Genetic diversity of the Australian National Mango Genebank

Scientia Horticulturae 150 (2013) 213–226

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Genetic diversity of the Australian National Mango Genebank Natalie L. Dillon a,∗ , Ian S.E. Bally a , Carole L. Wright a , Louise Hucks a , David J. Innes b , Ralf G. Dietzgen b,1 a

State of Queensland, Department of Agriculture, Fisheries and Forestry, Agri-Science Queensland, Centre for Tropical Agriculture, P.O. Box 1054, Mareeba, QLD 4880, Australia State of Queensland, Department of Agriculture, Fisheries and Forestry, Agri-Science Queensland, Queensland Agricultural Biotechnology Centre, The University of Queensland, P.O. Box 6097, St. Lucia, QLD 4067, Australia b

a r t i c l e

i n f o

Article history: Received 7 March 2012 Received in revised form 5 October 2012 Accepted 6 November 2012 Keywords: Mangifera Simple sequence repeats Microsatellite markers Genetic variability Fingerprinting Bayesian method

a b s t r a c t Assessment of genetic diversity is an essential component in germplasm characterisation and utilisation. In this study the genetic diversity of mango was determined among 254 Mangifera indica L. accessions and related Mangifera species originating from 12 diverse geographic areas using eleven known simple sequence repeat (SSR) markers from mango. A total of 133 alleles were detected, ranging from eight (LMMA12) to 16 (MIAC-5) alleles per locus with a mean value of 12.36 and an average polymorphism information content (PIC) of 0.72. The mean number of alleles (8.45) was highest in the South East Asian accessions (Indonesia/Malesia) and lowest in the accessions from the Philippines (2.55). Diversity analysis divided the accessions into four major nodes broadly representing their geographical origins. The genetic diversity of ‘Kensington Pride’ was confirmed as being very low and no parents for this cultivar were identified. No association could be established between SSR markers analysed and embryony. Ten synonymous accessions were identified with matching genetic identity with at least one other accession at all SSR loci examined. Twenty-two unique genotypes were identified for 50 trees previously assigned different accession names. The remaining accessions were genetically distinct from each other. This increased understanding of genetic diversity in the Australian National Mango Genebank will assist breeders to better select parents with the potential to contribute desired genes to the progeny and thus more rapidly deliver improved cultivars to industry to meet consumer demand. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Most cultivated mangoes belong to the species Mangifera indica, one of 600 species in the family Anacardiaceae. Other cultivated species in the family include pistachio (Pistacia vera L.) and cashew (Annacardium occidentale L.) (Stevens, 2001). The genus Mangifera contains around 70 species, which can be divided into two subgenera, Limus and Mangifera (Kostermans and Bompard, 1993) with at least 26 species producing edible fruits (Mukherjee and Litz, 2009). These include Mangifera caesia Jack, Mangifera foetida Lout., M. indica L., Mangifera kemanga Bl., Mangifera laurina Bl., Mangifera odorata Griff., Mangifera pajang Kostermans, and Mangifera sylvatica Roxb. (Tanaka, 1976). Thirteen Mangifera species are maintained

∗ Corresponding author at: Department of Agriculture, Fisheries and Forestry, Agri-Science Queensland, 28 Peters Street, Mareeba, QLD 4880, Australia. Tel.: +61 7 4048 4652; fax: +61 7 4092 3593. E-mail addresses: [email protected] (N.L. Dillon), [email protected] (I.S.E. Bally), [email protected] (C.L. Wright), [email protected] (L. Hucks), [email protected] (D.J. Innes), [email protected] (R.G. Dietzgen). 1 Current address: Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, St. Lucia, QLD 4067, Australia.

in the Australian National Mango Genebank (ANMG) collection in Queensland, Australia (Bally, 2009). The common mango (M. indica L.) can be divided into two groups distinguished by the embryology of their seeds and their respective centres of diversity in the North-Eastern Indian subcontinent and South East Asia (Mukherjee, 1997). Polyembryony in mango occurs from the production of multiple somatic embryos of nucellar origin and results in the development of clonal seedlings with a maternal genetic background. The practical impact of polyembryony for horticultural crops, such as mango, is the ability to fix hybrid vigour and clonally propagate superior lines through seed (Sauer, 1993). Polyembryonic-derived nucellar seedlings are used for uniform rootstock while recombinant zygotic seedlings in monoembryonic cultivars, are of interest to breeding programmes. Aron et al. (1998) suggests that polyembryony is genetically controlled by a single dominant gene in mango while López-Valenzuela et al. (1997) reported a single RAPD marker associated with the polyembryonic trait. From the two centres of diversity, mango cultivation spread throughout many tropical and subtropical regions of the world along trading routes. In the 10th century the Arabs are thought to have spread mango cultivation from India to East Africa (Mukherjee, 1997). A further expansion of mango cultivation occurred in the 15th and 16th centuries with the European voyagers

0304-4238/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.11.003

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to South America and West Africa (Nakasone and Paull, 1998). In the 18th century mango cultivation was taken to the Philippines and the Pacific Islands, Central America and the Caribbean (Singh, 1978; Duval et al., 2006). In the 19th and 20th centuries varieties from the Caribbean, India, Philippines and Africa were imported to Florida USA, where natural hybridisation and selection gave rise to the popular commercial varieties currently grown in many countries (Knight and Schnell, 1994). The distribution of mangoes that originated in South East Asia are less well documented, however, Popenoe (1920) suggested that M. laurina and other species may have contributed to commercial varieties. The density of Mangifera species in South East Asia and the fact that many have locally used edible fruit suggests that they may have had a role in the ancestry of some of the current South East Asian cultivated varieties. The long period of domestic cultivation, alloploidy and outcrossing have contributed to the wide genetic diversity and highly heterozygous nature within M. indica (Mukherjee, 1950; Singh, 1960). However, since the arrival of the Portuguese in India in the 15th century and the introduction of grafting, the genetic stability of many superior selections of monoembryonic cultivars has been maintained through vegetative propagation (Mukherjee and Litz, 2009). There is a relatively poor understanding of the pedigree and genetic relatedness of many M. indica cultivars. The distribution of mango cultivars in ancient and recent times, their adaption to different regions and adoption of local vernacular names makes tracing their origins and ancestry difficult. Traditional classification of the genus Mangifera has been based on phenological and morphological characteristics of flowers, leaves, fruits and seed. However, this mode of identification is exceedingly complicated with environmental effects on these characters and parallel selection for similar desired traits often being misleading (Smith and Smith, 1992). Often plants will need to be grown to full maturity prior to identification and evaluation of these traits, which can take up to 10 years. As a result several different classification systems have been proposed (Mukherjee, 1953; Ding Hou, 1978; Kostermans and Bompard, 1993). Molecular genotyping has been a useful tool in establishing cultivar identity and relationships. Recent application of molecular markers and nucleotide sequence analyses to determine phylogenetic relationships among Mangifera species (Hidayat et al., 2011; Gálvez-López et al., 2009; Díaz-Matallana et al., 2009; Pandit et al., 2007; Duval et al., 2006; Schnell et al., 2006; Yamanaka et al., 2006; Yonemori et al., 2002; Eiadthong et al., 2000) are now beginning to reveal some of the genetic relationships between mango cultivars, nonetheless the taxonomic classification of Mangifera remains uncertain. Over the last two decades genetic relationships of mango cultivars have been analysed using enzyme polymorphisms and numerous PCR-based marker techniques. Initial studies were undertaken using protein or isozyme analyses (Degani et al., 1990, 1992; Schnell and Knight, 1992b; Knight and Schnell, 1994) but these are influenced by the environment and reveal low polymorphisms. Later, studies using random amplified polymorphic DNA (RAPD) markers (Schnell and Knight, 1992a; Lavi et al., 1993; Schnell et al., 1995; Bally et al., 1996; López-Valenzuela et al., 1997; Jayasankar et al., 1998; Lowe et al., 2000; Ravishankar et al., 2000; Kumar et al., 2001; Karihaloo et al., 2003; Cordeiro et al., 2006a,b; Rahman et al., 2007; Srivastava et al., 2007; Rajwana et al., 2008; Díaz-Matallana et al., 2009), restriction fragment length polymorphism (RFLP) analysis (Eiadthong et al., 1999a); amplified fragment length polymorphism (AFLP) markers (Eiadthong et al., 2000; Kashkush et al., 2001; Capote et al., 2003; Yamanaka et al., 2006) and inter simple sequence repeat (ISSR) markers (González et al., 2002; He et al., 2005; Pandit et al., 2007; Srivastava et al., 2007; Santos et al., 2008; Huang et al., 2008) were utilised. Many of these studies have been undertaken on small numbers of

