©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (3): 1942-1948 (2012) Brief Note Mendelian inheritance, linkage and genotypic disequilibrium in microsatellite loci isolated from Hymenaea courbaril (Leguminosae) F.S. Carneiro1, A.E.B. Lacerda2, M.R. Lemes3,6, R. Gribel3,6, M. Kanashiro4 and A.M. Sebbenn5 1Faculdade de Engenharia de Ilha Solteira, Universidade Estadual de São Paulo “Júlio de Mesquita Filho”, Ilha Solteira, SP, Brasil 2Embrapa Florestas, Curitiba, PR, Brasil 3Laboratório de Genética e Biologia Reprodutiva de Plantas (LARGEN), Instituto Nacional de Pesquisa da Amazônia, Manaus, AM, Brasil 4Embrapa Amazônia Oriental, Belém, PA, Brasil 5Instituto Florestal de São Paulo, São Paulo, SP, Brasil 6Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro, RJ, Brasil Corresponding author: A.M. Sebbenn E-mail: alexandresebbenn@yahoo.com.br Genet. Mol. Res. 11 (3): 1942-1948 (2012) Received February 28, 2012 Accepted April 27, 2012 Published July 19, 2012 DOI http://dx.doi.org/10.4238/2012.July.19.13 ABSTRACT. The Neotropical tree Hymenaea courbaril, locally known as Jatobá, is a valuable source of lumber and also produces comestible and medicinal fruit. We characterized Mendelian inheri- tance, linkage and genotypic disequilibrium at nine microsatellite 1943 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (3): 1942-1948 (2012) Hymenaea microsatellite inheritance loci isolated from H. courbaril, in order to determine if they would provide accurate estimates of population genetic parameters of this important Amazon species. The study was made on 250 open-pol- linated offspring originated from 14 seed trees. Only one of nine loci presented significant deviation from the expected Mendelian segregation (1:1). Genotypic disequilibrium between pairwise loci was investigated based on samples from 55 adult and 56 juvenile trees. No genetic linkage between any paired loci was observed. After Bonferroni’s corrections for multiple tests, we found no evidence of genotypic disequilibrium between pairs of loci. We conclude that this set of loci can be used for genetic diversity/ structure, mating system, gene flow, and parentage analyses in H. courbaril populations. Key words: Brazilian Amazon; Hymenaea courbaril; SSR loci; Mendelian inheritance; Neotropical tree; Population genetics INTRODUCTION Hymenaea courbaril L. (Leguminosae) is one of the most important timber spe- cies currently logged in the Brazilian Amazon. In general this large tree occurs in very low population densities in natural stands (<1 tree/ha). Owing to its intense exploitation in the Amazon, populations of H. courbaril have been significantly reduced (Lacerda et al., 2008a). Investigations about the impacts of forest logging on the genetic diversity, mating system and gene flow using genetic markers, are very important for the development of species’ ap- propriate sustainable logging practices. For H. courbaril, such studies have recently started (Dunphy et al., 2004; Lacerda et al., 2008a,b; Sebbenn et al., 2008) after the development and characterization of a set of polymorphic microsatellite loci for the species (Ciampi et al., 2000). However, to ensure the appropriate use of these loci for genetic analyses of H. courbaril populations, it is important to verify if the microsatellite loci display Mendelian inheritance and if they are associated. Here, we characterized the Mendelian