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21 June 2013 Development of Microsatellite Loci in Artocarpus altilis (Moraceae) and Cross-Amplification in Congeneric Species
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The genus Artocarpus J. R. Forst. & G. Forst. (Moraceae) contains several agriculturally significant species, breadfruit (A. altilis (Parkinson) Fosberg) and jackfruit (A. heteropyhllus Lam.) being the most widely cultivated (Zerega et al., 2010). Breadfruit is a traditional staple in Oceania, and its progenitor species are A. camansi Blanco and A. mariannensis Trécul; hybrids also exist (Zerega et al., 2004). The origin of jackfruit is unclear, but it is likely native to the Indian subcontinent (Jarrett, 1959). Even with morphological descriptors, accurate breadfruit cultivar identification remains difficult (Jones et al., 2013), and little is known about jackfruit genetic diversity (Shyamalamma et al., 2008). Despite the fact that breadfruit and jackfruit are in different Artocarpus subgenera (Zerega et al., 2010), several primers cross-amplified well and showed polymorphisms, suggesting they might be useful in additional Artocarpus species.

METHODS AND RESULTS

To construct the genomic library, leaf tissue was obtained from an A. altilis individual (voucher DR468 deposited at the National Tropical Botanical Garden [NTBG] Herbarium [PTBG], Kalaheo, Hawaii) collected from Fiji (Living Accession 900261.002 at the Breadfruit Institute, NTBG, Hawaii). DNA was extracted using the QIAGEN DNeasy Plant Mini Kit (QIAGEN, Valencia, California, USA) following standard protocol. Microsatellite libraries were developed by Genetic Identification Services (Chatsworth, California, USA), following the methods of Jones et al. (2002). The libraries were enriched for four repeat motifs—(GA)n, (CA)n, (ATG)n, and (TAGA)n· Thirty-eight clones containing microsatellites were sequenced. Using DesignerPCR version 1.03 (Research Genetics, Huntsville, Alabama, USA), 75 primer pairs were designed (approximately two pairs for each clone) and screened across 25 A. altilis and 12 A. heterophyllus individuals to test for amplification and polymorphisms. Nineteen primers for breadfruit (A. altilis) and related species (A. camansi, A. mariannensis, and A. altilis × mariannensis) and 10 primers for A. heterophyllus yielded either a single band, or two that were consistently different in size, when separated and visualized under UV light on an agarose gel stained with SYBR Green (Proligo, Hamburg, Germany). Three of these primer pairs amplified two loci of differing lengths (Tables 1 and 2). Loci MAA54a/b have conserved sequences flanking similar microsatellite sequences and may represent historical duplications that have since diverged (Karhu et al., 2000). The same is true for loci MAA178a/b. In loci MAA196a/b the microsatellite flanking sequences are not identical, but share enough similarities to allow for cross amplification. For all three primers, the two loci amplified were straightforward to score and consistently amplified in all samples. Loci 178a and 196a do not demonstrate linkage to 178b and 196b, respectively; loci 54a/b did deviate from the null hypothesis that the loci were not linked (P = 0.04), as tested in GENEPOP (Raymond and Rousset, 1995). Nineteen primers (amplifying 21 loci) were then used to evaluate 241 A. altilis, 34 A. camansi, 15 A. mariannensis, and 64 A. altilis × mariannensis samples collected from trees growing in private and public collections (Table 2, Appendix 1), and representing original collections from throughout Oceania as well as Indonesia, the Philippines, and the Seychelle Islands. Ten primers (amplifying 13 loci) were used to evaluate 426 A. heterophyllus samples collected from public collections with provenance from Thailand, Indonesia, Malaysia, Jamaica, Singapore, Australia, India, and Miami, and from private holdings in Bangladesh (Table 2, Appendices 1 and 2). Herbarium vouchers were made from representative sites in Bangladesh; accession numbers are given for samples from public living collections (Appendix 2).

TABLE 1.