accessions from within limited geographical origins and did not reveal the wider extent of diversity among mangoes. Further studies on mango genetic diversity and identification of traits of interest using SSRs (also known as microsatellites markers) include those undertaken by Eiadthong et al. (1999b), Honsho et al. (2005), Duval et al. (2005, 2006), Schnell et al. (2005, 2006), Viruel et al. (2005), Ukoskit (2007), Gálvez-López et al. (2009) and most recently start codon targeted (SCoT) markers (Luo et al., 2010). Among the SSR studies, those undertaken by Duval et al. (2006) and Schnell et al. (2006) have examined the genetic diversity of a wider cross-section of mangoes. Australia does not have any indigenous Mangifera species, with all mangoes having been introduced since the 1800s. The first evidence of mangoes introduced into Australia was in an 1827 Sydney Botanic Gardens plant catalogue, which listed a mango introduced from the East Indies in 1823 and another cultivar, ‘Mangograle’, from Mauritius in 1825 (Johnson, 2000). Later introductions were from boats trading between Australia and South East Asia arriving at ports throughout the country. Many of the earlier introduced cultivars were green skinned, stringy-fleshed, turpentine types, collectively known as ‘Commons’. A notable exception to the common types, introduced between 1885 and 1889, was the cultivar ‘Kensington Pride’ (Tree, 1959) that became the iconic Australian cultivar that is still the mainstay of the Australian industry today. The pre-Australian origins of ‘Kensington Pride’ are uncertain with both Indian and South East Asian origins advocated (Johnson, 2000). Stephens (1963) suggests ‘Kensington Pride’ was introduced as seed from the East Indies or India on trading ships that visited the port of Bowen (formerly Port Denison) in the late 19th century. ‘Kensington Pride’ is polyembryonic, typical of many cultivars derived from South East Asia. Although ‘Kensington Pride’ has dominated commercial production in Australia, its shortfalls, such as irregular bearing, variability of fruit type and short shelf life, have long been recognised (Stephens, 1963; Beal, 1976; Watson, 1984). Since the 1960s Australian breeders have systematically attempted to find alternative cultivars suited to Australian growing conditions through various selection and breeding programmes (Bally et al., 2006). The earliest of these mango improvement programmes recommended cultivars such as ‘Haden’, ‘Kent’, ‘Nam Doc Mai’, and ‘Irwin’ for commercial cultivation (Winston, 1984; Wright and Bally, 1984). Later Australian breeding programmes released cultivars such as ‘Delta R2E2’ in 1991 (Anonymous, 1991), ‘B74’ (CalypsoTM ) in 2000 (Whiley, 2000), ‘Honeygold’ in 2002 (Anonymous, 2002), and ‘NMBP 1243’, ‘NMBP 1201’ and ‘NMBP 4069’ in 2009 (Bally, 2008). Although these cultivars have contributed to the development of the Australian industry, breeders have recognised the need for the continual development of new cultivars to remain competitive in domestic and international markets. Recently, genetic improvement has been undertaken through the Australian Mango Breeding Program (AMBP) and associated projects such as the mango fruit genomics initiative that aims to develop molecular tools to improve breeding efficiency (Dietzgen et al., 2009). Like mango cultivars worldwide, the diversity or relatedness of mangoes introduced into Australia since the early 1800s is generally not well documented. The largest collection of mango cultivars and related species in Australia is held in the ANMG (Bally, 2009), which consists of accessions from early introductions, selections of ‘Kensington Pride’ and other Australian cultivars as well as prominent international cultivars and several Mangifera species. The phenotypic diversity of the genebank collection has been partly documented (Winston, 1984; Wright and Bally, 1984; Winston and Schaffer, 1993) however the genetic diversity of many of the accessions is still poorly understood. In this communication we have assessed the genetic variability, diversity and ancestry of a large collection of mango accessions

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2. Materials and methods

reduction in the annealing temperature (60–50 ◦ C). This was followed by 30 cycles of 94 ◦ C for 30 s, 50 ◦ C for 1 min and 72 ◦ C for 90 s, followed by a final extension at 72 ◦ C for 10 min. The TD50–40 profile was 94 ◦ C for 3 min, followed by 10 cycles of 94 ◦ C for 30 s, 50 ◦ C for 1 min and 72 ◦ C for 90 s. Each cycle had a 1 ◦ C reduction in the annealing temperature (50–40 ◦ C). This was followed by 30 cycles of 94 ◦ C for 30 s, 40 ◦ C for 1 min and 72 ◦ C for 90 s, followed by a final extension at 72 ◦ C for 10 min. The PCR products were pooled into two multiplex sets (A and B, see Table 3) prior to capillary gel electrophoresis.

2.1. Plant material

2.5. Capillary electrophoresis

Over 300 mango accessions were used in the studies, 254 samples mainly belonging to the species M. indica but also including other Mangifera species, 46 selections of the cultivar ‘Kensington Pride’ selected for their superior fruit quality and production traits (Bally et al., 1996) and eight hybrid progeny of ‘Kensington Pride’. The collection is held at the ANMG maintained at Southedge Research Station in Mareeba (16◦ 45 S, 145◦ 16 E) and at Ayr Research Station (19◦ 31 S, 147◦ 22 E), Queensland, Australia. Trees are grafted onto the polyembryonic ‘Kensington Pride’ rootstock, uniform because of their nucellar embryo origin. The species, origin and embryony of accessions used in the first diversity study are listed in Table 1. The second study includes four old Australian ‘Common’ types, 46 selections of ‘Kensington Pride’, eight hybrid progeny of ‘Kensington Pride’ and their parents consisting of seven Florida, USA cultivars. These are listed in Table 2.

PCR amplicons were separated by capillary gel electrophoresis on a CEQTM 8800 Genetic Analysis System (Beckman Coulter Inc., Fullerton, CA, USA); 0.25–1 ␮l of PCR product was mixed with 25 ␮l of Sample Loading Solution (Beckman Coulter) and 0.3 ␮l of CEQ 400 size standard (Beckman Coulter) prior to a 35 min separation at 6 kV.

representing a broad geographic area using an optimised selection of established mango SSR markers. These molecular marker tools have significantly improved our understanding of the pedigree and genetic relatedness of many mango cultivars and Mangifera species, validated accessions and identified diverse Mangifera species. In future, this information will be important to select suitable breeding parents to introgress traits involved in disease resistance, tree architecture and fruit quality.

2.2. Genomic DNA extraction Where possible, young fresh flushing leaf buds were collected for genomic DNA extraction. Where leaf buds were not available, the youngest leaf material available at the time was collected. DNA extractions were performed using DNeasy® Plant extraction kits (Qiagen GmbH, Germany) according to the manufacturer’s instructions. The DNA concentration and integrity was assessed by 1% (w/v) agarose/TBE gel electrophoresis. Extracted DNA was stored at 4 ◦ C and diluted to a final concentration of 10 ng/␮l prior to PCR amplification. 2.3. Microsatellite markers Eleven previously published microsatellite markers were selected for these studies, with one primer reported by Honsho et al. (2005), six by Viruel et al. (2005), two by Schnell et al. (2005) and two by Duval et al. (2005). All microsatellite markers had reportedly produced clear polymorphic amplification patterns and at least one allele size specific for a country of origin. Forward primers were labelled with a fluorescent dye at the 5 end and were synthesised by Proligo (Lismore, NSW, Australia). Details of the microsatellite markers used in these studies are listed in Table 3. 2.4. PCR amplification PCR reactions (20 ␮l) were carried out in a PTC-100TM Programmable Thermal Controller (MJ Research Inc., Waltham, MA, USA) using thermal cycling conditions for each marker listed in Table 3. A 20 ␮l reaction mix was prepared for the PCR assay, containing 10–20 ng of template DNA, 150–250 ␮mol/L dNTPs, 2–4 mmol/L MgCl2 , 0.1–0.3 ␮mol/L forward and reverse SSR primers, 0.5–1 U Taq DNA polymerase in conjunction with reaction buffer containing (NH4 )2 SO4 (MBI Fermentas, Ontario, Canada). Two thermal cycling profiles were used for DNA amplification. The TD60–50 profile was 94 ◦ C for 3 min, followed by 10 cycles of 94 ◦ C for 30 s, 60 ◦ C for 1 min and 72 ◦ C for 90 s. Each cycle had a 1 ◦ C

