- Top of page
Cashew (Anacardium occidentale L.) is the most economically important tropical nut crop in the world, and yet there are no sequence tagged site (STS) markers available for its study. Here we use an automated, high-throughput system to isolate cashew microsatellites from a non-enriched genomic library blotted onto membranes at high density for screening. Sixty-five sequences contained a microsatellite array, of which 21 proved polymorphic among a closely related seed garden population of 49 genotypes. Twelve markers were suitable for multiplex analysis. Of these, 10 amplified in all three related tropical tree species tested: Anacardium microcarpum, Anacardium pumilum and Anacardium nanum.
Microsatellite markers are valuable for crop improvement programmes because of their ability to anchor linkage maps and their utility in paternity exclusion analyses. Microsatellites are also the marker of choice for population genetic studies. However, no microsatellite markers have been described for any Anacardium species. We developed 21 polymorphic microsatellite markers for cashew using an approach based on high-throughput screening of non-enriched genomic libraries. The main benefit of working from a non-enriched library is that it provides flexibility to rescreen the library for a wide diversity of repeat motifs, including tri- and tetranucleotide repeats. While microsatellites containing dinucleotide motifs are inevitably the most abundant, those comprising longer repeat motifs are favoured for fingerprinting purposes because of the increased ease of allele assignment and a reduced propensity to produce artefact ‘stutter’ products.
To construct the genomic library, DNA was first extracted from Anacardium occidentale var. AC10 using a cetyltrimethyl ammonium bromide (CTAB)-based method (Doyle & Doyle 1987). Following digestion with Sau3A I (Promega) and Hind III (Gibco-BRL), the DNA was fractionated by electrophoresis through a 1% w/v agarose gel, and fragments 300–800 bp in length were gel-extracted and purified using a NucleoSpin Extract column (Machery-Nagel). Purified DNA fragments were then ligated into the pGEM-3Z vector (Promega) cut with Hind III (Gibco-BRL) and BamH I (Promega). The ligated vector was transformed into competent XL1-Blue Escherichia coli cells (Stratagene) and cultured on 22.2 × 22.2 cm square agar plates. The Qpix2 Robotic Colony Picker (Genetix) was used to select and transfer transformed colonies directly into 384 well plates containing 60 µL of LB/ampicillin broth. A genomic library of 18 432 clones was then gridded onto Hybond N+ nylon membranes (Amersham) using a BioGrid Microgrid II arrayer (BioRobotics version 1.21).
Hybridization probing was carried out in rotating incubator tubes similar to that described by Armour et al. (1994) using 11 repeat probe sequences: AC15, AG15, AAC8, AAG8, AAT8, ACC8, AGG8, ATC8, AAAC6, AAAG6 and ACAT6. Each oligonucleotide probe was 5′ end-labelled with [γ32P]-ATP (Amersham), and hybridization was performed at 42 °C using Rapid-Hyb Buffer (Amersham). Following hybridization, membranes were exposed to X-ray film (Kodak), and positive colonies were diagnosed and isolated by comparing the signal to a set of reference points in a gridded standard that details the location of the positives in the genomic library. A polymerase chain reaction (PCR) test was then implemented to ensure the presence of a microsatellite within the insert of a colony identified, and importantly, also to determine the approximate location of the microsatellite within the insert. The PCR screen employed primers that targeted the appropriate microsatellite, but also contained a short anchor at the 5′ or 3′ termini (Charters et al. 1996) in association with the M13 universal primers designed from the vector sequence. From this screen, 79 clones were selected for sequencing, of which 65 were shown to contain a microsatellite of significant length (> 5 repeat units). The positive bacterial clones were sequenced using a 373A ABI automated sequencer (PerkinElmer) using the ABI PRISM BigDye Sequencing Ready Reaction Kit (Applied Biosystems). Primers were designed flanking the 59 identified microsatellite sequences using the primer 3 software (Rozen & Skaletsky 2000).
