* The following is derived from the White Papers

Introduction

The use of DNA markers and molecular breeding shortens the time to market of new crop lines and lowers
the overall cost of breeding. Molecular markers also contribute to faster and cheaper seed quality assurance
practices by substituting expensive and time-consuming grow-outs with early lab testing (for genetic purity,
trait confirmation, and adventitious presence). Presently, the most efficient and commonly used molecular
markers are Single Nucleotide Polymorphisms (SNPs).


SNPs are single nucleotide changes that are heritable, codominant, and abundant throughout eukaryotic
genomes. The advent of next generation sequencing technologies and genotyping by targeted sequencing
provides an attractive method for mid-density SNP genotyping; Agriplex Genomics’ PlexSeq is such a
platform that enables effective mid-density SNP panel by high level multiplexing.


The International Rice Research Institute (IRRI) designed the first version of the 1K-Rice Custom Amplicon
(1k-RiCA) panel. The 955 SNP markers were multiplexed in-to a PlexSeq™ panel as the 1k-RiCA v2. We
describe here the next generation of the panel: RiCA v4, the way it evolved and list some of its new features
as a molecular breeding and seed QA tool.

Characterizing the 1K Rice Custom Amplicon (RiCA) V.2 Panel

Version 4 of the IRRI Rice Custom Amplicon SNP panel is made of 1,040 markers and maintains the basic
structure of the original panel:

  • Genomic screen: 797 SNPs originating from the Cornell 6KArray Infinium Rice chip [2] and the 3,000

Rice Genomes Project [5, 6, 7]. The markers are evenly distributed throughout the genome and
were selected based on call rate higher than 95% and high minor allele frequencies (MAF≥0.4).

  • Trait-related markers: 205 SNPs associated with different important trait genes/QTLs.

  • Purity Markers: 22 SNPs chosen for their discriminatory ability amongst elite indica material, used

in determination of genetic purity, identity, and hybridity.


The revision of the panel included a performance analysis of RiCA version 2: the results of 5 projects adding
up to 5,187 individuals were analyzed. Markers were scored on their success rate and robustness. Based on
these criteria 48 lowest performing SNPs were removed from the panel.


In addition, the panel was augmented with more trait-associated markers (see Table 1). Altogether 133 new
SNPs were added to the revised panel, which now targets over 90 genes and QTLs of interest.

irri v4 chart.PNG

Table 1. List of Traits and genes represented on the AgriPlex RiCA panel

IRRI designed the first version of the 1K-Rice Custom Amplicon (1k-RiCA) panel [4]. The panel was designed using Illumina’s proprietary TruSeq Custom Amplicon (TSCA) 384 Index Kit workflow. The process identified SNPs that are evenly distributed across the rice genome with high-quality scores and validation status. In the multiplexed AgriPlex Genomics version of the panel, an expanded set of trait markers and genetic purity diagnostic markers (Fig. 1) augmented and retained much of the genome-wide SNPs. The resulting panel combines SNPs selected from the following resources:

 

  • 838 SNPs originated from the Cornell 6K Array InfiniumRice (or C6AIR) chip [2] and the 3,000 Rice Genomes Project [5, 6, 7]. The markers were selected based on a call rate higher than 95% and high minor allele frequencies (MAF≥0.4) determined from genotypic data available on 1,172 IRRI indica rice breeding lines and indica released varieties genotyped with the C6AIR.

  • 96 trait-related markers (see summary in Table 1) linked to 58 different important trait-associated
    genes/QTLs as reported in the literature.

  • 22 purity SNPs chosen for their discriminatory ability amongst elite indica material, used in determination of genetic purity, identity, and hybridity.

IRRI designed this panel as a tool for interrogating genetic variation in rice varieties, offering sufficient SNP density for genomic selection, fingerprinting, and assessment of allele frequencies for 58 key traits. The panel supports breeding germplasm, classification of rice germplasm, QTL analysis for bi-parental populations, and assessment of background recovery in marker-assisted backcrossing applications.

irri v2 image.PNG

Applications

Genomic Selection

The panel enables genomic selection in indica breeding programs. On average, any two elite indica parents will display between 300 – 500 polymorphic markers distributed across the genome (Figure 2), a density sufficient to enable imputation back tothe full genome level. The combination of rapid turnaround time of 2 weeks and low cost enables cost-effective genotyping of a prediction population during the last generation of line fixation, saving money on the cost of field space for seed increase andallowing rapid recycling of progeny as parents.