Characteristics of 25 microsatellite loci amplified in Artocarpus species.

t01_01.gif

All Bangladeshi jackfruit samples were processed at the Chicago Botanic Garden (CBG), while all breadfruit and some jackfruit samples were processed at U.S. Department of Agriculture (USDA) laboratories. A subset of the jackfruit samples were processed at both laboratories, yielding identical results. For samples processed at CBG, DNA was extracted as described above. For PCR reactions, forward primers had an M13 tail (5′-CACGACGTTGTAAAA-3′), and M13 primer labeled with WellRED Dye D2, D3, or D4 (Beckman Coulter, Brea, California, USA) was added to each reaction (Schuelke, 2000). PCR reactions used a two-step process. First, 10-µL reactions contained 5 µL of Master Mix (Promega Corporation, Madison, Wisconsin, USA), 0.5 µL of 10 mg/mL bovine serum albumin (BSA), 0.25 µL of 10 µM forward primer with the M13 tail, 0.25 µL of 10 µM reverse primer, 3 µL of H2O, and 1 µL of template DNA. PCR conditions for the first step were 94°C for 3 min; 13 cycles at 94°C for 30 s, 59.8°C for 30 s, and 72°C for 1 min; and a final extension of 72°C for 10 min. To each 10-µL reaction was added 2.5 µL Master Mix (Promega Corporation), 0.25 µL of 10 mg/mL BSA, 0.125 µL of 2.5 µM MgCl2, 0.25 µL of 10 µL labeled M13 primer, and 1.875 µL of H2O. PCR conditions for the second step were 94°C for 3 min; 27 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and a final extension of 72°C for 10 min. PCR product (0.5 µL of WellRED Dye D4-labeled product, 1 µL of WellRED Dye D3-labeled product, or 2.5 µL of WellRED Dye D2-labeled product [Beckman Coulter]) was added to 30 µL of HiDi formamide (Azco Biotech, San Diego, California, USA) and 3.3 µL of 400-bp size standard ladder (Beckman Coulter) and analyzed on a Beckman Coulter CEQ 8000 Genetic Analysis System. Alleles were scored using the CEQ 8000 software version 9.0 (Beckman Coulter). For samples processed at USDA laboratories, DNA was extracted at the USDA–Agricultural Research Service (ARS) Tropical Agriculture Research Station (TARS) following manufacturer's protocol using MP Biomedicals FastDNA Spin Kit (MP Biomedicals, Solon, Ohio, USA). DNA samples were then shipped to the USDA-ARS Genomics and Bioinformatics Research Unit in Stoneville, Mississippi. PCR reactions were carried out with 10 ng of DNA using the QIAGEN Multiplex PCR Kit (QIAGEN) in 5.0-µL reactions. Reactions contained 2.5 µL of 2× QIAGEN Multiplex PCR Master Mix, 0.1 µL each of a 10 mM forward and reverse primer labeled with HEX fluorescent dye, 0.1 µL each of a 10 mM forward and reverse primer labeled with FAM fluorescent dye, 10 ng of DNA, and water to equal 5 µL. PCR conditions were 95°C for 15 min; 40 cycles of 94°C for 30 s, 55°C for 90 s, and 72°C for 1 min; and a final extension of 60°C for 30 min. PCR fragments were analyzed on an ABI 3730xl DNA Analyzer and data processed using GeneMapper version 3.7 (Applied Biosystems, Foster City, California, USA).

TABLE 2.

Genetic diversity results for 25 microsatellite loci in Artocarpus species.a,b

t02_01.gif

Many breadfruit cultivars are known to be triploid based on chromosome counts (Ragone, 2001; Zerega et al., 2004), and this was confirmed with microsatellite data. The data revealed additional cultivars, which were thought to be triploid due to sterile fruits, to indeed be triploid. GenoDive (Meirmans and Van Tienderen, 2004) was used for diversity statistics. The number of alleles was evaluated for all primers. Methods for assessing allele dosage in triploids were only partially successful (Esselink et al., 2004), so observed and expected heterozygosities and deviation from Hardy–Weinberg equilibrium (HWE) were only analyzed in diploids, while percent heterozygosity was evaluated in triploids. All loci are polymorphic for at least one species. The allele numbers across loci and species range from one to 15, with the average number within a species ranging from two to nine (Table 2). GenoDive also assigned clones to determine if individuals could be differentiated. Genotypes across all loci were unique for A. heterophyllus and diploid A. altilis × mariannensis samples. In the remaining species, multiple samples share genotypes (number of unique genotypes/total samples): triploid A. altilis (53/162), triploid A. altilis × mariannensis (27/33), diploid A. altilis (46/79), A. mariannensis (14/15), and A. camansi (33/34). Several loci showed significant departure from HWE, although the loci varied between taxa (Table 2). Given that these are cultivated species and they violate several assumptions for HWE, this is not a surprising result and could be due to selection, nonrandom mating, or asexual reproduction.

