Next-Generation Sequencing (NGS) technologies, by reducing the cost and increasing the throughput of sequencing, have opened doors to generate genomic data in a range of previously poorly studied species. In this study, we propose a method for the rapid development of a large-scale molecular resources for orphan species. We studied as an example the true lavender (Lavandula angustifolia Mill.), a perennial sub-shrub plant native from the Mediterranean region and whose essential oil have numerous applications in cosmetics, pharmaceuticals, and alternative medicines. The heterozygous clone “Maillette” was used as a reference for DNA and RNA sequencing. We first built a reference Unigene, compound of coding sequences, thanks to de novo RNA-seq assembly. Then, we reconstructed the complete genes sequences (with introns and exons) using an Unigene-guided DNA-seq assembly approach. This aimed to maximize the possibilities of finding polymorphism between genetically close individuals despite the lack of a reference genome. Finally, we used these resources for SNP mining within a collection of 16 commercial lavender clones and tested the SNP within the scope of a genetic distance analysis. We obtained a cleaned reference of 8, 030 functionally in silico annotated genes. We found 359K polymorphic sites and observed a high SNP frequency (mean of 1 SNP per 90 bp) and a high level of heterozygosity (more than 60% of heterozygous SNP per genotype). On overall, we found similar genetic distances between pairs of clones, which is probably related to the out-crossing nature of the species and the restricted area of cultivation. The proposed method is transferable to other orphan species, requires little bioinformatics resources and can be realized within a year. This is also the first reported large-scale SNP development on Lavandula angustifolia. All the genomics resources developed herein are publicly available and provide a rich pool of molecular resources to explore and exploit lavender genetic diversity in breeding programs.
No abstract
32Next-Generation Sequencing (NGS) technologies, by reducing the cost and increasing the throughput of 33 sequencing, have opened doors of research efforts to generate genomic data to a range of previously 34 poorly studied species. In this study, we proposed a method for the rapid development of a large scale 35 molecular resources for orphan species. We studied as an example Lavandula angustifolia, a perennial 36 sub-shrub plant native from the Mediterranean region and whose essential oil have numerous applications 37 in cosmetics, pharmaceuticals, and alternative medicines. 38We first built a 'Maillette' reference Unigene, compound of coding sequences, thanks to de novo RNA-39 seq assembly. Then, we reconstructed the complete genes sequences (with exons and introns) using a 40 transcriptome-guided DNA-seq assembly approach in order to maximize the possibilities of finding 41 polymorphism between genetically close individuals. Finally, we used these resources for SNP mining 42 within a collection of 16 lavender clones and tested the SNP within the scope of a phylogeny analysis. 43We obtained a cleaned reference of 8, 030 functionally annotated 'genes' (in silico annotation). We found 44 up to 400K polymorphic sites, depending on the genotype analyzed, and observed a high SNP frequency 45 (mean of 1 SNP per 90 bp) and a high level of heterozygosity (more than 60% of heterozygous SNP per 46 genotype). We found similar genetic distances between pairs of clones, related to the out-crossing nature 47 of the species, the restricted area of cultivation and the clonal propagation of the varieties. 48The method propose is transferable to other orphan species, requires little bioinformatics resources and 49 can be realized within a year. This is the first reported large-scale SNP development on Lavandula 50 angustifolia. All this data provides a rich pool of molecular resource to explore and exploit biodiversity 51 in breeding programs. 52 53 54 55 3 3 5 introns in our reference sequences through a pipeline developed by Aluome et al. [27], increasing the 101 opportunity of SNP detection in case of low genetic distance between individuals . 102 The results presented herein offer a solid base for the initiation of population genetics studies, DNA 103 fingerprinting and the development of efficient genomic-assisted selection strategies for Lavandula 104 angustifolia and the related species of the Lamiaceae family. 105 106 Material and methods 107
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