inheritance, linkage and genotypic disequilib- rium in nine specific microsatellite loci previously developed for H. courbaril, an important timber species in the Brazilian Amazon, for robust application in population genetic studies such as genetic diversity and structure, mating system, gene flow, and parentage analyses in this species. METHODS AND RESULTS Mendelian inheritance and linkage disequilibrium between loci were examined based on 250 open-pollinated offspring collected from 14 seed trees (13 to 20 seeds per seed tree) in a 546-ha plot in the Tapajós National Forest, Pará State, Brazil. We also sam- pled 55 adult and 56 juvenile trees to study the genotypic disequilibrium between pairwise 1944 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (3): 1942-1948 (2012) F.S. Carneiro et al. loci. For DNA extraction, two cambium samples were collected per adult and/or juvenile trees. The cambium samples were stored in microtubes (1.5 mL) containing a DNA extrac- tion buffer solution [CTAB buffer (1/3) and ethanol (2/3)], and stored at -20°C until DNA extraction. From nursed saplings (ca. 45 cm heigh), two leaflets were collected, dried on silica gel, and maintained at -20°C until DNA extraction. Total genomic DNA was extracted following a standard CTAB protocol (Doyle and Doyle, 1987). Nine nuclear microsatellite markers previously developed for H. coubaril (Ciampi et al., 2000) were fluorescently labeled for multiplexing analysis as described by Carneiro et al. (2011). The in vitro amplification of the microsatellite loci was carried out in a total volume of 10 µL containing 1X PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2), 200 µM dNTPs, 2.5 mg/mL bovine serum albumin, 1.25 µM of each forward and reverse primer, 1 U Taq DNA polymerase and 5.0 ng genomic DNA using a Veriti thermal cycler (Life Technologies). The PCR conditions consisted of an initial heating at 95°C for 15 min followed by 30 cycles of 94°C for 1 min, annealing at the melting temperature of each primer pair for 1 min, and 72°C for 1 min, and a final extension at 72°C for 30 min. The PCR products were visualized on 1.5% agarose gels stained with ethidium bromide under UV light and sized using 1 kB Plus DNA Ladder (Gibco, USA). After dilution the PCR products were sized using GeneScan ROX 500 sizing standard in a 3130XL platform (Life Technologies). The amplified fragments (alleles) were analyzed using GeneMapper v. 1.0 (Life Technologies). Mendelian inheritance of the microsatellite loci was determined according to Gillet and Hattermer (1989). The genetic linkage between loci was tested against the hypothesis that maternal seed trees, considering heterozygous at two loci (AiAjBiBj) and in the absence of linkage, would result in equal proportions (test of 1:1:1:1) of the different maternal ga- metic types (AiBi, AiBj, AjBi, and AjBj). Thus, the observed genotypes of maternal families from seed trees heterozygous at two loci were compared to the expected gametic propor- tion based on no linkage (1:1:1:1). This hypothesis was evaluated by a maximum likeli- hood G-test carried out for each seed tree. We also tested the genotypic disequilibrium between pairwise loci in adults and juveniles, using the FSTAT program (Goudet, 1995) and a Bonferroni’s correction (α = 0.05) to avoid false positives. The results showed a significant deviation from the expected 1:1 Mendelian