2.6. Data analysis Data analysis was done using the CEQTM 8000 Genetic Analysis System software version 8.0.52 (Beckman Coulter) for internal standard and fragment size determination and for allelic designations. Pairwise distances among all population pairs were calculated from allele frequency data using three genetic distance methods: Reynolds distance (Reynolds et al., 1983), Cavalli-Sforza’s chord distance (Cavalli-Sforza and Edwards, 1967) and Nei’s genetic distance (Nei, 1972; Nei et al., 1983). Evaluation of the three analysis methods was based on the degree of congruence among tree topologies as well as the ability to detect geographical groupings. The best results were obtained with Reynold’s distance, a measure that assumes that genetic differences arise due to genetic drift only (Reynolds et al., 1983). An un-rooted dendrogram was constructed using the neighbour-joining (NJ) method (Saitou and Nei, 1987) and 1000 bootstrap replications were obtained (justified following random number seed replications to determine meaningful bootstrap estimates) where possible. Distance calculations, tree construction and bootstrapping were all performed in PowerMarker V3.0 (Liu and Muse, 2005). The dendrogram for diversity of the ANMG was rooted with Buchanania arborescens as the outgroup, while dendrograms for ‘Kensington Pride’ diversity analysis and country of origin analysis were rooted on the midpoint. Polymorphism information content (PIC) values for diversity analysis were calculated for each locus according to the formula: PIC = 1 −  Pi2, where Pi is the frequency of the ith allele (Liu, 1998). Distance matrices were translated to NEXUS format and used as input for SplitsTree4, version 4.11.3 (Huson and Bryant, 2006). Phylogenetic network analysis was conducted with SplitsTree4 decomposition, demonstrating an alternative route of descent in the tree to that of the NJ model. This analysis does not force the SSR marker data into a bifurcating tree and allows for numerous parallel paths indicative of the presence of phylogenetic incompatibilities in the divergence of SSRs. Allele data from all accessions in the first study were examined using CERVUS© Version 3.0.3 parentage analysis software to investigate parental identification (Kalinowski et al., 2007). This software runs simulations to produce log-likelihood scores (LODs) and produces a confidence statistic for assigning paternity. Simulations were run with 100,000 replications and a 1% default type error rate. Morphological embryony data and molecular SSR data was evaluated by estimating cophenetic correlation using Mantel’s matrix correspondence test (Mantel, 1967). The program STRUCTURE version 2.3.3 (Falush et al., 2003, 2007; Pritchard et al., 2000) was used to investigate gene flow

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Table 1 List of 254 mango accessions their classification, origin and embryony. Accession no.a

Cultivar

Species

Origin (COO coded )

Embryonyb

207 22 28

Sabrec Batawic Boriboc

M. indica M. indica M. indica

Africa (East) (1) Africa (Kenya) (1) Africa (Kenya) (1)

P P M&P

226 234 154

84 89 94 8

Heidi Hood Isis Andreoli

M. indica M. indica M. indica M. indica

Africa (South) (1) Africa (South) (1) Africa (South) (1) Australia (2)

M M M P

5 14 118 145

17 18 26

Banana Callo Banana Long Blue

M. indica M. indica M. indica

Australia (2) Australia (2) Australia (2)

P P M&P

147 86 39

29

Bosworth 5

M. indica

Australia (2)

P

30

BOT

M. indica

Australia (2)

31 35 36 37 45

Bowen Earlyc Bullocks Heart Bundaberg Late Bundy Special Cedar Bay

M. indica M. indica M. indica M. indica M. indica

51

Common Maltby/Borne Common Top End Ruralc Delta R2E2 Dray Fred Roosc

M. indica M. indica M. indica

Australia (2) Australia (2) Australia (2)

M&P P U

G. Allan Golden tropicc Goldsworthyc Gullivers Triumphc Hinchinbrook Honey Gem Ingham Latec Kamerunga White Kensington Mackay Kensington Mono Kensington Pridec Kyal Larman Lemon Mag B Millaroo 6c Mullumbimby Gold Nixons Special Peach CG Pineapple Predojevich Pulella late Roberts 3c Roberts Special Rockhampton Large S113 NT Summerlee ii Tekin Thomas Trusso Whilley 42 Yorkey’s Knob Rosa Santa Alexandrina Alphonso Ic Anopan

M. indica M. indica M. indica M. indica

Australia (2) Australia (2) Australia (2) Australia (2)

P U P P

M. indica M. indica M. indica M. indica

Australia (2) Australia (2) Australia (2) Australia (2)

M. indica

Australia (2)

52 192 56 65 66 71 72 77 85 87 92 100 104 105 106 115 117 120 141 156 159 169 185 187 190 191 198 199 200 205 220 225 229 233 238 250 201 209 4 10

Accession no.a

Cultivar

Species

Origin (COO coded )

Embryonyb

Telok Anson Tung Chi Manzanillo ˜ Nunez Alphonso Pc Bagan Palli Pc Late Sindhric Mahmood Khan Walac Malda Sabzc Hindi Khas Carabaoc

M. indica M. indica M. indica

Malesia (10) Malesia (10) Mexico (11)

M P M

M. indica M. indica M. indica M. indica

Pakistan (12) Pakistan (12) Pakistan (12) Pakistan (12)

M M M M

M. indica M. indica M. indica

M U P

40

Carabao 1c

M. indica

U

41

M. indica

Australia (2) Australia (2) Australia (2) Australia (2) Australia (2)

P P P U P

24 50 113 203 235

M. indica M. indica M. indica M. indica M. indica

M. indica

Australia (2)

P

237

M. indica

Sri Lanka (14)

U

M. indica

Australia (2)

P

239

Carabao Lamaoc Betti Amberc Coconutc Kuruc Rupee Vallai Kolambanc Vella Colomban 1c Willardc

Pakistan (12) Pakistan (12) Philippines (13) Philippines (13) Philippines (13) Sri Lanka (14) Sri Lanka (14) Sri Lanka (14) Sri Lanka (14) Sri Lanka (14)

M. indica

Sri Lanka (14)

M

Kopu reva OH’Ure Pioc Rapac

M. indica M. indica M. indica

P P P

48 49 62 88

Chokananc Cobra Tonguec Falanc Hong Sac

M. indica M. indica M. indica M. indica

Tahiti (15) Tahiti (15) Tahiti (Cook Islands) (15) Thailand (16) Thailand (16) Thailand (16) Thailand (16)

P M P P

101 108 109 144

Keawc Keow Savoey Khomc Maha Chanook

M. indica M. indica M. indica M. indica

Thailand (16) Thailand (16) Thailand (16) Thailand (16)

P P P M

P

158

Muhn Kohm

M. indica

Thailand (16)

U

M. indica

Thailand (16)

P

112 171 195

c

c

P P P P P P P

P P P P

M. indica

Australia (2)

M

162

Nam Doc Mai

M. indica

Australia (2)

P

163

M. indica

Thailand (16)

P

M. indica M. indica M. indica M. indica M. indica M. indica

Australia (2) Australia (2) Australia (2) Australia (2) Australia (2) Australia (2)

P P P P P P

170 172 180 193 208 213

Nan Klang Wanc Nong Sang Ok Rong Papa Land Raetc Sam Pi Siphon

M. indica M. indica M. indica M. indica M. indica M. indica

Thailand (16) Thailand (16) Thailand (16) Thailand (16) Thailand (16) Thailand (16)

P P U P P P

M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica

Australia (2) Australia (2) Australia (2) Australia (2) Australia (2) Australia (2) Australia (2) Australia (2)

P P P M M P P M

227 232 194 251 3 7 9 33

Thai Green Tong Dumc Rajah Yusof Akbar Anderson Ann Brooksc

M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica

Thailand (16) Thailand (16) Unknown Unknown USA (17) USA (17) USA (17) USA (17)

U P P M M M M M

M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica

Australia (2) Australia (2) Australia (2) Australia (2) Australia (2) Australia (2) Australia (2) Brazil (3) Brazil (3)

