Dataset Information

Deep sampling and pooled amplicon sequencing reveals hidden genic variation in heterogeneous rye accessions.

ABSTRACT: Background: Loss of genetic variation negatively impacts breeding efforts and food security. Genebanks house over 7 million accessions representing vast allelic diversity that is a resource for sustainable breeding. Discovery of DNA variations is an important step in the efficient use of these resources. While technologies have improved and costs dropped, it remains impractical to consider resequencing millions of accessions. Candidate genes are known for most agronomic traits, providing a list of high priority targets. Heterogeneity in seed stocks means that multiple samples from an accession need to be evaluated to recover available alleles. To address this we developed a pooled amplicon sequencing approach and applied it to the out-crossing cereal rye (Secale cereale L.).

Results: Using the amplicon sequencing approach 95 rye accessions of different improvement status and worldwide origin, each represented by a pooled sample comprising DNA of 96 individual plants, were evaluated for sequence variation in six candidate genes with significant functions on biotic and abiotic stress resistance, and seed quality. Seventy-four predicted deleterious variants were identified using multiple algorithms. Rare variants were recovered including those found only in a low percentage of seed.

Conclusions: We conclude that this approach provides a rapid and flexible method for evaluating stock heterogeneity, probing allele diversity, and recovering previously hidden variation. A large extent of within-population heterogeneity revealed in the study provides an important point for consideration during rye germplasm conservation and utilization efforts.

SUBMITTER: Hawliczek A

PROVIDER: S-EPMC7706248 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Deep sampling and pooled amplicon sequencing reveals hidden genic variation in heterogeneous rye accessions.

Hawliczek Anna A Bolibok Leszek L Tofil Katarzyna K Borzęcka Ewa E Jankowicz-Cieślak Joanna J Gawroński Piotr P Kral Adam A Till Bradley J BJ Bolibok-Brągoszewska Hanna H

BMC genomics 20201130 1

<h4>Background</h4>Loss of genetic variation negatively impacts breeding efforts and food security. Genebanks house over 7 million accessions representing vast allelic diversity that is a resource for sustainable breeding. Discovery of DNA variations is an important step in the efficient use of these resources. While technologies have improved and costs dropped, it remains impractical to consider resequencing millions of accessions. Candidate genes are known for most agronomic traits, providing ...[more]

PMID: 33256606

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Project description:Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Dataset Information

Deep sampling and pooled amplicon sequencing reveals hidden genic variation in heterogeneous rye accessions.

Publications

Deep sampling and pooled amplicon sequencing reveals hidden genic variation in heterogeneous rye accessions.

Similar Datasets

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