seg- regation in only one family in the locus Hc33 (Table 1). However, when we grouped the families with the same maternal genotype and re-analyzed the segregation, there was no observed deviation from the expected 1:1 segregation. After Bonferroni’s correction only eight (0.7%) of 106 linkage tests performed (Table 2) were significant, suggesting linkage between some pairs of loci. In all cases in which significant linkage was observed, it oc- curred in different pairs of loci of different samples and in only one family. On the other hand, the majority of families adhered to the expected 1:1:1:1 Mendelian inheritance for the same pairs of loci analyzed. Hence, we conclude that the nine microsatellite loci ana- lyzed are not linked. A small number of pairwise loci showed linkage, which probably can be an effect of the small sampling size within the families. After Bonferroni’s correction, the results showed no significant evidence of genotypic disequilibrium between pairwise loci (Table 3). 1945 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (3): 1942-1948 (2012) Hymenaea microsatellite inheritance Locus-seed tree Genotype N nij : nii + njj nik : njk G1 G2 Hc6 400131 5864 17 4:13 0:0 5.02 - 400440 6468 19 3:6 1:9 1.02 7.36 500182 5864 15 8:7 0:0 0.07 - 700119 5864 19 11:8 0:0 0.48 - 702357 5864 10 4:6 0:0 0.40 - Hc12 401048 158160 20 8:12 0:0 0.81 - 401169 158160 17 13:4 0:0 5.02 - 501785 158160 14 8:6 0:0 0.29 - 602696 158160 20 13:7 0:0 1.83 - 700119 158160 20 12:8 0:0 0.81 - 702357 158160 13 6:7 0:0 0.08 - Hc42 400131 124140 20 2:6 12:0 2.09 - 400440 124126 19 2:5 6:6 1.33 - 401048 124140 18 1:1 10:6 - 1.01 501383 124144 14 1:3 3:7 1.05 1.65 606001 122124 12 5:7 0:0 0.33 - Hc14 400131 118122 20 4:16 0:0 7.71 - 501383 118122 16 4:12 0:0 4.19 - 502348 118122 12 4:8 0:0 1.36 - 700119 118122 20 12:8 0:0 0.81 - Hc40 400131 170174 20 1:5 7:7 2.91 - 400440 174182 20 1:2 5:12 0.34 2.97 401048 166188 20 0:1 9:10 - 0.05 401169 160184 17 0:0 6:11 - 1.49 500182 170180 17 2:2 7:6 - 0.08 501383 180188 13 1:2 3:7 0.34 1.65 501785 168172 13 2:2 6:3 - 1.02 600959 168188 17 20 4:11 - 3.40 602696 174184 18 0:0 12:6 - 2.04 700119 168184 14 1:2 6:5 0.34 0.09 Hc34 400131 188190 19 2:5 1:11 1.33 9.75 400440 190192 18 2:1 5:10 0.34 1.70 401048 164190 10 4:6 0:0 0.40 - 602696 190192 16 5:3 7:1 0.51 5.06 700119 190192 16 3:6 2:5 1.02 1.33 702357 190192 12 0:0 8:4 - 1.36 Hc33 501785 108110 13 5:8 0:0 0.70 - 606001 108110 18 2:16 0:0 12.40* - Grouped 108110 31 7:24 0:0 9.86 Hc17 400131 106112 20 5:2 7:6 1.33 0.08 400440 110112 16 11:5 0:0 2.31 - 500182 106114 16 2:5 2:7 1.33 2.94 501383 106110 15 5:3 1:6 0.51 3.96 501785 106110 18 0:4 12:2 - 7.92 602696 106110 20 8:12 0:0 0.81 - 700119 110114 20 4:4 7:5 0.00 0.33 Hc25 400131 120152 20 1:7 6:6 5.06 - 401048 122152 18 2:7 1:8 2.94 6.20 600959 122154 17 2:5 1:9 1.33 7.36 602696 120154 16 1:9 2:4 7.36 0.68 606001 122150 15 0:2 8:5 - 0.70 N = sample size; G1 and G2 = maximum likelihood G statistics for the hypothesis of nij = nii + njj and nik = njk, respectively. Probability of Bonferroni’s corrections α = 0.001 (χ2 Table = 10.83). Table 1. Mendelian inheritance tests for nine microsatellite loci in Hymenaea courbaril. 1946 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (3): 1942-1948 (2012) F.S. Carneiro et al. Lo cu s Se ed -tr ee G Lo cu s Se ed -tr ee G Lo cu s Se ed -tr ee G Lo cu s Se ed -tr ee G Lo cu s Se ed -tr ee G L oc us S ee d- tre e G H c6 xH c1 2 70 01 19 7 .1 4 H c1 2x H c4 2 40 10 48 1. 