The Development of Quality Control Genotyping Approaches: A Case Study Using Elite Maize Lines

Jiafa Chen,Denise E Costich,Juan Burgueño,Noemi Ortega,Cesar Petroli,Sarah J Hearne,Cristian Zavala,Jorge Franco,Mukesh Jain

doi:10.1371/journal.pone.0157236

Abstract

Quality control (QC) of germplasm identity and purity is a critical component of breeding and conservation activities. SNP genotyping technologies and increased availability of markers provide the opportunity to employ genotyping as a low-cost and robust component of this QC. In the public sector available low-cost SNP QC genotyping methods have been developed from a very limited panel of markers of 1,000 to 1,500 markers without broad selection of the most informative SNPs. Selection of optimal SNPs and definition of appropriate germplasm sampling in addition to platform section impact on logistical and resource-use considerations for breeding and conservation applications when mainstreaming QC. In order to address these issues, we evaluated the selection and use of SNPs for QC applications from large DArTSeq data sets generated from CIMMYT maize inbred lines (CMLs). Two QC genotyping strategies were developed, the first is a “rapid QC”, employing a small number of SNPs to identify potential mislabeling of seed packages or plots, the second is a “broad QC”, employing a larger number of SNP, used to identify each germplasm entry and to measure heterogeneity. The optimal marker selection strategies combined the selection of markers with high minor allele frequency, sampling of clustered SNP in proportion to marker cluster distance and selecting markers that maintain a uniform genomic distribution. The rapid and broad QC SNP panels selected using this approach were further validated using blind test assessments of related re-generation samples. The influence of sampling within each line was evaluated. Sampling 192 individuals would result in close to 100% possibility of detecting a 5% contamination in the entry, and approximately a 98% probability to detect a 2% contamination of the line. These results provide a framework for the establishment of QC genotyping. A comparison of financial and time costs for use of these approaches across different platforms is discussed providing a framework for institutions involved in maize conservation and breeding to assess the resource use effectiveness of QC genotyping. Application of these research findings, in combination with existing QC approaches, will ensure the regeneration, distribution and use in breeding of true to type inbred germplasm. These findings also provide an effective approach to optimize SNP selection for QC genotyping in other species.

Highlights

The CIMMYT (International Maize and Wheat Improvement Center) Maize Lines (CMLs) are a set of 577 elite inbred lines, which have been developed over the last 25 years
In order to address these issues, we evaluated the selection and use of Single nucleotide polymorphism (SNP) for Quality control (QC) applications from large DArTSeq data sets generated from CIMMYT maize inbred lines (CMLs)
SNPs were initially filtered to remove SNPs with missing rate >40%, SNPs with minor allele frequency (MAF) < 5% and heterogeneity >10%, resulting in 18,082 markers used for subsequent analyses (S2 Table), these genotypic data are available in: http://hdl.handle.net/11529/10431

Summary

Introduction

The CIMMYT (International Maize and Wheat Improvement Center) Maize Lines (CMLs) are a set of 577 elite inbred lines, which have been developed over the last 25 years. The CMLs represent one of the most widely distributed sources of publically generated elite lines, which are freely available to both public and private sector breeders, research and growers, worldwide. Distributed under the Standard Material Transfer Agreement (SMTA) of the International Treaty on Plant Genetic Resources for Food and Agriculture (ITPGRFA), these lines have become the most important public tropical maize germplasm source globally [1]. Since 2005 the conservation, regeneration and distribution of CMLs has been the responsibility of the CIMMYT Germplasm Bank (CGB). The CMLs are the most requested accessions that the CGB holds and as such are subject to the most frequent regenerationsTherefore, during which, the possibility for contamination is always present. In order for CGB to maintain these materials as true to type, pure and stable lines, we determined that a stringent QC genotyping system should be implemented

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Conclusion