC) values ranged from 0.185 to 0.920 with a mean of 0.687, and the majority of them (21) showed high PIC values (> 0.700). The Probability of Identity (PI) was calculated using IDENTITY 1.0 (Wagner and Sefc, 1999). The total value of the PI for all the 36 microsatellite loci was 2.42 10-31. Twenty-eight microsatellite markers amplied the related species used here (Table 2). Table 2.

CONCLUSIONS The SSR markers developed in this study showed relatively higher values of heterzygosity and PIC values compared to earlier studies (Duval et al., 2005; Honsho et al., 2005). Low PI values for these markers indicate their usefulness for ngerprinting mango cultivars. These markers have also shown their compatibility with the related species M. odorata, M. anadamanica, M. zeylanica, M. camptosperma, and M. grifthii. Using these SSR markers, studies to characterize a large mango germplasm collection at the Indian Institute of Horticultural Research, Bangalore and the Central Institute of Subtropical Horticulture, Lucknow, are in progress. The data generated will be used for genetic diversity and pedigree analysis. This would help in efcient management of germplasm and conservation. The data generated from these SSR markers will be useful in analyzing population structure, development of linkage map, and association analysis. LITERATURE CITED
Duval, M. F., J. Bunel, C. Sitbon, and A. M. Risterucci. 2005. Development of microsatellite markers of mango. Molecular Ecology Notes 5: 824826. Honsho, C., K. Nishiyama, W. Eiadthong, and K. Yonemori. 2005. Isolation and characterizaton of new microsatellites markers in mango. Molecular Ecology Notes 5: 152154. Glenn, T. C., and T. C. Schable. 2005. Isolating microsatellite DNA loci. Methods in Enzymology 395: 202222. Kalinowski, S. T., M. L. Taper, and T. C. Marshall. 2007. Revising how the computer program CERVUS accommodates genotyping error increases success in paternity assignment. Molecular Ecology 16: 10991106. Mukherjee, S. K. 1972. Origin of mango. Economic Botany 26: 260264. Ravishankar, K. V., L. Anand, and M. R. Dinesh. 2000. Assessment of genetic relatedness among a few Indian mango cultivars using RAPD markers. The Journal of Horticultural Science & Biotechnology 75: 198201. Ravishankar, K. V., P. Chandrashekara, S. A. Sreedhara, M. R. Dinesh, L. Anand, and G. V. S. Saiprasad. 2004. Diverse genetic bases of Indian polyembryonic and monoembryonic mango (Mangifera indica L) cultivars. Current Science 87: 870871. Rozen, S., and H. J. Skaletsky. 2000. Primer 3 on the WWW for general users and for biologist programmers. In S. Krawetz and S. Misener [eds.], Bioinformatics methods and protocols: Methods in molecular biology, pp. 365386. Humana Press, Totowa, New Jersey, USA. Wagner, H. W., and K. M. Sefc. 1999. IDENTITY 1.0 Centre for Applied Genetics. University of Agricultural Sciences, Vienna, Austria (http://www.boku.ac.at/zag/forsch/identity.htm).

Cross-species amplication of microsatellite markers developed for Mangifera indica.

Mangifera Mangifera Mangifera Mangifera Mangifera andamanica odorata zeylanica camptosperma grifthi A A A A A NA A NA A A A NA A A NA A A NA A A A A A A A NA A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A NA NA A A A NA NA A A NA A A A A A A A A A A A A A A A A A A A A NA A A A A A NA A A A A NA NA A A NA A A A NA A A A A A A A A A A A A A A A A

Locus MiIIHR01 MiIIHR02 MiIIHR03 MiIIHR04 MiIIHR05 MiIIHR06 MiIIHR07 MiIIHR08 MiIIHR09 MiIIHR10 MiIIHR11 MiIIHR12 MiIIHR13 MiIIHR14 MiIIHR15 MiIIHR16 MiIIHR17 MiIIHR18 MiIIHR19 MiIIHR20 MiIIHR21 MiIIHR22 MiIIHR23 MiIIHR24 MiIIHR25 MiIIHR26 MiIIHR27 MiIIHR28 MiIIHR29 MiIIHR30 MiIIHR31 MiIIHR32 MiIIHR33 MiIIHR34 MiIIHR35 MiIIHR36

A, amplied; NA, not amplied

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Appendix 1. Mangifera species and mango cultivars used in this study (with IIHR Accession numbers) No. 1 2 3 4 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Species M. andamanica M. odorata M. zeylanica M. camptosperma M. griffthii Cultivars Alphonso Amrapali Bennet Alphonso Bhutto Bombay Bombay Alphonso Chinnarasam Dashehari Eldon Gulaliya Guruvam Janardhan Pasand Kensington Kerala Dwarf Kesar Kurukkan Langra Muvandan Nam Dok Moi Neelum Osteen Padari Raspuri Ratna Royal Special Rumani Sensation Starch Suvarna Rekha Tommy Atkins Totapuri IIHR Accession no. 21038 20007 20007a 20005 22122 345 19880 341 19907 19910 19922 452 19931 19941 19942 19962 19982 20096 20095 19994 441 20067 22121 439 20026 347 436 20046 20051 20052 20059 20080 435 20088 8753