irri v2 image 2.PNG

QTL Profiling

The array at AgriPlex contains 96 trait markers targeting58 high-value trait targets, covering a range of traits related to disease resistance, grain quality, abiotic stress tolerance, heading date, hybrid rice production and others (Table 1). These markers are designed to give accurate profiling of the targeted genes and QTLs across all Oryza sativagenomic diversity, including both indica and japonica. The same, or part of these QTLs and traits can be interrogated by specific,

single plex assays, providing a mechanism for initial selection based on major loci followed by validation and full-genome profiling on the RiCA array.

Seed Purity and Hybridity

The RiCA v. 2 panel includes 22 markers specifically chosen for their ability to distinguish amongst elite material. Combined with the genome-wide and trait markers, this makes the panel an excellent choice for applications related to genetic purity, some examples are, purity testing (uniformity), F1 hybridity tests, and varietal identification identity fingerprinting. The same markers are implemented at Intertek as individual SNP assays, providing a mechanism for cross-validation of results.

Marker-assisted Backcrossing

The RiCA v. 2 panel includes 22 markers specifically chosen for their ability to distinguish amongst elite material. Combined with the genome-wide and trait markers, this makes the panel an excellent choice for applications related to genetic purity, some examples are, purity testing (uniformity), F1 hybridity tests, and varietal identification identity fingerprinting. The same markers are implemented at Intertek as individual SNP assays, providing a mechanism for cross-validation of results.

QTL Mapping

Although not the primary target application, it is possible to use the RiCA v. 2 panel for biparental mapping purposes. Density of polymorphic markers may in some cases be lower than desirable, depending on the parentsinvolved, leading to gaps in the linkage map. However, the panel does provide a cost-effective optionfor genotyping much of the genome, and any remaining gaps could then be filled in with other marker systems, or by further panel customization.

The 1K IRRI RiCA V4 Custom Amplicon Assay SNP Panel - An IRRI/AgriPlex Collaboration

Molecular breeding and modern seed Quality Assurance (QA) practices require the use of molecular markers. Molecular markers have evolved in the last 80 years as a means of sampling and comparing genomes. As the technologies for interrogating genetic variation have progressed, and so too has the resolution of the genomic picture the markers are able to depict.


For a breeder, the use of molecular markers in the course of breeding programs enables accurate and early selection of individuals of interest, thus, shortening the number of selection cycles needed, reducing the time to market of newlines, and lowering the overall cost of breeding. To seed producers, molecular markers provide the means to assess seed quality parameters such as genetic purity, trait confirmation, and adventitious presence without the need for grow-outs; thus, it is faster and cheaper QA than traditional methods.

Over time, researchers have developed many DNA molecular marker systems, but Single Nucleotide Polymorphisms (SNPs) have emerged as the ultimate molecular marker; SNPs are single nucleotide changes that are heritable, codominant, and distributed with relatively high frequency throughout eukaryotic genomes. The desire to create high-density marker chips able to interrogate a large number of SNPs per DNA sample has dominated the evolution of SNP genotyping. Research has led to many high-resolution SNP arrays for rice [1, 2, 3]. Less effort has been made in developing informative, high-throughput, and cost-effective mid-density genotyping solutions for applied molecular breeding programs and seed production QA. The advent of Next Generation Sequencing technologies and genotyping by targeted sequencing provides an attractive method for mid-density SNP genotyping.


We describe here the 1040 SNP markers that were developed by IRRI and multiplexed in a PlexSeqTM panel by AgriPlex Genomics. We introducePlexSeqTM, a targeted sequencing methodology, and list various possible applications for the panel as a general molecular breeding and seed QA tool.

Conclusions

The RiCA V4 panel implemented at AgriPlex provides an excellent, cost-effective alternative for applications requiring a moderate to high sample throughput at modest SNP density. While it primarily enables genomic selection, the substantially expanded suite of trait markers and the addition of purity markers expand its usefulness in a range of other applications. The panel fits neatly in with rapid line fixation protocols, thanks to its low cost-per-sample and fast turnaround time, enabling major-locus selection and genomic selection to occur before field amplification of seed,
saving both time and money. The flexibility of the platform also enables continual revision and upgrading of the marker system, ensuring it keeps pace with current trait needs.