PCRs were conducted in 10 µL mixtures comprising 1× PCR buffer (Bioline −160 mm (NH4)2SO4, 670 mm Tris-Cl (pH 8.8), 0.1% Tween 20), 0.3 mm MgCl2, 0.4 mm dNTPs, 0.5 µm of each microsatellite primer pair and 1 U of Taq DNA polymerase (Bioline). PCRs were performed on a GeneAmp PCR System 2700 (Applied Biosystems) thermal cycler using an initial 94 °C denaturing step for 1 min followed by 35 cycles at 94 °C for 1 min, 1 min annealing (see Table 1 for optimized temperatures) and 72 °C extension for 1 min, then a final extension step at 72 °C for 7 min. The level of marker polymorphism was assessed on nine trees present in the seed gardens at Naliendele Research Station, Tanzania, using the ligated-pooling strategy described by Cryer et al. (2005). FAM and HEX-labelled M13 universal forward primers were used together with the locus-specific microsatellite reverse primer to amplify each locus from the template pools. Amplicons produced were separated on an ABI PRISM 3100 capillary sequencer and assessed against a ROX-labelled standard (Applied Biosystems) to determine the number of alleles in the pooled sample.
Table 1. Characteristics of the 21 polymorphic microsatellite loci isolated from Anacardium occidentale when analysed on a population of 49 individuals, including optimal annealing temperature (Ta), cloned allelic size ranges, number of alleles (Na), observed heterozygosity (HO), expected heterozygosity (HE) and HWE probabilities. The 12 markers used for multiplex analyses are depicted with the respective fluorescent labels used: a, NED; b, HEX; and c, FAM
|Locus||EMBL Accession no.||Primer sequence (5′−3′)||Repeat motif||Ta (°C)||Allelic size range (bp)||Na||HO||HE||P value|
|mAoR2||AM085800||F: GGCCATGGGAAACAACAA R: GGAAGGGCATTATGGGTAAG||(CA)10(TA)6||58.2||366–375||3||0.612||0.593||0.578|
|mAoR3a||AM085801||F: CAGAACCGTCACTCCACTCCR: ATCCAGACGAAGAAGCGATG||(AC)12(AAAAT)2||60.3||241–247||2||0.551||0.484||0.328|
|mAoR6c||AM085802||F: CAAAACTAGCCGGAATCTAGC R: CCCCATCAAACCCTTATGAC||(AT)5(GT)12||58.2||143–157||3||0.633||0.587||0.650|
|mAoR7b||AM085803||F: AACCTTCACTCCTCTGAAGC R: GTGAATCCAAAGCGTGTG||(AT)2(GT)5AT(GT)5||58.2||178–181||2||0.480||0.501||0.850|
|mAoR11c||AM085804||F: ATCCAACAGCCACAATCCTC R: CTTACAGCCCCAAACTCTCG||(AT)3(AC)16||60.3||234–236||2||0.200||0.210||0.890|
|mAoR12||AM085805||F: TCACCAAGATTGTGCTCCTG R: AAACTACGTCCGGTCACACA||(AC)12ATAC(AT)4||58.2||324–336||3||0.653||0.535||0.110|
|mAoR16c||AM085806||F: GGAGAAAGCAGTGGAGTTGC R: CAAGTGAGTCCTCTCACTCTCA||(GT)8(TA)17(GT)3||60.3||256–268||2||0.449||0.495||0.471|
|mAoR17b||AM085807||F: GCAATGTGCAGACATGGTTC R: GGTTTCGCATGGAAGAAGAG||(GA)24||56.1||124–159||5||0.898||0.667||0.029|
|mAoR26||AM085808||F: TCCACAAAATCAGCCTCCAC R: GAGCGCTCGTGTCCTGTACT||(TA)5CA(TG)6||60.3||414–416||2||0.061||0.059||0.857|
|mAoR29c||AM085809||F: GGAGAAGAAAAGTTAGGTTTGAC R: CGTCTTCTTCCACATGCTTC||(TG)10||58.2||316–320||3||0.143||0.134||0.620|
|mAoR33||AM085810||F: CATCCTTTTGCCAATTAAAAACA R: CACGTGTATTGTGCTCACTCG||(CT)18(AT)19||56.1||354–356||2||0.061||0.060||0.857|