CONCLUSIONS

The newly developed microsatellite loci show high levels of polymorphism in several Artocarpus species, and the success in cross-amplification is promising for broad use of these markers in congeners. These markers provide an excellent resource to quantify levels of genetic variation, patterns of population structure, and evolutionary and domestication history. They should also prove useful in future studies for cultivar identification, establishing crop germplasm conservation strategies, helping to determine ploidy level, and understanding origins and dispersal of breadfruit and jackfruit.

LITERATURE CITED

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Appendices

APPENDIX 1. Location data for Artocarpus samples used to characterize microsatellite loci.

  • Below is the list of samples from public germplasm collections. For each sample, the following information is given: germplasm accession number, the collection provenance or cultivar name, and the name of the germplasm collection, in that order. Abbreviations: CHIC = Nancy Poole Rich Herbarium at the Chicago Botanic Garden; FTG-H = Fairchild Tropical Botanic Garden, Homestead, Florida; FTG-M = Fairchild Tropical Botanic Garden, Miami, Florida ( http://www.fairchildgarden.org/livingcollections/ tropicalfruitprogram/jackfruit/); NTBG = National Tropical Botanic Garden, Kalaheo, Hawaii ( http://ntbg.org/breadfruit/collection/); TARS = Tropical Agriculture Research Station; USDA-NGR-H = U.S. Department of Agriculture—National Germplasm Repository, Hilo, Hawaii ( http://www.ars-grin.gov/cgi-bin/npgs/html/site_holding.pl?HILO); USDA-NGR-M= U.S. Department of Agriculture–National Germplasm Repository, Mayagüez, Puerto Rico ( http://www.ars-grin.gov/cgi-bin/npgs/html/site_holding.pl?MAY).

  • Artocarpus altilis (Parkinson) Fosberg, diploid samples: HART 26, unknown, USDA-NGR-H; HART 1, Samoa, USDA-NGR-H; HART 21, Marquesas Islands, USDA-NGR-H; HART 50, Solomon Islands, USDANGR-H; 020354.001, Samoa, NTBG; 910278.001, Vanuatu, NTBG; 890470.002, Rotuma, NTBG; 890470.001, Rotuma, NTBG; 900248.001, Society Islands, NTBG; 890457.001, Rotuma, NTBG; 900265.001, Fiji, NTBG; 890469.002, Solomon Islands, NTBG; 890469.001, Solomon Islands, NTBG; 70659.021, Samoa, NTBG; 70659.022, Samoa, NTBG; 770517.001, Samoa, NTBG; 900259.001, Vanuatu, NTBG; 890473.001, Vanuatu, NTBG; 900263.001, Samoa, NTBG; 890461.001, Marquesas Islands, NTBG; 890461.002, Marquesas Islands, NTBG; 070246.002, ‘Meiuhpw en Samoa’, NTBG; 770519.001, Samoa, NYBG; 960575.001, ‘Mos en Samoa’, NTBG; 890454.001, Cook Islands, NTBG; 900231.001, Cook Islands, NTBG; 890471.002, Fiji, NTBG; 890153.001, Cook Islands, NTBG; 910266.001, Society Islands, NTBG; 790492.001, Society Islands, NTBG; 900233.002, Rotuma, NTBG; 900233.001, Rotuma, NTBG; 030039.001, ‘Puou’, NTBG; 770520.001, Samoa, NTBG; 890474.001, Samoa, NTBG; 910275.001, Vanuatu, NTBG; 880691.001, Tonga, NTBG; 900257.001, Rotuma, NTBG; 890475.002, Samoa, NTBG; 900234.001, Fiji, NTBG; 900261.001, Fiji, NTBG; 900261.002, Fiji, NTBG; 910279.001, Vanuatu, NTBG; 910279.002, Vanuatu, NTBG; 910276.001, Vanuatu, NTBG; 890156.002, Cook Islands, NTBG; 890156.001, Cook Islands, NTBG; 910277.001, Vanuatu, NTBG; 900281.001, Solomon Islands, NTBG; 900281.002, Solomon Islands, NTBG; 890456.001, Solomon Islands, NTBG; 770521.001, Samoa, NTBG; 040063.001, ‘Ulu Fiti’, NTBG; 890258.001, Rotuma, NTBG; 020356.001, ‘Ulu Fiti’, NTBG; 890458.001, Rotuma, NTBG; 890458.002, Rotuma, NTBG; 900368.001, Rotuma, NTBG; 900260.002, Fiji, NTBG; 900260.001, Fiji, NTBG; 890155.001, Samoa, NTBG; 890155.002, Samoa, NTBG; 900228.001, Samoa, NTBG; 900235.001, Solomon Islands, NTBG; 020498.001, Solomon Islands, NTBG; 020500.001, Solomon Islands, NTBG; 890471.001, Fiji, NTBG; 900264.001, Fiji, NTBG; 890477.001, Fiji, NTBG; 970236.001, Rotuma, NTBG; 910289.001, unknown, NTBG; 900226.001, unknown, NTBG;900224.001, unknown, NTBG;020347.001, unknown, NTBG; 020348.001, unknown, NTBG; 020352.001, unknown, NTBG; 020353.002, unknown, NTBG; 020353.001, unknown, NTBG; 020355.001, unknown, NTBG