U U P P U U P P P

42 43 55 57 58 59 63 64 67

Carrie Casino Gold Davis Hadenc Duncan Early Gold Edward Fascell Florigon Gail

M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica

USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17)

M P M M M M M P M

M. indica M. indica

India (5) India (5)

M P

69 70

Glenn Golden Delight

M. indica M. indica

USA (17) USA (17)

M M

N.L. Dillon et al. / Scientia Horticulturae 150 (2013) 213–226

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Table 1 (Continued) Accession no.a

Cultivar

Species

Origin (COO coded )

Embryonyb

M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica

India (5) India (5) India (5) India (5) India (5) India (5) India (5) India (5) India (5) India (5) India (5) India (5)

M M P P M P P M M M M P

M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica

India (5) India (5) India (5) India (5) India (5) India (5) India (5) India (5)

148 152

Bangalorac Bangampallic Bappakai Bombay Greenc Brindabin Cathamia Chandrakaran Chereku Rasam Creeping Dasheharic Fajari Hamayat Pasand Hybrid 10 Hybrid 17 Kalapady Kishenbhogc Kurukan Langrac Magovar Mahmuda Vikarabad Malika Manjeera

M. indica M. indica

153

Manoranjan

160

Mundapa

19 20 21 27 32 44 46 47 53 54 61 80 90 91 99 111 114 116 142 146

c

Accession no.a

Cultivar

Species

Origin (COO coded )

Embryonyb

78 83 93 95 97 102 103 107 121 122 179 183

Hadenc Hatcher Irwinc Jakarta Jewel Keittc Keitt Red Kentc Lily Lippens Palmerc Parvin

M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica

USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17)

M M M M M M U M M M M M

M M M M P M M M

211 214 215 216 217 231 236 253

Sensation Smith Spirit of 76 Springfels Sri Jaya Tommy Atkinsc Van Dykec Zillc

M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica

USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17) USA (17)

M M M M M M M M

India (5) India (5)

M M

254 61

Zillate Excellc

M. indica M. indica

M M

M. indica

India (5)

M

74

Gouvieac

M. indica

M. indica

India (5)

P

Mapulevuc

M. indica

USA (17) USA (Hawaii) (18) USA (Hawaii) (18) USA (Hawaii) (18) USA (Hawaii) (18) USA (Hawaii) (18) USA (Hawaii) (18) USA (Hawaii) (18) USA (Hawaii) (18) Viet Nam (19) Viet Nam (19) Viet Nam (19) Viet Nam (19) Viet Nam (19) Viet Nam (19)

P M U P P P

155

c

161

Mylepania

M. indica

India (5)

P

157

Momi K

M. indica

164

Navaneelumc

M. indica

India (5)

M

174

Onoc

M. indica

165

Neelum

M. indica

India (5)

M

181

Paris

173

Olour

M. indica

India (5)

P

189

M. indica c

Pope

M. indica c

175

Padiri

M. indica

India (5)

M

197

Repozo

M. indica

176 177 178 188 196 202

Pahiri Pairi Pajo Pirie Red Mulgoa Royal Special

M. indica M. indica M. indica M. indica M. indica M. indica

India (5) India (5) India (5) India (5) India (5) India (5)

M U M M M M

210 240 241 242 243 244

M. indica M. indica M. indica M. indica M. indica M. indica

204 212 218 222 252 81

S.B. Chowsa Sings Late Sufaida Suvarnarekhac Zardalu Arumanisc

M. indica M. indica M. indica M. indica M. indica M. indica

India (5) India (5) India (5) India (5) India (5) Indonesia (6)

M M&P M M M P

245 246 247 248 249 25

Sapa Xoài Bouic Xoài Buc Xoài Cat #7c Xoài Cat Chuc Xoài Cat Hoa Locc Xoài Cat Thomc Xoài Nhoc Xoài Queóc Xoài Thanh Cac Xoài Tuongc Black Jamaican

Bahadar hhandi Bali Applec

M. indica

Indonesia (6)

M

75

Grahamc

M. indica

M. indica

Indonesia (6)

P

96

Jamaica Blackc

M. indica

15 16

P M M U P P P P P

166

Nelsonc

M. indica

P

182

Parric

M. indica

Indonesia (6) Indonesia (6) Indonesia (6) Indonesia (6)

P M&P U U

126 127 128 129

Binjaic Unknown Casturi Round Bogor #2c

M. caesia M. casturi M. casturi M. foetida

M. indica M. indica M. indica M. indica

Indonesia (6) Indonesia (6) Indonesia (6) Indonesia (6)

M P U P

130 131 123 138

#1 Laleejeewoc Lomboc 97-864

M. lalijiwo M. lalijiwo M. laurina M. rubropetala

Indonesia (6) Indonesia (6) Indonesia (6) Indonesia (6)

U U P M

M. indica

Indonesia (6)

P

136

Unknown

M. pentandra

Malaysia (9)

U

M. indica

Indonesia (6)

M

73

Golek

M. indica

Indonesia (6)

P

76

Gudang

M. indica

Indonesia (6)

Kimba Manalagi Mangga Maduc New Guinea Long Pilikpisan Sabah Sulawasi Tang Ki Panjang Tanjang Pinang Pinkc

M. indica M. indica M. indica M. indica

224

M

M. indica

Gedong

186 206 219 223

M

Julie

68

110 149 151 167

M

Viet Nam (19) Viet Nam (19) Viet Nam (19) Viet Nam (19) Viet Nam (19) West Indies, Jamaica (8) West Indies (20) West Indies, Jamaica (8) West Indies (20) West Indies (20) West Indies (20) Indonesia (6) Indonesia (6) Indonesia (6) Indonesia (6)

98

c

M. indica M. indica M. indica M. indica M. indica M. indica

M

M P M M M M P P M

218

N.L. Dillon et al. / Scientia Horticulturae 150 (2013) 213–226

Table 1 (Continued) Accession no.a

Cultivar

Species

Origin (COO coded )

Embryonyb

230 1 168 11 23 143 2 6 82

Tiwi 13-1 Nimrod Applec Batu Ferrungic Maha 165c Ah-toy-long Ampalam Arumanis Red

M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica M. indica

Indonesia (6) Israel (7) Israel (7) Malaysia (9) Malaysia (9) Malaysia (9) Malesia (10) Malesia (10) Malesia (10)

P P M M M P P P P

Asam Kombangc Asam Ramuk Haem Wangi Mangga Dodalc Sungi Siput

M. indica

Malesia (10)

M. indica M. indica M. indica M. indica

Malesia (10) Malesia (10) Malesia (10) Malesia (10)

12 13 79 150 221

Accession no.a

Cultivar

Species

Origin (COO coded )

Embryonyb

125 132 133 134 135 137 139 39 124

Unknown Delphi IPOH #1 Kweni Kerri #1 Unknown Burma Mango Unknownc

M. applanata M. laurina M. lauirna M. odorata M. odorata M. quadrifida M. torquenda Unknown M. altissima

P P P M P U U P P

U

184

Pauc

M. altissima

P U P P

140 228 119 34

Unknownc Thai Wild LBG 98-314 Satinwood

M. zeylanica Unknown Unknown Buchanania arborescens

Malesia (10) Malesia (10) Malesia (10) Malesia (10) Malesia (10) Malesia (10) Malesia (10) Myanmar (4) Philippines (13) Philippines (13) Sri Lanka (14) Thailand (16) Unknown (22) (21)

U M U U U

a

Accession no., numerical code designated for cultivar for STRUCTURE analysis. Embryony, M, monoembryonic; P, polyembryonic; U, unknown. c Accessions used in the country of origin analysis. d COO code, numerical code designated for country of origin for STRUCTURE analysis. ‘Malesia’ covers the areas including Malay Peninsula, Sumatra, Java, Borneo, and Papua New Guinea. b

and the spatial genetic structure of mango accessions. This programme employs model-based clustering in which a Bayesian approach identifies clusters (populations) based on a fit to the Hardy–Weinberg equilibrium and linkage equilibrium. This software can infer direct genetic relationships based on molecular data. In the different simulations, no prior information was used to define the clusters. The first step of the analysis consisted of estimating K, the number of sub-populations or clusters. As a preliminary analysis, STRUCTURE was run independently five times for each number of sub-population (K) values, ranging from one to 20, using correlated allele frequencies and the admixture model with 100,000 replicates for burn-in and 1,000,000 Markov chain Monte Carlo (MCMC) iterations during analysis. Based on the likelihood plot of these models and germplasm information of the studied accessions STRUCTURE was re-run three times for each number of subpopulation (K) value, ranging from four to seven, using the admixture model with 1,000,000 replicates for burn-in and 5,000,000 MCMC iterations during analysis. For each value of K, the log-likelihood values were averaged and standard deviation calculated. The final population subgroups were then inferred based on stability of grouping patterns across runs and by calculating the K statistic (Evanno et al., 2005). After placing samples Table 2 Parentage of Australian and Floridian mango cultivars. Maternal parent

