Genotyping Analysis by RAD-Seq Reads Is Useful to Assess the Genetic Identity and Relationships of Breeding Lines in Lavender Species Aimed at Managing Plant Variety Protection

Abstract

Lavender species are widely distributed in their wild forms around the Mediterranean Basin and they are also cultivated worldwide as improved and registered clonal varieties. The economic interest of the species belonging to the Lavandula genus is determined by their use as ornamental plants and important source of essential oils that are destinated to the production of cosmetics, pharmaceuticals and foodstuffs. Because of the increasing number of cases of illegal commercialization of selected varieties, the protection of plant breeders' rights has become of main relevance for the recognition of breeding companies' royalties. With this aim, genomic tools based on molecular markers have been demonstrated to be very reliable and transferable among laboratories, and also much more informative than morphological descriptors. With the rising of the next-generation sequencing (NGS) technologies, several genotyping-by-sequencing approaches are now available. This study deals with a deep characterization of 15 varietal clones, belonging to two distinct Lavandula species, by means of restriction-site associated DNA sequencing (RAD-Seq). We demonstrated that this technology screens single nucleotide variants that enable to assess the genetic identity of individual accessions, to reconstruct genetic relationships among related breeding lines, to group them into genetically distinguishable main subclusters, and to assign their molecular lineages to distinct ancestors. Moreover, a number of polymorphic sites were identified within genes putatively involved in biosynthetic pathways related to both tissue pigmentation and terpene production, useful for breeding and/or protecting newly registered varieties. Overall, the results highlighted the presence of pure ancestries and interspecific hybrids for the analyzed Lavandula species, and demonstrated that RAD-Seq analysis is very informative and highly reliable for characterizing Lavandula clones and managing plant variety protection.

Keywords: Lavandula; NGS; ancestry reconstruction; chloroplast DNA barcoding; flavonoids; genotyping by RAD sequencing; interspecific crosses; plant breeder’s rights; terpenes.

Figure 1

(a) UPGMA dendrogram based on the pair-wise genetic similarity matrix highlighting five main “Clusters” for the no missing values containing dataset. (b) STRUCTURE software histogram for K = 3 of 15 individuals of Lavandula with a no missing values containing dataset (“red star” symbol labels individuals with homozygosity >80%).

Figure 2

Principal Coordinate Analysis (PCoA) [24], based on the eigenvectors calculated starting from the genetic similarity matrix and highlighting the 5 mains “Clusters” (A to E) identified for the 15 analysed samples of Lavandula.

Figure 3

(a) Neighbour Joining tree based on the polymorphic sites among ITS nuclear region, and matK, trnH-psbA and rbcL chloroplast barcoding regions. Bootstrap values are reported. (b) LOGO representation of polymorphic sites identified among the 15 Lavandula accessions analysed for the DNA barcoding.