ddRAD-seq

Partial results are shown below:

1. How long does it take to get results from ddRAD-seq?

The typical turnaround time for ddRAD-seq results varies depending on project size and complexity but usually ranges from 4 to 8 weeks.

2. Can we customize the ddRAD-seq service for specific needs?

Yes, we offer tailored solutions to meet unique project requirements, including custom protocol adjustments and data analysis options.

3. What types of samples are suitable for ddRAD-seq?

ddRAD-seq can be applied to various sample types, including blood, tissue, and other genomic DNA sources from plants, animals, and microorganisms.

4. Can ddRAD-seq be used without a reference genome?

Yes, ddRAD-seq does not require a reference genome, making it suitable for species where reference genomes are not available.

5. How to choose the appropriate reduced-representation genome technology?

When selecting the appropriate simplified genomics technology, consider the following four factors to design your research strategy:

Reference Genome

• With Reference Genome: Using a reference genome helps reduce errors due to homologous or repetitive sequences, facilitates LD analysis, selection analysis, and GWAS. This is suitable for conventional RAD sequencing and PE151 sequencing.
• Without Reference Genome: ddRAD-seq is recommended.

Sequencing Strategy

• Double Digest: For short fragments, paired-end (PE) sequencing may result in significant adapter overlap and is not ideal for long read lengths; single-end (SE) sequencing is recommended for short fragments.
• Long Fragments: Long reads can detect more variant information but require sufficient coverage.
• Short Reads: Improves sequencing depth per restriction site, enhancing SNP detection accuracy.
• Non-Reference Species: SE sequencing is recommended to avoid data wastage.

Number of Markers

• High Marker Density: Conventional RAD sequencing is suitable for analyses requiring high-density markers.
• Complex Genomes and Large Sample Sizes: GBS sequencing is ideal for complex genomes and large sample volumes.

PCR Amplification Artifacts

• PCR Bias: May lead to heterozygous sites being misidentified as homozygous or introduce errors. Conventional RAD sequencing can remove PCR duplicates using fragment sequence information, while GBS and ddRAD-seq cannot.

ddRAD sequencing-based genotyping for population structure analysis in cultivated tomato provides new insights into the genomic diversity of Mediterranean 'da serbo' type long shelf-life germplasm Horticulture Research

Journal: Horticulture Research

Impact factor: 5.404

Published: 01 September 2020

Background

Tomato (Solanum lycopersicum L.) stands as a vital economic vegetable crop with global cultivation. One can trace the beginnings of tomato domestication to the Andean region of South America, from where it spread throughout the Americas. During the 16th century, tomatoes were introduced to Europe, with Spain and Italy as major entry points. This introduction sparked further domestication efforts, contributing to the emergence of substantial local genetic diversity. Consequently, the Mediterranean Basin in Europe is recognized as a secondary center for the diversification of tomatoes. In the context of this investigation, the authors harnessed ddRAD-seq technology to methodically explore the genetic diversity present within various tomato varieties.

Materials & Methods

Results

In this investigation, the genetic diversity of 288 tomato specimens was examined, encompassing 152 samples of the long shelf life (LSL) variety 'da serbo,' primarily originating from Italy and Spain, as well as other common varieties from diverse countries. Beyond the LSL trait, 'da serbo' varieties also display stress tolerance characteristics. In order to do non-parametric hierarchical clustering and model ancestral population structure, the study found 32,799 high-quality single nucleotide polymorphisms (SNPs). six distinct genetic subpopulations were delineated, effectively segregating the majority of 'da serbo' varieties, reflecting population substructure related to both varietal type and geographical origin. Linkage disequilibrium (LD) exhibited rapid decay within a genomic span of less than 5kb. Investigation of SNPs featuring low-frequency alleles (MAF) in 'da serbo' materials unveiled a high-frequency mutation in genes associated with stress tolerance, such as CTR1 and JAR1, which are implicated in fruit ripening. Finally, leveraging a selection of 58 core materials representing a significant portion of the genetic diversity, essential traits were further developed. The genetic signatures of the 'da serbo' germplasm, which was cultivated with selection in the Mediterranean Basin, are revealed in this work. Furthermore, it provides new perspectives on the long-lived "da serbo" germplasm, establishing it as a rich source of stress-tolerance genes.

Fig. 1 Estimate of genetic diversity in 288 tomato accessions using ddRAD.

Fig. 2 Linkage disequilibrium (LD) decay and comparison.

Fig. 3 Loading plot of the first (F1) and second (F2) principal components showing the variation for main fruit traits in accessions of the mini-core set developed from ddRAD SNP data of 288 cultivated tomato genotypes.

Conclusion

Using ddRAD-seq and the latest tomato genome, the authors identified 2,297 new target genes with SNPs in the Mediterranean 'da serbo' gene pool. This study reveals a geographic distinction among Mediterranean landraces and highlights SNPs in novel gene regions related to stress and hormone pathways. A mini-core collection of diverse genotypes was provided for breeding, offering a valuable gene reservoir for future precision breeding and genome-wide association studies.

Reference

1. Esposito, S., Cardi, T., Campanelli, G. et al. ddRAD sequencing-based genotyping for population structure analysis in cultivated tomato provides new insights into the genomic diversity of Mediterranean 'da serbo' type long shelf-life germplasm. Hortic Res 7, 134 (2020).