45 H c1 4x H c4 0 40 01 31 5. 38 H c4 0x H c3 4 40 11 69 0 .5 7 H c4 2x H c1 4 50 13 83 7 .6 5 H c3 4x H c1 7 6 02 69 6 1 .5 5 H c6 xH c1 2 70 23 57 4 .9 5 H c1 2x H c4 2 40 11 69 0. 25 H c1 4x H c4 0 50 23 48 4. 87 H c4 0x H c3 4 40 04 40 3 .2 9 H c4 2x H c1 4 40 01 31 1 6. 74 * H c3 4x H c1 7 7 00 11 9 3 .7 2 H c6 xH c4 2 40 04 40 3 .2 7 H c1 2x H c4 2 70 01 19 0. 78 H c1 4x H c4 0 70 01 19 0. 39 H c4 0x H c3 4 60 26 96 2 .1 6 H c4 2x H c4 0 40 04 40 2 .4 5 H c3 4x H c1 7 4 00 44 0 5 .7 1 H c6 xH c4 2 50 01 82 4 .0 3 H c1 2x H c4 2 70 23 57 0. 68 H c1 4x H c3 4 40 01 31 16 .8 0* H c4 0x H c3 4 70 01 19 0 .5 1 H c4 2x H c4 0 40 10 48 3 .4 0 H c3 4x H c1 7 4 00 13 1 9 .9 0 H c6 xH c4 2 70 01 19 2 .7 0 H c1 2x H c1 4 70 01 19 3. 18 H c1 4x H c3 4 50 23 48 0. 90 H c4 0x H c3 4 40 01 31 9 .8 6 H c4 2x H c4 0 60 60 01 4 .5 2 H c3 4x H c2 5 4 01 04 8 4 .6 4 H c6 xH c4 2 70 23 57 3 .4 9 H c1 2x H c4 0 40 10 48 0. 82 H c1 4x H c3 4 70 01 19 1. 68 H c4 0x H c3 4 40 10 48 5 .8 8 H c4 2x H c4 0 50 13 83 4 .9 3 H c3 4x H c2 5 6 02 69 6 4 .7 3 H c6 xH c1 4 50 01 82 3 .8 5 H c1 2x H c4 0 40 11 69 4. 68 H c1 4x H c1 7 40 01 31 7. 30 H c4 0x H c3 4 50 01 82 3 .5 5 H c4 2x H c3 4 50 13 83 4 .7 2 H c3 4x H c2 5 4 00 44 0 7 .1 8 H c6 xH c1 4 70 01 19 4 .7 9 H c1 2x H c4 0 50 17 85 0. 17 H c1 4x H c1 7 50 23 48 8. 54 H c4 0x H c3 4 60 09 59 5 .7 6 H c4 2x H c3 4 40 04 40 7 .7 6 H c6 xH c4 0 40 01 31 10 .6 2 H c1 2x H c4 0 60 26 96 3. 80 H c1 4x H c1 7 70 01 19 3. 67 H c4 0x H c3 3 50 17 85 3 .5 1 H c4 2x H c3 4 40 10 48 11 .2 3 H c3 3x H c2 5 6 06 00 1 8 .2 6 H c6 xH c4 0 40 04 40 6 .0 7 H c1 2x H c4 0 70 01 19 0. 78 H c1 4x H c2 5 40 01 31 8. 26 H c4 0x H c1 7 40 01 31 0 .7 8 H c4 2x H c3 4 40 01 31 1 7. 32 * H c6 xH c4 0 50 01 82 2 .0 5 H c1 2x H c4 0 70 23 57 7. 22 H c1 4x H c2 5 50 23 48 6. 59 H c4 0x H c1 7 40 04 40 10 .7 1 H c4 2x H c3 3 60 60 01 1 2. 69 * H c1 7x H c2 5 4 00 13 1 2 .9 4 H c6 xH c4 0 70 01 19 3 .7 5 H c1 2x H c3 4 70 01 19 1. 65 H c1 4x H c2 5 70 01 19 15 .7 6* H c4 0x H c1 7 40 10 48 2 .9 7 H c4 2x H c1 7 50 13 83 3 .9 6 H c1 7x H c2 5 5 01 38 3 3 .3 1 H c6 xH c4 0 70 23 57 8 .3 7 H c1 2x H c3 4 40 10 48 5. 38 H c4 0x H c1 7 40 11 69 3. 68 H c4 2x H c1 7 40 04 40 4 .8 2 H c1 7x H c2 5 60 26 96 4 .8 1 H c6 xH c3 4 40 04 40 3 .8 8 H c1 2x H c3 4 40 11 69 0. 81 H c4 0x H c1 7 50 01 82 0. 16 H c4 2x H c1 7 40 10 48 5 .9 6 H c1 7x H c2 5 40 04 40 8 .9 5 H c6 xH c3 4 70 01 19 1 .1 1 H c1 2x H c3 4 70 23 57 3. 96 H c4 0x H c1 7 50 13 83 2. 60 H c4 2x H c1 7 40 01 31 9 .6 6 H c1 7x H c2 5 70 01 19 1 5. 22 * H c6 xH c3 4 50 01 82 6 .3 1 H c1 2x H c3 4 60 26 96 0. 91 H c4 0x H c1 7 60 26 96 4. 11 H c4 2x H c2 5 60 60 01 3 .2 9 H c6 xH c1 7 40 04 40 0 .4 5 H c1 2x H c3 3 50 17 85 2. 67 H c4 0x H c1 7 70 01 19 2. 37 H c4 2x H c2 5 40 10 48 6 .4 9 H c6 xH c1 7 50 01 82 1 .2 1 H c1 2x H c1 7 40 10 48 1. 41 H c4 0x H c2 5 40 01 31 1. 94 H c4 2x H c2 5 70 01 19 7 .4 7 H c6 xH c1 7 40 01 31 9 .4 8 H c1 2x H c1 7 40 11 69 0. 51 H c4 0x H c2 5 40 04 40 10 .6 4 H c4 2x H c2 5 40 01 31 9 .6 8 H c6 xH c1 7 70 01 19 3 .7 6 H c1 2x H c1 7 50 17 85 0. 52 H c4 0x H c2 5 40 10 48 0. 40 H c6 xH c1 7 70 23 57 3 .0 6 H c1 2x H c1 7 60 26 96 2. 61 H c4 0x H c2 5 50 13 83 1. 73 H c6 xH c2 5 40 01 31 10 .6 4 H c1 2x H c1 7 70 01 19 3. 32 H c4 0x H c2 5 60 09 59 1. 30 H c6 xH c2 5 70 01 19 1 5. 