Computational protocol: Development of multiplex microsatellite PCR panels for the seagrass Thalassia hemprichii (Hydrocharitaceae)1

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Protocol publication

[…] Recently and have also published microsatellites for T. hemprichii. A total of 49 polymorphic microsatellite loci are available if all loci are counted. To enhance genotyping outcomes for this species, we developed three sets of highly informative multiplex PCR panels. Utility of loci to be used in multiplex PCR reactions relates to amplicon size, dimer formation, stutter noise, annealing temperature, and primer complexity. Due to so many loci being available to select from, only perfect microsatellite repeats (i.e., no compound repeats) were chosen for multiplex development. As a result, loci published by were excluded from consideration, as they were all compound microsatellites. From the remaining loci, initial selection for inclusion was made based on amplicon length, number of alleles (high allelic diversity preferred), and inbreeding coefficient (loci with extreme deviation of Hardy–Weinberg equilibrium were avoided). Using Multiplex Manager version 1.2 (), initial test multiplexes were developed and new forward primers were synthesized. Loci that were difficult to amplify were removed from the multiplexes (), resulting in three panels containing seven, six, and five loci per PCR (). Two loci were repeated in multiplexes to account for occasional dropouts, resulting in a 16-loci genotype in only three PCRs.Multiplex PCRs were conducted using Type-it (QIAGEN) in 10-μL reactions using 1 μL of primer mix () and ∼2 ng of genomic DNA. To assess population genetic diversity, two populations of T. hemprichii were screened: 47 samples from Hammond Island and 47 samples from Pulau Semakau Reef, Singapore (). Both populations’ samples were collected from an area of approximately 50 × 50 m. The distance between samples was greater than 2 m. These two populations are widely separated and were selected to reveal the potential of the developed loci. Thalassia has a high dispersal capacity (), so very distant sites are necessary to uncover this potential. Products were analyzed as above and alleles scored using Genetic Profiler Suite version 2.2 (GE Healthcare).Analysis of allelic data involved the initial identification of clones using GenoDive version 2.0b25 (). Clonal genotypes were removed from the data set before calculation to avoid pseudo-replicas that originate from the same plant. Genetic and marker diversity statistics were calculated using GenoDive. The 16-loci genotypes were analyzed for genotypic identity (Pgen) and sibling probability (Psex) using GenClone version 2 (). Linkage disequilibrium between loci was calculated using the log-likelihood-ratio statistic of GENEPOP version 4.2 (; web version).The samples screened for both populations with the multiplex panels readily amplified, providing high-probability identification of genotypes. A total of 16 genets (unique genotypes) were identified among 47 samples from Hammond Island and 42 genets among 47 samples from Singapore. Intermediate to low levels of clonality were detected in these two widely separated populations. The resolving power of the combined panels resulted in a Pgen(f) [the probability of obtaining a common genotype from the allele pool] of 3.3 × 10−10 – 1.4 × 10−8 for Hammond Island and 3.7 × 10−5 – 1.8 × 10−12 for Singapore and a Psex(f) [probability that a shared genotype originated from a seed] of 2.0 × 10−3 – 2.8 × 10−3 for Hammond Island and 5.49 × 10−5 for Singapore. The level of expected heterozygosity was generally low for most loci (), particularly when compared to T. testudinum (). A solid explanation is not possible at this stage, but it might be likely that the populations screened are the result of a recent radiation from a relatively small refugia. Linkage disequilibrium was detected between loci THH-5 and TH52. Near presence of these two loci on the same chromosome could lead to the association of alleles. But, random factors or linkage to genes under similar selective pressure could also lead to linkage disequilibrium. […]

Pipeline specifications

Software tools Genodive, Genepop
Application Population genetic analysis