  • Artocarpus altilis (Parkinson) Fosberg, triploid samples: TARS 6732 plot 22, Barbados, USDA-NGR-M; TARS plot 11, unknown, USDA-NGR-M; TARS 7990 plot 5, Samoa, USDA-NGR-M; TARS plot 18, unknown, USDA-NGR-M; HART 11, Marquesas Islands, USDA-NGR-H; HART 16, Society Islands, USDA-NGR-H; HART 18, Society Islands, USDANGR-H; HART 23, Marquesas Islands, USDA-NGR-H; HART 28, Marquesas Islands, USDA-NGR-H; HART 29, Society Islands, USDANGR-H; HART 30, Society Islands, USDA-NGR-H; HART 32, Society Islands, USDA-NGR-H; HART 33, Society Islands, USDA-NGR-H; HART 34, Society Islands, USDA-NGR-H; HART 35, Palau, USDANGR-H; HART 33, Chuuk, USDA-NGR-H; HART 38, Northern Mariana Islands, USDA-NGR-H; HART 40, Society Islands, USDA-NGR-H; HART 42, Society Islands, USDA-NGR-H; HART 44, Society Islands, USDA-NGR-H; HART 45, Society Islands, USDA-NGR-H; HART 33, Palau, USDA-NGR-H; HART 49, Pohnpei, USDA-NGR-H; HART 51, Chuuk, USDA-NGR-H; HART 52, Pohnpei, USDA-NGR-H; HART 53, Pohnpei, USDA-NGR-H; HART 54, Society Islands, USDA-NGR-H; HART 56, unknown, USDA-NGR-H; HART 57, unknown, USDA-NGR-H; 900232.001, Cook Islands, NTBG; 890153.002, Cook Islands, NTBG; 900256.001, Cook Islands, NTBG; 890472.002, Cook Islands, NTBG; 890472.001, Cook Islands, NTBG; 890476.002, Fiji, NTBG; 890162.002, Northern Mariana Islands, NTBG; 890162.001, Northern Mariana Islands, NTBG; 900241.001, Marquesas Islands, NTBG; 900240.001, Marquesas Islands, NTBG; 900238.001, Marquesas Islands, NTBG; 900242.001, Marquesas Islands, NTBG; 900239.001, Marquesas Islands, NTBG; 890462.001, Marquesas Islands, NTBG; 900237.001, Marquesas Islands, NTBG; 890159.001, Palau, NTBG; 890159.002, Palau, NTBG; 890183.003, Palau, NTBG; 900266.002, Pohnpei, NTBG; 900266.001, Pohnpei, NTBG; 890478.001, Pohnpei, NTBG; 890478.002, Pohnpei, NTBG; 000534.001, Pohnpei, NTBG; 790497.002, Pohnpei, NTBG; 000531.001, Pohnpei, NTBG; 890167.001, Pohnpei, NTBG; 890167.002, Pohnpei, NTBG; 890479.002, Pohnpei, NTBG; 890479.001, Pohnpei, NTBG; 790493.001, Pohnpei, NTBG; 910273002, Pohnpei, NTBG; 910271.001, Pohnpei, NTBG; 910274.001, Pohnpei, NTBG; 900262.001, Samoa, NTBG; 770524.001, Samoa, NTBG; 890455.001, Samoa, NTBG; 810290.001, Seychelles, NTBG; 810290.002, Seychelles, NTBG; 810290.003, Seychelles, NTBG; 810289.002, Seychelles, NTBG; 780332.001, Society Islands, NTBG; 40051.001, Society Islands, NTBG; 780325.001, Society Islands, NTBG; 910267.001, Society Islands, NTBG; 780333.001, Society Islands, NTBG; 900249.001, Society Islands, NTBG; 900249.002, Society Islands, NTBG; 890157.001, Society Islands, NTBG; 890158.001, Society Islands, NTBG; 890158.002, Society Islands, NTBG; 900243.001, Society Islands, NTBG; 890147.001, Society Islands, NTBG; 890147.002, Society Islands, NTBG; 780335.001, Society Islands, NTBG; 780330.002, Society Islands, NTBG; 780330.001, Society Islands, NTBG; 890151.001, Society Islands, NTBG; 780291.001, Society Islands, NTBG; 790487.001, Society Islands, NTBG; 900245.001, Society Islands, NTBG; 890150.001, Society Islands, NTBG; 800269.001, Society Islands, NTBG; 890459.001, Society Islands, NTBG; 890459.002, Society Islands, NTBG; 890149.001, Society Islands, NTBG; 890154.001, Society Islands, NTBG; 30035.001, Society Islands, NTBG; 780327.001, Society Islands, NTBG; 890464.001, Society Islands, NTBG; 890463.002, Society Islands, NTBG; 890463.001, Society Islands, NTBG; 790489.001, Society Islands, NTBG; 910266.002, Society Islands, NTBG; 030037.001, Society Islands, NTBG; 900244.001, Society Islands, NTBG; 780328.001, Society Islands, NTBG; 890460.001, Society Islands, NTBG; 790485.001, Society Islands, NTBG; 890152.002, Society Islands, NTBG; 890152.001, Society Islands, NTBG; 780329.001, Society Islands, NTBG; 790486.001, Society Islands, NTBG; 780338.001, Society Islands, NTBG; 890465.001, Society Islands, NTBG; 790488.001, Society Islands, NTBG; 790491.001, Society Islands, NTBG; 900246.001, Society Islands, NTBG; 900247.001, Society Islands, NTBG; 890186.001, Society Islands, NTBG; 890186.002, Society Islands, NTBG; 890148.001, Society Islands, NTBG; 900236.001, Solomon Islands, NTBG; 880690.001, Tonga, NTBG; 890165.001, Chuuk, NTBG; 070883.001, unknown, NTBG; 30033.001, ‘Mei Tehid’, NTBG; 40518.001, ‘Rare Autia’, NTBG; 780345.001, ‘Raumae’, NTBG; 030042.001, ‘Toneno’, NTBG; 070882.001, ‘Ulu’, NTBG; 070882.002, ‘UIu’, NTBG; 090739.001, ‘Ulu’, NTBG; 100346.001, ‘Ulu’, NTBG; 100347.001, ‘Ulu’, NTBG; 100348.001, ‘Ulu’, NTBG; 100349.001, ‘Ulu’, NTBG; 100369.001, ‘Ulu’, NTBG; 100370.001, ‘Ulu’, NTBG; 970274.001, ‘Ulu’, NTBG; 070101.001, ‘Ulu’, NTBG; 070132.001, ‘Ulu’, NTBG; 080439.001, ‘Ulu’, NTBG; 080440.001, ‘Ulu’, NTBG; 080858.001, ‘Ulu’, NTBG; 080859.001, ‘Ulu’, NTBG; 080863.001, ‘Ulu’, NTBG; 080864.001, ‘Ulu’, NTBG; 080864.002, ‘Ulu’, NTBG; 080881.001, ‘Ulu’, NTBG; 030044.001, ‘Ulu Tala’, NTBG; 900268.001, unknown, NTBG; 910290.001, unknown, NTBG; 900267.001, unknown, NTBG; 910288.001, unknown, NTBG; 910287.001, unknown, NTBG; 900225.001, unknown, NTBG; 910286.001, unknown, NTBG; 030028.001, ‘White’, NTBG; 810290.004, ‘White‘, NTBG; 810289.001, ‘White’, NTBG

