Identification of accession-specific variants and development of KASP markers for assessing the genetic makeup of <i>Brassica rapa</i> seeds

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Abstract

Background

Most crop seeds are F1 hybrids. Seed providers and plant breeders must be confident that the seed supplied to growers is of known, and uniform, genetic makeup. This requires maintenance of pure genotypes of the parental lines and testing to ensure the genetic purity of the F1 seed. Traditionally, seed purity has been assessed with a grow-out test (GOT) in the field, a time consuming and costly venture. Early in the last decade, seed testing with molecular markers was introduced as a replacement for GOT, and Kompetitive allele specific PCR (KASP) markers were recognized as promising tools for genetic testing of seeds. However, the markers available at that time could be inaccurate and applicable to only a small number of accessions or varieties due to the limited genetic information and reference genomes available.

Results

We identified 4,925,742 SNPs in 50 accessions of the Brasscia rapa core collection. From these, we identified 2,925 SNPs as accession-specific, considering properties of flanking region harboring accession-specific SNPs and genic region conservation among accessions by the Next Generation Sequencing (NGS) analysis. In total, 100 accession-specific markers were developed as accession-specific KASP markers. Based on the results of our validation experiments, the accession-specific markers successfully distinguised individuals from the mixed population including 50 target accessions from B. rapa core collection and the outgroup. Additionally, the marker set we developed here discriminated F1 hybrids and their parental lines with distinct clusters.

Conclusions

This study provides efficient methods for developing KASP markers to distinguish individuals from the mixture comprised of breeding lines and germplasms from the resequencing data of Chinese cabbage ( Brassica rapa spp. pekinensis).

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-022-08567-9.

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Most cited references 29

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Trimmomatic: a flexible trimmer for Illumina sequence data

Anthony M. Bolger, Marc Lohse, Bjoern Usadel (2014)

Motivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. Results: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic Contact: usadel@bio1.rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.

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The Sequence Alignment/Map format and SAMtools

Heng Li, Bob Handsaker, Alec Wysoker … (2009)

Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk

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Fast and accurate short read alignment with Burrows–Wheeler transform

Heng Li, Richard Durbin (2009)

Motivation: The enormous amount of short reads generated by the new DNA sequencing technologies call for the development of fast and accurate read alignment programs. A first generation of hash table-based methods has been developed, including MAQ, which is accurate, feature rich and fast enough to align short reads from a single individual. However, MAQ does not support gapped alignment for single-end reads, which makes it unsuitable for alignment of longer reads where indels may occur frequently. The speed of MAQ is also a concern when the alignment is scaled up to the resequencing of hundreds of individuals. Results: We implemented Burrows-Wheeler Alignment tool (BWA), a new read alignment package that is based on backward search with Burrows–Wheeler Transform (BWT), to efficiently align short sequencing reads against a large reference sequence such as the human genome, allowing mismatches and gaps. BWA supports both base space reads, e.g. from Illumina sequencing machines, and color space reads from AB SOLiD machines. Evaluations on both simulated and real data suggest that BWA is ∼10–20× faster than MAQ, while achieving similar accuracy. In addition, BWA outputs alignment in the new standard SAM (Sequence Alignment/Map) format. Variant calling and other downstream analyses after the alignment can be achieved with the open source SAMtools software package. Availability: http://maq.sourceforge.net Contact: rd@sanger.ac.uk

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Author and article information

Contributors

Yong-Min Kim: ymkim@kribb.re.kr

Journal

Journal ID (nlm-ta): BMC Genomics

Journal ID (iso-abbrev): BMC Genomics

Title: BMC Genomics

Publisher: BioMed Central (London )

ISSN (Electronic): 1471-2164

Publication date (Electronic): 25 April 2022

Publication date PMC-release: 25 April 2022

Publication date Collection: 2022

Volume: 23

Electronic Location Identifier: 326

Affiliations

[1 ]GRID grid.249967.7, ISNI 0000 0004 0636 3099, Genome Editing Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), ; Daejeon, Republic of Korea

[2 ]GRID grid.254230.2, ISNI 0000 0001 0722 6377, Molecular Genetics and Genomics Laboratory, Department of Horticulture, College of Agriculture and Life Science, , Chungnam National University, ; Daejeon, Republic of Korea

[3 ]XENOTYPE Inc., 118 Jungang-ro, Jung-gu, Daejeon, 34912 Republic of Korea

[4 ]Seed Industry Promotion Center, Foundation of Agri. Tech. Commercialization & Transfer (FACT), Gimje, Republic of Korea

[5 ]Dayi International Seed Co., Ltd., 16-35 Ssiat-gilJeollabuk-do, Baeksan-myeon, Gimje, 54324 Korea

[6 ]GRID grid.249967.7, ISNI 0000 0004 0636 3099, Plant Systems Engineering Research Center, , Korea Research Institute of Bioscience and Biotechnology, ; Daejeon, Republic of Korea

Article

Publisher ID: 8567

DOI: 10.1186/s12864-022-08567-9

PMC ID: 9036802

PubMed ID: 35468724

SO-VID: 226a48be-d76b-4b75-ab32-e82b8b1367d7

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 27 November 2021

Date accepted : 19 April 2022

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100014189, Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry;

Award ID: 213006-05-5-SBG30

Award ID: 821026-03

Award ID: 213006-05-5-SBG30

Award Recipient : Seongmin Hong Suk-Yoon Kwon Yong Pyo Lim

Funded by: FundRef http://dx.doi.org/10.13039/501100003664, Korea Forest Service;

Award ID: 2014071H10-2122-AA04

Award Recipient : Seongmin Hong Yong Pyo Lim

Custom metadata

ScienceOpen disciplines: Genetics

Keywords: seed purity assessment,kasp marker,accession-specific marker,brassica rapa,b. rapa breeding

Data availability:

ScienceOpen disciplines: Genetics

Keywords: seed purity assessment, kasp marker, accession-specific marker, brassica rapa, b. rapa breeding

Identification of accession-specific variants and development of KASP markers for assessing the genetic makeup of Brassica rapa seeds

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Abstract

Background

Results

Conclusions

Supplementary Information

Related collections

Genome Integrity

Most cited references 29

Trimmomatic: a flexible trimmer for Illumina sequence data

The Sequence Alignment/Map format and SAMtools

Fast and accurate short read alignment with Burrows–Wheeler transform

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