Genomic exploration and molecular marker development in a large and complex conifer genome using RADseq and mRNAseq

Area of Science:

• Genomics
• Molecular Biology
• Forestry

Background:

• Developing molecular markers is crucial for understanding and managing complex genomes, particularly in conifer species like Cedrus atlantica.
• Previous genomic studies in conifers have been limited by genome size and complexity.

Purpose of the Study:

• To develop molecular markers for the 16 gigabase genome of Cedrus atlantica using restriction site associated DNA sequencing (RADseq) and messenger RNA sequencing (mRNAseq).
• To assess the utility of RADseq for generating genetic markers in complex conifer genomes.
• To develop a script for estimating ascertainment bias in single nucleotide polymorphism (SNP) detection.

Main Methods:

• Restriction site associated DNA sequencing (RADseq) with a hypomethylation-sensitive enzyme and messenger RNA sequencing (mRNAseq) were employed for de novo genome assembly.
• Single nucleotide polymorphisms (SNPs) and simple sequence repeats (SSRs) were identified from the generated assemblies.
• A script was developed to estimate ascertainment bias in SNP detection, considering pooling and sampling effects.

Main Results:

• RADseq and mRNAseq assemblies covered 0.1% and 0.6% of the genome, respectively, with mRNAseq yielding a higher proportion of protein-coding regions.
• 17,348 SNPs were identified using RADseq and 5,714 SSRs from the transcriptome.
• A subset of 282 SNPs was validated with a 50.4% conversion rate, confirming the utility of RADseq for conifer genomes. Increasing sample size effectively reduced ascertainment bias.

Conclusions:

• RADseq is a valuable approach for developing molecular markers in highly complex conifer genomes.
• The developed SNP markers are suitable for genetic studies in Cedrus atlantica.
• The ascertainment bias script aids in accurate SNP discovery in population studies.