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Abstract
Chloroplast DNA sequences are of great interest for population genetics and phylogenetic studies. However, only a small set of markers are commonly used. Most of them have been designed for amplification in a large range of Angiosperms and are located in the Large Single Copy (LSC). Here we developed a new set of 100 primer pairs optimized for amplification in Monocotyledons. Primer pairs amplify coding (exon) and non-coding regions (intron and intergenic spacer). They span the different chloroplast regions: 72 are located in the LSC, 13 in the Small Single Copy (SSC) and 15 in the Inverted Repeat region (IR). Amplification and sequencing were tested in 13 species of Monocotyledons: Dioscorea abyssinica, D. praehensilis, D. rotundata, D. dumetorum, D. bulbifera, Trichopus sempervirens (Dioscoreaceae), Phoenix canariensis, P. dactylifera, Astrocaryum scopatum, A. murumuru, Ceroxylon echinulatum (Arecaceae), Digitaria excilis and Pennisetum glaucum (Poaceae). The diversity found in Dioscorea, Digitaria and Pennisetum mainly corresponded to Single Nucleotide Polymorphism (SNP) while the diversity found in Arecaceae also comprises Variable Number Tandem Repeat (VNTR). We observed that the most variable loci (rps15-ycf1, rpl32-ccsA, ndhF-rpl32, ndhG-ndhI and ccsA) are located in the SSC. Through the analysis of the genetic structure of a wild-cultivated species complex in Dioscorea, we demonstrated that this new set of primers is of great interest for population genetics and we anticipate that it will also be useful for phylogeny and bar-coding studies.
Highlights
The knowledge of the chloroplast genome structure and sequence variation in Monocotyledons is still partial and unbalanced
Primers were designed to amplify a wide range of monocotyledons species and we tested them on various species of different genera (D. abyssinica, D. praehensilis, D. rotundata, D. dumetorum, D. bulbifera, T. sempervirens, P. canariensis, P. dactylifera, A. scopatum, A. murumuru, C. echinulatum, D. excilis, P. glaucum)
Amplification success was 85% (Table S1) which was very similar to the expected mean amplification of 88% derived from the sequences deposited in GenBank used to design the primers (95% for A. calamus, 95% for D. elephantipes, 97% for L. minor, 80% for O. nivara, 88% for P. aphrodite and 80% for Z. mays)
Summary
The knowledge of the chloroplast genome structure and sequence variation in Monocotyledons is still partial and unbalanced. We used the newly defined primer pairs to study intra-specific cpDNA diversity of three different yam species (Dioscorea spp.) Due to high inter-generic divergence in Dioscoreaceae [51] causing alignment difficulties in non-coding regions with T. sempervirens, nucleotide diversity was only estimated between two distant species of Dioscorea, D. abyssinica and D. elephantipes.
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