RAD-seq as an effective strategy for heterogenous variety identification in plants—a case study in Italian Ryegrass (Lolium multiflorum)

Abstract

The primary approach for variety distinction in Italian ryegrass is currently the DUS (distinctness, uniformity and stability) test based on phenotypic traits. Considering the diverse genetic background within the population and the complexity of the environment, however, it is challenging to accurately distinguish varieties based on DUS criteria alone. In this study, we proposed the application of high-throughput RAD-seq to distinguish 11 Italian ryegrass varieties with three bulks of 50 individuals per variety. Our findings revealed significant differences among the 11 tested varieties. The PCA, DAPC and STRUCTURE analysis indicated a heterogeneous genetic background for all of them, and the AMOVA analysis also showed large genetic variance among these varieties (ΦST = 0.373), which were clearly distinguished based on phylogenetic analysis. Further nucleotide diversity (Pi) analysis showed that the variety ‘Changjiang No.2’ had the best intra-variety consistency among 11 tested varieties. Our findings suggest that the RAD-seq could be an effectively alternative method for the variety distinction of Italian ryegrass, as well as a potential tool for open-pollinated varieties (OPVs) of other allogamous species.

Pipeline to Design Inbred Lines and F1 Hybrids of Leaf Chicory (Radicchio) Using Male-sterility and Genotyping-by-sequencing

“…The analysis performed in this study confirmed the suitability of the GBS method used. As supported by previous research [31,32,34,35], the restriction site-associated DNA sequencing method is a reliable tool for genotyping crop species. Genotyping-by-sequencing through RADseq and SNP markers has many possible applications for breeders in MAB, MAS, or PVP purposes.…”

“…The RADseq approach proved to be suitable for reconstructing the genetic structure of a breeding core collection, highlighting genomic information about the ancestral genotypes of different lines, and analysing interspecific hybrids. Moreover, through the use of related species' assembled genomes, crop comparative analyses can be performed to exploit new genetic resources, which can be adopted for many breeding and traceability purposes, as already demonstrated in other studies [16,32,46,47]. Moreover, the CAPS marker for male-sterility characterization discussed here has proven its reliability and its possible application in breeding planning, as well as its associated RADtag_2329, which can be used in future wide-spectrum GBS approaches in this species.…”

“…There are many GBS-related techniques that differ in the sequencing platform, the chemistry they are based on, the type of output, data throughput and other aspects [24][25][26][27][28][29]. Among the available GBS approaches, restriction-site associated DNA sequencing (RADseq) [30] has demonstrated its potential use in different crop species for breeding, PVP and traceability purposes [16,31,32]. Among the several advantages provided by the adoption of this approach is the unnecessity of an already sequenced genome because a comparative analysis between the sequenced reads can be made even without mapping them onto chromosomes.…”

Pipeline to Design Inbred Lines and F1 Hybrids of Leaf Chicory (Radicchio) Using Male Sterility and Genotyping-by-Sequencing

“…There are many GBS-related techniques that differ in the sequencing platform, the chemistry they are based on, the type of output, data throughput and other aspects [ 24 , 25 , 26 , 27 , 28 , 29 ]. Among the available GBS approaches, restriction-site associated DNA sequencing (RADseq) [ 30 ] has demonstrated its potential use in different crop species for breeding, PVP and traceability purposes [ 16 , 31 , 32 ]. Among the several advantages provided by the adoption of this approach is the unnecessity of an already sequenced genome because a comparative analysis between the sequenced reads can be made even without mapping them onto chromosomes.…”

Assessment of the Genetic Distinctiveness and Uniformity of Pre-Basic Seed Stocks of Italian Ryegrass Varieties

“…Finally, the SSR assay is easily performed by PCR, a method that does not require specific and expensive laboratory equipment. Despite the recent development of next-generation sequencing (NGS) techniques, particularly restriction site-associated DNA sequencing (RAD-seq) [ 15 ] and genotyping-by-sequencing (GBS) technologies, such as genotyping by random amplicon sequencing and direct (GRAS-Di) [ 16 ], we want to provide a molecular assay that is less costly and time-consuming and that could be practically applied by small laboratories of seed companies. From the literature, the use of molecular markers in the evaluation of the genetic diversity of L. multiflorum accessions has been reported by few studies using sequence-related amplified polymorphisms (SRAPs) [ 17 ], randomly amplified polymorphic DNA (RAPD) [ 18 ], and SSRs combined with bulk strategies [ 19 ].…”