Paternal parent

Australian cultivars A67 B74 (CalypsoTM ) Bundy Special Delta R2E2 Honeygold NMBP1201 NMBP1243 NMBP4069

Sensation Sensation Keitt (?) Kent Kensington Pride Irwin Irwin Van Dyke

Kensington Pride Kensington Pride Kensington Pride Kensington Pride Unknown Kensington Pride Kensington Pride Kensington Pride

Florida cultivars Brooks Haden Irwin Keitt Kent Sensation Van Dyke

Sandersha Mulgoba Lippens Mulgoba Brooks Haden Haden

Unknown Terpentine Unknown Brooks Haden Brooks Unknown

into the cluster for which they showed the highest percentage of membership (q), STRUCTURE-assigned individuals were assessed in comparison to the geographical congruence of the clusters. Based on this information, the optimal grouping value was selected. 3. Results 3.1. Genetic diversity of the Australian National Mango Genebank 3.1.1. Allelic diversity Eleven microsatellite markers were selected to generate polymorphic markers for genetic diversity analysis of 254 mango accessions from the ANMG (Table 1). They provided discrete and consistent polymorphic banding patterns on which to base the genetic diversity of accessions. Single loci were amplified with all 11 microsatellite markers with only two alleles detected for any one marker. A total of 133 alleles were detected with a maximum of 16 alleles per locus in MIAC-5, a minimum of eight in LMMA12 and an average of 12.09 per locus (Table 3). Five SSR markers had a PIC value higher than 0.75, with an average PIC value of 0.72. The observed heterozygosity (HO ) was slightly below the expected heterozygosity (HE ), indicating a tendency towards inbreeding, most likely due to population isolation. The mean genetic diversity (or observed heterozygosity) was 0.69 while the mean expected heterozygosity was 0.75. Among the 133 polymorphic alleles detected, 67 were present and one was unique to mango accessions selected from Australia. Specific alleles were also identified for other mango origins. One unique allele was detected for accessions from Africa, Israel, Sri Lanka and USA Florida, and two unique alleles for USA Hawaii, with frequencies of 0.002. Indian accessions contained 11 unique alleles at seven loci with frequencies below 0.008. South East Asian (Indonesia/Malesia) accessions had 10 unique alleles at six loci, at a frequency of 0.004 or lower. Nineteen accessions, of which 12 were M. indica, six were other Mangifera spp. and one B. arborescens, showed one or more unique alleles. B. arborescens had the highest number of unique alleles (five at five loci). Of the Mangifera spp. M. caesia var. ‘Binjai’ had three unique alleles at three loci, and Mangifera sp. var. ‘Sabah’ had two unique alleles at two loci. Of the M. indica accessions, four had two unique alleles at two loci and the remainder had one unique allele.

Table 3 Details of the 11 SSR markers used to analyse diversity of 254 Mangifera accessions (study one) from the Australian National Mango Genebank and the 10 SSR markers used to analyse diversity of Australian and Floridian cultivars and accessions (study two). Locus

GenBank accession no.

AY628373

LMMA8

AY628380

LMMA10

AY628382

LMMA11

AY628383

LMMA12

AY628384

LMMA15

AY628387

MIAC-5

AB190348

MiSHRS-18

AY942819

MiSHRS-39

AY942829

mMiCIR010

AJ635172

mMiCIR020

AJ635182

Viruel et al. (2005) Viruel et al. (2005) Viruel et al. (2005) Viruel et al. (2005) Viruel et al. (2005) Viruel et al. (2005) Honsho et al. (2005) Schnell et al. (2005) Schnell et al. (2005) Duval et al. (2005) Duval et al. (2005)

Multiplex set

PCR program

254 Mangifera accessions (study one)

Australian and Floridian accessions (study two)

Allele no.

Allelic size range (bp)

HO

HE

PIC values

Departure from HWE

Allele no.

Allelic size range (bp)

HO

HE

PIC values

Departure from HWE

**

5

198–208

0.9385

0.6054

0.5286

**

4

258–270

0.9077

0.5592

0.4652

**

5

151–179

0.9385

0.6122

0.5372

**

A

TD60–50

14

192–220

0.6838

0.8406

0.8191

B

TD60–50

11

254–276

0.7402

0.8035

0.774

A

TD50–40

15

151–189

0.7205

0.8105

0.7876

B

TD50–40

14

227–257

0.7874

0.8267

0.8015

6

233–253

0.3281

0.3986

0.3805

*

A

TD50–40

8

172–216

0.7205

0.7649

0.7261

5

198–210

0.8308

0.5368

0.4361

**

A

TD60–50

11

196–228

0.5754

0.6038

0.5628

3

210–220

0.2000

0.1814

0.1675

ND

B

TD50–40

16

113–167

0.7689

0.8656

0.8498

7

123–159

0.9385

0.6556

0.5928

**

A

TD60–50

9

93–123

0.6719

0.7216

0.6697

5

96–114

0.9385

0.6315

0.5574

**

A

TD60–50

14

333–387

0.6324

0.6948

0.6437

B

TD60–50

11

272–302

0.5534

0.5501

0.5268

4

282–296

0.1692

0.2073

0.1915

ND

A

TD50–40

10

155–175

0.7756

0.7642

0.7253

5

161–173

0.9692

0.6275

0.5554

**

*

*

**

N.L. Dillon et al. / Scientia Horticulturae 150 (2013) 213–226

LMMA1

Source reference

Significant after Bonferroni correction at P ≤ 0.05. Significant after Bonferroni correction at P ≤ 0.001. ND, not done; HE , expected heterozygosity; HO , observed heterozygosity; PIC, polymorphic information content; HWE, Hardy–Weinberg equilibrium. *

**

219

220

N.L. Dillon et al. / Scientia Horticulturae 150 (2013) 213–226

3.1.2. Embryony analysis The Mantel test analysis did not indicate a significant relationship between morphological embryony data and molecular SSR data in mango. No unique alleles were identified that could be used for differentiation of monoembryonic and polyembryonic types. Within the 254 accessions, 106 were monoembryonic, 112 polyembryonic, and five showed both mono- and poly-embryonic characteristics (Table 1). 3.1.3. Cultivar identification Ten accessions shared identical microsatellite genotypes and identical plant accession names. These were considered the same and grouped in the NJ dendrogram as a single accession. For example, the accession named ‘Carabao’ in this analysis represents six accessions, including ‘Carabao Flav’, ‘Carabao Harbon’, ‘Carabao Los Banos’, ‘Carabao Mindinao’, ‘Carabao Pep’ and ‘Carabao Super Manila’. This group of accessions could not be distinguished from each other with the 11 markers used in this study, yielding one unique genotype. Twenty-two unique microsatellite genotypes were also identified for 50 single plant accessions with different names. These individuals were included in the NJ dendrogram with their designated accession names. As a result, 226 unique genotypes were identified from the 254 accessions screened in this study. The selected set of validated SSR-based markers forms a mango genetic identity kit (Magik) that can be utilised to distinguish diversity among accessions. 3.1.4. Diversity analysis of mango accessions The allele data from the 11 SSR markers were used to generate a large NJ dendrogram, based on Reynolds genetic distance, for the 254 M. indica and related Mangifera accessions (Online Resource 1). The NJ dendrogram shows a high level of variability among accessions, forming four major nodes. Node one contains the majority of the Mangifera species analysed, including M. foetida, M. caesia, M. odorata, Mangifera torquenda, Mangifera pentandra, and Mangifera applanata. This node also includes several M. indica accessions and the related genus Buchanania. The second node contains polyembyonic South East Asian accessions originating from Thailand, the Philippines and Malesia/Indonesia. The third node includes accessions mainly from Viet Nam, India, Sri Lanka and Pakistan, with individuals from South East Asia, Florida, USA, Africa and the Pacific Islands. The fourth node is the largest of the nodes including the majority of accessions from Florida, USA and Australia. This node also includes accessions from India. 3.1.5. Genetic relationships of mango accessions using SplitsTree4 analysis Distance matrices for the 254 accessions were used for SplitsTree4 analysis (data not shown). This tree shows a slightly different grouping of accessions to that of the NJ dendrogram. The grouping of B. arborescens, M. foetida, M. caesia var. ‘Binjai’, M. casturi, and other possible Mangifera spp., with the polyembyonic Asian accessions (from Viet Nam, Thailand and the Philippines) are similar to the NJ dendrogram. However, M. lalijiwo, M. odorata and M. torquenda also cluster in this group in the SplitsTree4 analysis. The Florida, USA accessions cluster together similar to the NJ dendrogram analysis, as do the Australian and Indian accessions. 3.1.6. Geographic origin A NJ dendrogram, based on Reynolds genetic distance, was generated for twelve geographical regions with each region containing at least three genotypes of M. indica (Fig. 1). Accessions from the Philippines and Thailand group together, as do the accessions from Indonesia/Malaysia/Malesia; Kenya/South Africa; India and Pakistan; Florida and Hawaii/the Pacific Islands and accessions from Australia, Sri Lanka and the Caribbean. Accessions from Viet Nam are distinct from any other group.