  • Artocarpus altilis × mariannensis, diploid samples: 890452.001, Kiribati, NTBG; 890177.003, Tokelau, NTBG; 890178.001, Tokelau, NTBG; 890177.001, Tokelau, NTBG; 890172.001, Kiribati, NTBG; 890179.001, Tokelau, NTBG; 890257.001, Tokelau, NTBG; 890171.001, Tokelau, NTBG; 890176.001, Tokelau, NTBG; 890174.001, Tokelau, NTBG; 890172.002, Tokelau, NTBG; 890176.002, Tokelau, NTBG; 890168.002, Tokelau, NTBG; 890177.002, Tokelau, NTBG; 890171.002, Tokelau, NTBG; 890181.002, Tokelau, NTBG; 890181.001, Tokelau, NTBG; 890173.001, Tokelau, NTBG; 890173.002, Tokelau, NTBG; 900230.001, Tokelau, NTBG; 890175.001, Tokelau, NTBG; 890182.001, Tokelau, NTBG; 890182.002, Tokelau, NTBG; 890180.001, Tokelau, NTBG; 890170.002, Tolekau, NTBG; 900253.002, Chuuk, NTBG; 900253.001, Chuuk, NTBG; 890166.001, Chuuk, NTBG; 890466.002, Chuuk, NTBG; 890184.001, Yap, NTBG