|mAoR35||AM085811||F: CTTTCGTTCCAATGCTCCTC R: CATGTGACAGTTCGGCTGTT||(AG)14||58.2||165–169||3||0.714||0.595||0.183|
|mAoR41||AM085812||F: GCTTAGCCGGCACGATATTA R: AGCTCACCTCGTTTCGTTTC||(GGT)8||58.2||151–161||2||0.122||0.115||0.678|
|mAoR42c||AM085813||F: ACTGTCACGTCAATGGCATC R: GCGAAGGTCAAAGAGCAGTC||(CAT)9TAT(CTT)7||60.3||197–206||3||0.469||0.534||0.572|
|mAoR44||AM085814||F: CACGTTCGCATCATCCAA R: CGTCAGAGATTACGGCATTG||GTG(GT)3GCT(GGT)4||58.2||256–263||3||0.633||0.541||0.149|
|mAoR46c||AM085815||F: CGGCGTCGTTAAAGCAGT R: TCCTCCTCCGTCTCACTTTC||(ACC)7(AC)3||58.2||217–221||2||0.571||0.503||0.338|
|mAoR47||AM085816||F: AAGAGCTGCGACCAATGTTT R: CTTGAACTTGACACTTCATCCA||(TAAA)2(TA)7(AAT)5||58.2||161–173||2||0.265||0.236||0.306|
|mAoR48a||AM085817||F: CAGCGAGTGGCTTACGAAAT R: GACCATGGGCTTGATACGTC||(GAA)6(GA)3||58.2||172–178||2||0.449||0.503||0.446|
|mAoR52||AM085818||F: GCTATGACCCTTGGGAACTC R: GTGACACAACCAAAACCACA||(GT)16(TA)2||58.2||191–203||2||0.184||0.233||0.128|
|mAoR55b||AM085819||F: TGACTTTCAAATGCCACAAC R: CTCAAGCTTTCATGGGGATT||(AT)6CT(AC)5||58.2||104–114||3||0.551||0.579||0.717|
|mAoR59b||AM085820||F: TCCGCCCCTACTCCTATATT R: TGGTGTCGACTGCTTCTTGT||(AT)7(GT)14||51.8||317–327||2||0.061||0.060||0.857|
Twenty-one markers amplified two or more alleles in members of the seed garden. These were characterized on a seedling population of 49 plants to test Hardy–Weinberg parameters using the genepop version 3.4 program (Raymond & Rousset 1995). All loci except mAoR17 were in equilibrium (Table 1), although the excess of heterozygosities suggests that null alleles are not prevalent at this locus. A maximum of five alleles was amplified by marker mAoR17, with the remaining revealing two or three alleles (Table 1). The limited number of alleles detected is most probably due to the close genetic relationship of the trees (Mneney et al. 2001). Nonetheless, the relatively high level of observed heterozygosity (mean 0.42) of these markers illustrates their potential usefulness. In addition, no significant linkage disequilibrium was shown between the pairs of loci after Bonferroni correction for multiple comparisons (Sokal & Rohlf 1994), indicating the independent behaviour of all loci.
For the seed garden population, 12 of the polymorphic markers (mAoR3, mAoR6, mAoR7, mAoR11, mAoR16, mAoR17, mAoR29, mAoR42, mAoR46, mAoR48, mAoR52, mAoR55) were suitable for multiplex fractionation based on non-overlapping allelic size ranges. The multiplex reaction comprised of 5.0 µL of 2× QIAGEN multiplex PCR master mix, 40 ng of DNA template and 1.0 µL of 10× primer mix (containing 2.0 µm of each of the 24 primer–oligonucleotides). When the multiplex fractionation approach was performed on three related species (Anacardium microcarpum, Anacardium pumilum and Anacardium nanum), all loci amplified except mAoR11 in A. othonidium and mAoR16 in A. microcarpum. The markers developed here provide a valuable resource for future breeding efforts and map-based applications in cashew.