Fig. 1. Neighbour-joining dendrogram for twelve geographical regions, each containing at least three genotypes of M. indica, based on Reynolds genetic distance calculated from microsatellite data. Bootstrap values greater than 0.5 are shown.

3.1.7. Parentage analysis Parentage analysis did not identify any likely candidate parents for ‘Kensington Pride’, however it did identify ‘Malika’ as the progeny of ‘Dashehari’ and ‘Neelum’ with no mismatches at 95% confidence. Other likely parents identified, with no mismatches at 95% confidence, were ‘Brooks’ for ‘Keitt’ and ‘Keitt Red’, ‘Haden’ for ‘Kent’ and ‘Hatcher’, ‘Lippens’ for ‘Irwin’ and ‘Jewel’, ‘Irwin’ for ‘Repozo’, and ‘Zill’ for ‘Spirit of 76’. 3.1.8. Admixture analysis and genetic structure in mango A model-based analysis of mango genetic structure was performed that allowed the number of populations (clusters) and the degree of membership of each individual in each cluster (admixture or introgression) to be inferred. All eleven microsatellite markers were used to estimate the number of sub-populations (K). The highest value for K, the rate of change in the log probability of the data between successive clusters, was obtained for the number of sub-populations (K) = 5, suggesting five genetic clusters among the 254 mango accessions studied. In general, the Bayesian estimates of K were similar to the number of nodes observed in the distance based cluster analysis using the neighbour-joining algorithm. Cluster visualisation for the permuted average Q-matrix for the runs of STRUCTURE associated with the maximum likelihood value of 5 is presented in Fig. 2. Of a total of 254 accessions, 163 (64%) were assigned to one of the five clusters with a probability of membership qI of over 80%. Those individuals with a probability below the 80% threshold showed admixture with almost 36% of individuals showing assignation to two or more clusters. This indicates that all analysed populations of mango whatever their present distribution have ancestors in the five clusters. Admixture analysis identified individuals classified as Mangifera species, landraces and cultivars that showed gene flow from cross-pollination of mixed ancestral origin. Cluster four had the largest number of individuals (63 trees), followed by clusters one (57 trees), two (49 trees), three (44 trees), and five (41 trees). Cluster one contains predominately Australian

N.L. Dillon et al. / Scientia Horticulturae 150 (2013) 213–226

221

Fig. 2. Bayesian analysis. Numbers on the y-axis show the subgroup membership and the numbers on the x-axis show the accession number with country of origin code in parentheses. Accession numbers and country of origin code are listed in Table 1. Five sub-populations, or groups, are indicated by colour; sub-population one (red) accessions from Indonesia and Australia; sub-population two (green) accessions from the Philippines, Thailand and Viet Nam; sub-population three (blue) predominately Australian accessions but including Indian and Indonesian accessions; sub-population four (yellow) predominately Indian accessions; sub-population five (pink) accessions from USA Florida, USA Hawaii, Mexico and Africa. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

accessions (32 trees), and includes Indonesian (6 trees), and Indian (6 trees) accessions. Cluster two contains Indonesian (15 trees), Malesian (12) and Australian (11 trees) accessions. Cluster three is made up of accessions from USA Florida (29 trees), USA Hawaii (5 trees), Mexico (1 tree) and Africa (4 trees). The majority of cluster four consists of Indian accessions (28 trees), however, almost each country of origin has at least one accession in this grouping. Cluster five is predominantly Asian accessions from the Philippines (5 trees), Thailand (19 trees) and Viet Nam (10 trees). To confirm populations, all 91 individuals that showed admixture from gene flow (P < 80%) in the first analysis were removed from the data set and the analysis repeated. Again the highest value for K was 5, supporting five genetic clusters among the 163 mango accessions studied. In this data set 99% of individuals were assigned to one of the five clusters with a probability of membership qI of over 80%. 3.2. Genetic diversity of ‘Kensington Pride’ Ten microsatellite markers were used for this study with a total of 51 alleles detected from 65 accessions assessed (Table 3).

A maximum of seven alleles per locus for MIAC-5, a minimum of three alleles for LMMA15 and an average of 5.1 alleles per locus were detected. Five SSR markers had a PIC value higher than 0.5 with an average PIC value of 0.45. The NJ dendrogram based on Reynolds genetic distance, for Australian and Florida accessions and for the ‘Kensington Pride’ subset of accessions is presented in Fig. 3. The Florida cultivars form a clade clearly separated from the Australian cultivars, including ‘Kensington Pride’. The genetic diversity of the ‘Kensington Pride’ accessions is low with 41 of the 46 ‘Kensington Pride’ accessions sharing all alleles. The mean genetic diversity (or observed heterozygosity) for all accessions was 0.72. 4. Discussion The eleven microsatellite markers used in this study were applied to the ANMG collection to determine the genetic variability and diversity of M. indica accessions and related Mangifera species. The mean number of alleles per locus (12.09) generated from the selected 11 markers applied to 254 accessions compares favourably to those reported previously. Schnell et al. (2006)

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4.1. Identity and genetic relationships between M. indica and other Mangifera species

Fig. 3. Neighbour-joining dendrogram of a subset of accessions including old Australian common types (green circles), ‘Kensington Pride’ selections (red squares), hybrid progeny of ‘Kensington Pride’ (purple diamonds) and their maternal parent (yellow triangles, including related Floridian accessions), based on Reynolds genetic distance. Bootstrap values greater than 0.5 are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

generated 6.96 alleles per locus using 25 microsatellite markers across 203 accessions, Duval et al. (2005) generated 6.5 alleles per locus using 19 microsatellite markers across 204 accessions and Viruel et al. (2005) generated 5.5 alleles per locus using 16 microsatellite markers across 28 accessions. This improvement is most likely due to our pre-selection of highly polymorphic markers for screening in this study and the diversity of our genebank accessions, which included a number of related Mangifera species.