  • Artocarpus altilis × mariannensis, triploid samples: 890468.002, Kiribati, NTBG; 890163.002, Northern Mariana Islands, NTBG; 890163.001, Northern Mariana Islands, NTBG; 980210.001, Palau, NTBG; 890160.001, Palau, NTBG; 910652.001, Palau, NTBG; 890182.001, Tokelau, NTBG; 890183.001, Palau, NTBG; 890183.002, Palau, NTBG; 900250.001, Palau, NTBG; 890480.002, Pohnpei, NTBG; 890480.001, Pohnpei, NTBG; 910270.001, Pohnpei, NTBG; 890480.003, Pohnpei, NTBG; 790494.001, Pohnpei, NTBG; 910272.002, Pohnpei, NTBG; 910272.001, Pohnpei, NTBG; 900255.001, Pohnpei, NTBG; 890467.001, Pohnpei, NTBG; 790490.001, Society Islands, NTBG; 910265.001, Society Islands, NTBG; 890453.001, Tokelau, NTBG; 890185.001, Tokelau, NTBG; 910269.001, Chuuk, NTBG; 910268.001, Chuuk, NTBG; 890164.001, Chuuk, NTBG; 890164.002, Chuuk, NTBG; 890161.001, Yap, NTBG; 030034.001, ‘Pohnpei, NTBG; 030034.002, Pohnpei, NTBG; 030041.001, ‘Rotuma’, NTBG; 990781.001, unknown, NTBG; 30045.001, ‘Yap’, NTBG

  • Artocarpus camansi Blanco, all diploid: TARS 18009 plot 14, unknown, USDA-NGR-M; HART 63 N90-148, unknown, USDA-NGR-H; NTBG50, 910283.001, Indonesia, NTBG; 980212.001, Palau, NTBG; 000398.001, Papua New Guinea, NTBG; 000501.001, Papua New Guinea, NTBG; 000398.003, Papua New Guinea, NTBG; 000399.001, Papua New Guinea, NTBG; 000398.002, Papua New Guinea, NTBG; 000502.001, Papua New Guinea, NTBG; 000501.001, Papua New Guinea, NTBG; 000499.001, Papua New Guinea, NTBG; 000503.001, Papua New Guinea, NTBG; 000499.002, Papua New Guinea, NTBG; 000395.001, Papua New Guinea, NTBG; 000390.001, Papua New Guinea, NTBG; 000503.002, Papua New Guinea, NTBG; 000389.001, Papua New Guinea, NTBG; 000398004, Papua New Guinea, NTBG; 000500.001, Papua New Guinea, NTBG; 910281.001, Philippines, NTBG; 910280.001, Pohnpei, NTBG; 770444.001, Society Islands, NTBG; 960576.001, unknown, NTBG; 000390.002, Papua New Guinea, NTBG; 000394.002, Papua New Guinea, NTBG; 000395.002, Papua New Guinea, NTBG; 000398.005, Papua New Guinea, NTBG; 000499.003, Papua New Guinea, NTBG; 000500.002, Papua New Guinea, NTBG; 000501.004, Papua New Guinea, NTBG; 000501.005, Papua New Guinea, NTBG; 000502.002, Papua New Guinea, NTBG; 000503.004, Papua New Guinea, NTBG