26 * H c1 2x H c1 7 70 23 57 3. 33 H c4 0x H c2 5 60 26 96 5. 06 H c1 2x H c2 5 40 10 48 12 .8 9* H c4 0x H c2 5 70 01 19 4. 72 H c1 2x H c2 5 50 23 48 0. 67 H c1 2x H c2 5 60 26 96 5. 15 H c1 2x H c2 5 70 23 57 3. 49 *P ro ba bi lit ie s o f B on fe rr on i’s c or re ct io ns fo r α = 0 .0 5, 0 .0 00 47 (χ 2 Ta bl e = 1 2. 11 ). Ta bl e 2. M ax im um l ik el ih oo d G t es t fo r te st in g th e hy po th es is o f in de pe nd en t se gr eg at io n be tw ee n pa irs o f m ic ro sa te lli te l oc i (1 :1 :1 :1 ) in H ym en ae a co ur ba ri l. 1947 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (3): 1942-1948 (2012) Hymenaea microsatellite inheritance CONCLUSIONS The nine microsatellite loci tested adhered to Mendelian inheritance assumptions and showed no linkage or genotypic disequilibrium, indicating that these loci can precisely estimate important population genetic parameters applied to genetic diversity and struc- ture, mating system, and gene flow studies in H. courbaril. ACKNOWLEDGMENTS Research supported by Conselho Nacional de Desenvolvimento Científico e Tec- nológico (CNPq)/Brazil, the European Commission (SEEDSOURCE Project - Contract #003708), and the Dendrogene Project (DFID/UK and EMBRAPA/Brazil). F.S. Carneiro was supported by a CNPq scholarship (Grant #2006/04490-9). A.M. Sebbenn, M.R. Lem- es, and R. Gribel are recipients of CNPq research fellowships. Pairs of loci Juveniles (Dbh <48 cm) Adults (Dbh ≥48 cm) Hc6xHc12 0.85417 0.02083 Hc6xHc42 1.00000 0.16389 Hc6xHc14 0.81007 0.54931 Hc6xHc40 0.52049 1.00000 Hc6xHc34 0.06181 0.00104 Hc6xHc33 0.01146 0.26250 Hc6xHc17 0.62396 0.63889 Hc6xHc25 0.26285 0.70938 Hc12xHc42 0.14722 0.81076 Hc12xHc14 0.05313 0.74479 Hc12xHc40 0.89514 0.98090 Hc12xHc34 0.06632 0.05660 Hc12xHc33 0.96285 0.79479 Hc12xHc17 0.51840 0.61840 Hc12xHc25 0.43924 0.99688 Hc42xHc14 0.11354 0.82674 Hc42xHc40 1.00000 0.55278 Hc42xHc34 0.11215 0.12951 Hc42xHc33 0.46111 0.28160 Hc42xHc17 0.34931 0.36493 Hc42xHc25 1.00000 0.85104 Hc14xHc40 0.36076 0.25625 Hc14xHc34 0.14826 0.78125 Hc14xHc33 0.16528 0.60938 Hc14xHc17 0.05451 0.50208 Hc14xHc25 0.91563 0.60069 Hc40xHc34 0.34028 0.89861 Hc40xHc33 0.66042 0.61910 Hc40xHc17 0.76389 0.30104 Hc40xHc25 0.74340 1.00000 Hc34xHc33 0.31632 0.00104 Hc34xHc17 0.06389 0.00139 Hc34xHc25 0.32535 0.30972 Hc33xHc17 0.13472 0.77396 Hc33xHc25 0.75486 0.04896 Hc17xHc25 0.64653 0.45799 Table 3. Genotypic disequilibrium between pairwise microsatellite loci in juvenile and adult trees of Hymenaea courbaril. The values represent the probability of genotypic linkage after 1000 permutations of alleles among individuals. Probability of Bonferroni’s corrections: P = 0.00069 (α = 0.05). Dbh = diameter at breast height. 1948 ©FUNPEC-RP www.funpecrp.com.brGenetics and Molecular Research 11 (3): 1942-1948 (2012) F.S. Carneiro et al. REFERENCES Carneiro FS, Lacerda AEB, Lemes MR and Gribel R (2011). Effects of selective logging on the mating system and pollen dispersal of Hymenaea courbaril L. (Leguminosae) in the Eastern Brazilian Amazon as revealed by microsatellite analysis. Forest Ecol. Manag. 262: 1758-1765. Ciampi AY, Gaiotto F and Saganuma E (2000). Desenvolvimento e caracterização de marcadores microssatélites para Hymenaea courbaril. Genet. Mol. Biol. 23: 216. Doyle JJ and Doyle JL (1987). Isolation of plant DNA from fresh tissue. Focus 12: 13-15. 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