The majority of Mangifera species analysed clustered in node one of the NJ dendrogram. Our data support a close relationship between M. odorata, M. foetida and M. indica. A hybrid origin had been suggested for M. odorata Griff. (synonyms kuwini, kwini) by Ding Hou (1978). This was later verified as a cross between M. indica and M. foetida Lour. (Teo et al., 2002; Kiew et al., 2003) and is supported by our NJ dendrogram, SplitsTree4 and admixture analyses. The index of similarity in Teo et al. (2002) shows M. odorata closer to M. foetida (76% similarity) than to M. indica (66% similarity). When comparing phylogenetic relationships of ITS sequences of these species, M. odorata is more closely related to M. foetida than to M. indica (Yonemori et al., 2002). However, recently Hidayat et al. (2011) placed M. odorata closer to M. indica than to M. foetida based on variation of matK sequences. Our genetic analyses using SSR markers also raised questions about the identity of some accessions that had previously been classified based on phenotypic characters alone. Results from both the NJ dendrogram and SplitsTree4 analyses place our accession of M. torquenda unexpectedly in close relationship with M. odorata. With little genetic diversity seen in an intra-species study of M. odorata (Yamanaka et al., 2006) and morphological similarity of the flowers of M. torquenda and M. odorata (Kostermans and Bompard, 1993) it is likely that the accession of M. torquenda used in this study may have been mis-labelled. Kostermans and Bompard (1993) list ‘Pelipisan’ as a local name for M. applanata in the Banjarese language from south Kalamantan. ‘Pelipisan’ could not be genetically distinguished from M. applanata or M. rubropetala in our NJ dendrogram or SplitsTree4 analyses. Accession ‘Asam Kumbang’ groups with ‘Sabre’ from South Africa and ‘Rosa’ from Brazil in node three. Kostermans and Bompard (1993) list ‘Asam Kumbang’ as a vernacular name for M. quadrifida, however in our analysis as the accession M. quadrifida appears to be mis-labelled and is more likely to be M. pentandra. In both the NJ dendrogram and SplitsTree4 analyses M. odorata is grouped with a wild Mangifera sp. var. ‘Thai Wild’ and the Thai green eating cultivar ‘Keow Savoey’. This raises the question of its true identity as possibly a different species, an inter-specific hybrid or an artefact of insufficient markers used. Other M. indica accessions also group with the Mangifera species. These include ‘Tung Chi’ and ‘Chandrakaran’ with M. laurina IPOH, ‘Betti Amber’ with M. laurina Delphi, and ‘Hinchinbrook’ and ‘Gedong’ with M. applanata and M. rubropetala. These accessions all show admixture with gene flow from accessions of diverse ancestry. Further investigations towards the identity of these accessions are warranted to clarify their genetic background. The accession ‘Pau’ is genetically identical to M. altissima in our NJ dendrogram and SplitsTree4 analyses. Kostermans and Bompard (1993) list ‘Pau’ as a vernacular name for M. altissima, indicating our accession labelled ‘Pau’ belongs to M. altissima, not M. indica. Limited information regarding inter-specific crosses is available from the literature. Mukherjee et al. (1968) reported that successful crosses between M. odorata and M. zeylanica were made in India, while in Australia successful crosses between M. indica and M. laurina have been made (Bally et al., in press). Suspected natural hybrids have been reported in Kalimantan between M. gedebe with M. laurina and M. foetida, and M. kemans with M. panjang and M. caesia (Bompard, 2009). Certainly from the Bayesian method and admixture analysis a number of individuals identified as Mangifera species, landraces and cultivars of mango revealed possible mixed ancestry, indicating a more complex origin than initially expected. The second node of the NJ dendrogram contains polyembyonic South East Asian accessions. Accessions from (a) Thailand and the Philippines, (b) Indonesia/Malesia and (c) India form distinct

N.L. Dillon et al. / Scientia Horticulturae 150 (2013) 213–226

subgroups that are related, but which can be clearly differentiated. Our study has confirmed previous phenotypic classification (Kostermans and Bompard, 1993) of many of these accessions and provided new information on their genetic relationships. The accessions from Thailand and the Philippines are closely related. The Vietnamese accessions fall into a separate node in the NJ dendrogram, suggesting that accessions from these countries have diversified developmental paths. This separation is not apparent in the SplitsTree4 analysis. Our analyses support observations inferred by Bondad et al. (1984), that the Thai cultivar ‘Nam Doc Mai’ is more closely related to the Philippine cultivar ‘Carabao’ than to any of the Vietnamese accessions. Previously, Popenoe (1920) reported that Philippine and Vietnamese mangoes were of the same ‘race’, that is, a group of cultivated plants with well-marked differentiating characters that propagate true-to-seed, except for simple fluctuating variations. In refinement of these early findings, our NJ dendrogram, Bayesian method and admixture analysis suggests a common ancestry for mangoes from Viet Nam, Thailand and the Philippines with an early divergence of the Vietnamese cultivars (Fig. 1). Placement of accessions from Indonesia/Malesia indicates several accessions that appear synonymous. One such group includes ‘Rajah’, ‘Golek’ and ‘Arumanis’ that could not be genetically distinguished. ‘Arumanis Red’, ‘Haem Wangi’ and ‘Sungi Siput’ could also not be genetically distinguished, while ‘New Guinea Long’, ‘Sulawasi’, ‘Banana Long’ and ‘Cedar Bay’ are also similar. This group also contains several Mangifera spp. from Indonesia, M. lalijiwo, M. laurina and M. casturi. Similar groupings in the SplitsTree4 analysis indicate that possibly the accession ‘Mangga Madu’ belongs to M. lalijiwo and the accessions ‘Gudang’ and ‘Asam Ramuk’ belong to M. casturi. Further investigation of the morphology of these accessions is warranted to confirm these assumptions. Many of the popular cultivated mono-embryonic Indian varieties are closely related indicating low genetic diversity among these, while other Indian accessions are scattered throughout the NJ dendrogam and SplitsTree4 analyses indicating the wider diversity within M. indica from the sub-continent. Another distinct node includes accessions mainly from Viet Nam, India and Pakistan, with individuals from South East Asia, Florida, USA, Africa and the Pacific Islands. This is also seen in the SplitsTree4 analysis apart from accessions from Viet Nam, mentioned previously. Although the Vietnamese accessions cluster together several are morphologically diverse suggesting they may not all be M. indica or have possible inter-specific hybrid origins. For example, morphological inspection of ‘Xoài Queó’ flowers shows multiple fertile stamens which indicate it likely to be (or closely related to) M. caloneura Kurz. Species belonging to the section Euantherae, including M. caloneura and M. pentandra, have five fertile stamens in a flower and five petals, while species of section Mangifera usually only have one fertile stamen (Kostermans and Bompard, 1993). Le et al. (1999) shows that in Viet Nam M. caloneura Kurz. (Vernacular names ‘Queó’ and ‘Xoài Lua’) is distributed countrywide in wetlands and has flaming red fruits, as its Vietnamese name denotes. Further investigation is also warranted of the accession ‘Xoài #7 as morphologically the flowers appear to have three fertile stamens while the other ‘Xoài’ accessions have just one. Node four of the NJ dendrogram is the largest of the groups and can be split into two sub-nodes that also correspond to clusters in the SplitsTree4 analysis. The subgroups contain accessions originating from Florida, USA, and from Australia, respectively. Although Florida, USA is not an original centre of origin it has become a secondary centre of origin (Knight and Schnell, 1994). The genetic similarity of the Florida accessions arises from their common heritage that can be traced back to as few as four Indian accessions and the ‘Terpentine’ land race (Olano et al., 2005). Natural hybridisation

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of these introduced accessions produced many of the recognised Florida cultivars which are grown and widely distributed throughout the world today. Group three of the Bayesian method and admixture analysis has the highest number of accessions from Florida. Many of these cultivars have been developed since the 19th century with known parentage. The current population structure gives a good basis for determination of ancestry and is consistent with known cultivars with known parentage from diverse origins. Several accessions with different names were found to have identical microsatellite genotypes in these analyses. Some previous reports on the morphology of these accessions have found them to be indistinguishable (Winston and Schaffer, 1993; Watson, 1984) while others found slight differences (Campbell, 1992; Winston and Schaffer, 1993). For example, the Florida USA accession ‘Ann’ has higher fibre than ‘Haden’ and ‘Hatcher’, while ‘Hatcher’ has low fibre (Campbell, 1992; Winston and Schaffer, 1993). Such minor phenotypic differences may be due to somatic mutation. However, Schnell et al. (2006) were able to differentiate between ‘Haden’ and ‘Hatcher’ using 25 microsatellite markers. ‘Tommy Atkins’ and ‘Parri’ could not be genetically distinguished, nor could ‘Akbar’ and ‘Sensation’ which are morphologically similar (Winston and Schaffer, 1993). The fact that this study was unable to genetically distinguish between these accessions indicates that accessions may not be true to those of their original source, may be mislabelled or that an insufficient number of markers was used to separate them. The tight grouping of Australian accessions suggests a common heritage and may reflect common trade routes from which the accessions were originally sourced. Their positioning in the NJ dendrogram and SplitsTree4 analysis suggests their origins are not from Viet Nam, Philippines, and Thailand. The origins of many Australian accessions may be gleaned from the other closely linked accessions in this group originated from India and Sri Lanka. Six accessions originating from India, Indonesia and Australia, appear morphologically similar and belong to the ‘Common’ land race could not be genetically distinguished. Other accessions of the ‘Common’ land race (‘Common Top End Rural’ and ‘Blue’) also appear in this node of the NJ dendrogram. In the SplitsTree4 analysis all accessions of the ‘Common’ land race group together yet apart from the main cluster of Australian accessions. 4.2. Population structure The Bayesian model-based structure analysis revealed the presence of five populations among the mango accessions. The groupings were similar to those of the NJ dendrogram and SplitsTree4 analyses. Of the 254 accessions analysed, 64% were assigned to one of the five clusters with a probability of membership qI of over 80%. All clusters showed admixture with almost 36% of accessions assigned to two or more clusters. This means that all populations of mango whatever their present distribution have ancestors in the five clusters. The mixture is likely the result of breeding, domestication history, spread by human migration or through trade, all which have large effects on the diversity of population structure. 4.3. Geographic origin Genetic distances clearly separate M. indica populations into distinct nodes based on their geographical origin. Accessions from the Philippines, Thailand, and Viet Nam group separately from all the other accessions in this analysis (Fig. 1). This is also reflected in the NJ, SplitsTree4 and Bayesian method admixture analyses. The close relationship between accessions from India and Pakistan is shown and expected due to their adjacent geographical location (the two countries being united until 1947) and the fact that most cultivars grown in Pakistan have been developed by seedling selection