  • Artocarpus heterophyllus Lam., all diploid: TARS 1566 plot 5, Indonesia, USDA-NGR-M; TARS 1566 plot 9, Indonesia, USDA-NGR-M; TARS 1566 plot 10, Indonesia, USDA-NGR-M; TARS 1566 plot 19, Indonesia, USDA-NGR-M; TARS 18002 plot 16, Malaysia, USDA-NGR-M ; HART 27, Thailand, USDA-NGR-H; WB2-FTBG-F-21-A, unknown, FTG-M; WB2-FTBG-F-05-A, unknown, FTG-M; S2-01-07-07, unknown, FTG-M; WB2-FTBG-F-16-A, unknown, FTG-M; T#10 R4, ‘Black Gold’, FTG-H; T#5 R5, ‘Chenna’, FTG-H; T#15 R4, ‘Cochin’, FTG-H; T#5 R3, ‘Dang Rasimi’, FTG-H; T#l 1 R4, ‘J31’, FTG-H; T#6 R4, ‘L.”, FTG-H; T#2 R4, ‘L.”, FTG-H; T#12 R4, ‘NS1’, FTG-H; T#5 R4, ‘Pani Varaka’, FTG-H; WB2-FTBG-F-15-A, ‘Golden Nugget’, FTG-M; WB2-FTBG-F-01-A, ‘Kwai Mok’, FTG-M

  • Artocarpus mariannensis Trécul, all diploid: HART 67 N05-8, unknown, USDA-NGR-H; 900252.001, Northern Mariana Islands, NTBG; 900252.003, Northern Mariana Islands, NTBG; 900252.002, Northern Mariana Islands, NTBG; 000522.002, Northern Mariana Islands, NTBG; 000523.001, Northern Mariana Islands, NTBG; 000521.002, Northern Mariana Islands, NTBG; 000521.001, Northern Mariana Islands, NTBG; 000522.001, Northern Mariana Islands, NTBG; 000521.003, Northern Mariana Islands, NTBG; 970306.001, Pohnpei, NTBG; 970306.002, Pohnpei, NTBG; 000528.001, Pohnpei, NTBG; 000523.002, Northern Mariana Islands, NTBG; 940010.002, Northern Mariana Islands, NTBG

APPENDIX 2.

Artocarpus heterophyllus samples collected in Bangladesh by authors Witherup, Zuberi, and Zerega. A representative herbarium voucher was made at most sites, and a picture voucher exists for all samples.

tA01_01.gif

Notes

[1] The authors thank S. Hussein and I. Cole for assistance with specimen collection, J. Fant and D. Reitz for assistance in the laboratory, and J. Fant and three anonymous reviewers for thoughtful comments on the manuscript. This study was funded in part by the National Science Foundation (NSF DEB REVSYS 0919119), a Plant Biology and Conservation Award from Northwestern University, an American Society of Plant Taxonomists Graduate Student Research Grant, and U.S. Department of Agriculture– Agricultural Research Service (USDA-ARS) National Programs.

Colby Witherup, Diane Ragone, Tyr Wiesner-Hanks, Brian Irish, Brian Scheffler, Sheron Simpson, Francis Zee, M. Iqbal Zuberi, and Nyree J. C. Zerega "Development of Microsatellite Loci in Artocarpus altilis (Moraceae) and Cross-Amplification in Congeneric Species," Applications in Plant Sciences 1(7), (21 June 2013). https://doi.org/10.3732/apps.1200423
Received: 26 July 2012; Accepted: 16 December 2012; Published: 21 June 2013
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