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from Northern India (Rajwana et al., 2008). Basically, accessions from India and Pakistan have developed from a common genepool. Likewise, the close relationship between accessions from Florida with Hawaii/the Pacific Islands is expected with much germplasm having been exchanged between these areas (Schnell et al., 2006). The link between accessions from Australia and Sri Lanka may also give some insight into the pre-Australian origins of cultivars such as ‘Kensington Pride’. There is also a closer link with accessions from Indonesia/Malaysia with Australian accessions, rather than the more distantly related accessions from the Philippines, Thailand and Viet Nam, reflecting the early trading routes between Australia and South East Asian regions. 4.4. Parentage analysis The pre-Australian origin of ‘Kensington Pride’, before its introduction at Port Denison (now Bowen) between 1885 and 1889, is unknown and remains a mystery. This is due partly because it has distinctive flavour and aroma characteristics not common in other cultivars. It’s shape and red blush colour suggest it has an Indian sub-continent origin, while it’s polyembryonic nature suggests an South East Asian origin. While Johnson (2000) suggests ‘Kensington Pride’ is possibly a hybrid with Indian and South East Asian parentage this study did not find any significant candidate parents for this cultivar and cannot further the discussion on the origins of ‘Kensington Pride’. Parentage analysis did however confirm findings of previous studies. ‘Brooks’ was found to be the most likely parent for ‘Keitt’ (a seedling of ‘Mulgoba’) and ‘Keitt Red’ as previously suggested by Olano et al. (2005). Parentage analysis also identified ‘Irwin’ and ‘Jewel’ as seedlings of ‘Lippens’ (Campbell, 1992) ‘Repozo’ as a seedling of ‘Irwin’ (Hamilton et al., 1992) and ‘Spirit of 76 as a seedling of ‘Zill’ (Campbell, 1992). The NJ dendrogram and SplitsTree4 analyses also supported these relationships. Schnell et al. (2006) also confirmed these parental relationships. The hybrid accession ‘Mallika’ in our analysis is found to be closely linked to its maternal parent ‘Neelum’, and supports it’s origin as a hybrid between ‘Dashehari’ and ‘Neelum’ (Pandy, 1984). ‘Bangampalli’ can be confirmed as a parent of the accession ‘Hybrid 10’ but our analysis indicates the second parent is ‘Suvarnarekha’ rather than ‘Alphonso’ (Iyer and Subramanyam, 1992). 4.5. Genetic diversity of ‘Kensington Pride’ The genetic diversity of the ‘Kensington Pride’ accessions is low with up to 38 accessions sharing all alleles. This lack of genetic diversity within the ‘Kensington Pride’ accessions was not unexpected as similar genetic uniformity was found by Bally et al. (1996) using RAPD markers on many of the same accessions. Similarly, no significant phenotypic differences (yield, tree size, fruit number, average fruit weight or time of flowering) were measured between these accessions (Bally, pers. commun.). When Truscott et al. (1993) investigated variation in polyembryonic seedling populations using isozymes they found 12% of zygotic off types in ‘Kensington Pride’. This level of diversity was not found in this investigation or by Bally et al. (1996), suggesting that the methods of propagating polyembryonic seed in Australia favours the selection of the nucellar seedlings before the zygotic seedling. Bally et al. (1996) concluded from the lack of diversity in ‘Kensington Pride’ that little was to be gained from selection within the ‘Kensington Pride’ population and the introduction of genes from other cultivars may be the best way to improve the cultivar. Since the early 1990s the AMBP has been cross-breeding ‘Kensington Pride’ with a range of other mango cultivars to improve the fruit quality and productivity characteristics. In effect, the breeding programme has increased the genetic diversity of ‘Kensington

Pride’ style cultivars as demonstrated by the Kensington NJ dendrogram (Fig. 2) where the ‘Kensington Pride’ hybrids (‘Delta R2E2’, ‘A67 , ‘B74’, ‘Honeygold’, ‘NMBP 1243’, ‘NMBP 1201’, and ‘NMBP 4069’) fall between their maternal parent and the paternal parent ‘Kensington Pride’ (Table 2). 4.6. Application of genetic diversity data in mango breeding The genetic analysis of the diversity and ancestry of mango cultivars and Mangifera species held in the ANMG has confirmed many suspected genetic relationships among accessions and revealed new relationships that provide a better understanding of the origins of accessions. The analysis has revealed mis-identified and duplicate accessions within the collection and confirmed names as synonyms of many accessions. New genetic relationships among the group of accessions originating in South East Asia have been revealed and the genetic boundaries between commonly grown mango cultivars and wild Mangifera species have become blurred. Our analysis suggests that inter-specific hybridisation may be more common than previously reported. A closer examination of some of the suspected hybrid accessions is needed to clarify their status. The realisation that inter-specific hybridisation has occurred naturally as suggested by Bompard (2009) is encouraging for mango breeders looking to integrate genes form some of the wild Mangifera species into advanced M. indica lines. Natural hybridisation and introgression occurs widely in plants and plays an important role in their evolution (Arnold, 1997). Introgressive hybridisation is of great interest for plant evolutionary studies because it produces considerable numbers of new genotypes, thereby increasing genetic diversity, which may lead to new adaptations (Riesberg, 1991) and the formation of new ecotypes (Levin et al., 1996) or species (Soltis and Soltis, 1999) as with M. odorata. The improved understanding of cultivar identity, parentage genetic diversity and population structure that this study reveals is of great value to mango breeders in selection of breeding stock, and confirming parentage of progeny. The ability to identify the paternal parent in open pollinated crossing programmes allows extensive culling of unwanted progeny before field planting and tree maturity, saving time and resources growing and evaluating unwanted progeny. Understanding the genetic relatedness of potential parents enables the breeder to better select parents with the potential to contribute desired genes to the progeny. Genetic diversity is necessary for sustainable productivity of a crop with the introduction of new genes for such traits as yield, adaptation, disease resistance (Bally et al., in press), tree architecture, low temperature tolerance (Campbell and Ledesma, in press) or rootstock and interstock suitability (Campbell, 2007), and consumer preferences. Rich diversity currently exists but already two Mangifera species are listed on The IUCN Red List of Threatened Species (2011) as extinct in the wild, one is listed as critically endangered and seven as endangered. Conservation of Mangifera genetic resources is critical to the long term survival, improvement and sustainable production of the current commercial varieties. The application of molecular marker technologies as applied in this study can significantly improve breeding efficiency. Acknowledgments We thank Dr. Emma Mace and Kirsten Sakrewski (Hermitage Research Station, Warwick) and Dr. Mandy Christopher (Leslie Research Station, Toowoomba) for CEQ 8800 PCR product separation and analysis. We also acknowledge Dr. Katharina Shulte, Australian Tropical Herbarium, James Cook University, Cairns, for assistance with Splitstree4 analysis. We acknowledge funding for this work as part of the Mango Fruit Genomics Initiative supported

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