Using Next Generation Sequencing for Multiplexed Trait-Linked Markers in Wheat

<div><p>With the advent of next generation sequencing (NGS) technologies, single nucleotide polymorphisms (SNPs) have become the major type of marker for genotyping in many crops. However, the availability of SNP markers for important traits of bread wheat <b>(</b><i>Triticum aestivum</i> L.) that can be effectively used in marker-assisted selection (MAS) is still limited and SNP assays for MAS are usually uniplex. A shift from uniplex to multiplex assays will allow the simultaneous analysis of multiple markers and increase MAS efficiency. We designed 33 locus-specific markers from SNP or indel-based marker sequences that linked to 20 different quantitative trait loci (QTL) or genes of agronomic importance in wheat and analyzed the amplicon sequences using an Ion Torrent Proton Sequencer and a custom allele detection pipeline to determine the genotypes of 24 selected germplasm accessions. Among the 33 markers, 27 were successfully multiplexed and 23 had 100% SNP call rates. Results from analysis of "kompetitive allele-specific PCR" (KASP) and sequence tagged site (STS) markers developed from the same loci fully verified the genotype calls of 23 markers. The NGS-based multiplexed assay developed in this study is suitable for rapid and high-throughput screening of SNPs and some indel-based markers in wheat.</p></div

GBMAS two-step PCR.

<p>The first PCR is a multiplex and amplifies the different target regions containing SNP(s). The locus-specific forward primers were tailed with an M13 sequence at the 5’-end and the reverse primers were tailed with Ion truncated P1/B sequence. The generated amplicons contains an M13 tail and Ion truncated P1/B sequence. The second PCR involves the addition of barcodes and standard Ion A adapter to the amplicons.</p

Average number of sequence reads per marker using 17 ng (x-axis) and 141 ng DNA (y-axis) as template.

<p>The top figure used touch down (TD) and the bottom figure used non-TD PCR. The arrow points to the number of reads of CNL9 marker for <i>Sr35</i>.</p

Average number of reads as affected by different primer concentrations: 6.25 nM, 12.5 nM, 2 tiers of 2 nM and 25 nM, and 3 tiers of 2 nM, 15 nM and 30 nM.

<p>Standard errors are shown as error bars on top of columns.</p

Primer specificity and theoretical read percentages of expected favorable SNP allele (A) amplified from one to three genomes of wheat.

<p>The wild type SNP allele in this example is T.</p><p>Primer specificity and theoretical read percentages of expected favorable SNP allele (A) amplified from one to three genomes of wheat.</p

Average number of reads per marker analyzed using non-normalized (x-axis) and normalized (y-axis) DNA.

<p>The top figure used 100 nM primers for PCR and the bottom figure used 12.5 nM primers for PCR.</p

Average number of reads per marker in non-TD (blue column) and TD PCR (red column).

<p>Standard errors are shown as error bars on top of columns. Samples were run using the primer combination of 8 nM and 16 nM.</p

Minimum percentage of favorable allele reads from three GBMAS libraries constructed using the 2-tier primer pool of 8 nM and 16 nM primers and non-TD PCR.

<p>Minimum percentage of favorable allele reads from three GBMAS libraries constructed using the 2-tier primer pool of 8 nM and 16 nM primers and non-TD PCR.</p

Application of Genotyping-by-Sequencing on Semiconductor Sequencing Platforms: A Comparison of Genetic and Reference-Based Marker Ordering in Barley

<div><p>The rapid development of next-generation sequencing platforms has enabled the use of sequencing for routine genotyping across a range of genetics studies and breeding applications. Genotyping-by-sequencing (GBS), a low-cost, reduced representation sequencing method, is becoming a common approach for whole-genome marker profiling in many species. With quickly developing sequencing technologies, adapting current GBS methodologies to new platforms will leverage these advancements for future studies. To test new semiconductor sequencing platforms for GBS, we genotyped a barley recombinant inbred line (RIL) population. Based on a previous GBS approach, we designed bar code and adapter sets for the Ion Torrent platforms. Four sets of 24-plex libraries were constructed consisting of 94 RILs and the two parents and sequenced on two Ion platforms. In parallel, a 96-plex library of the same RILs was sequenced on the Illumina HiSeq 2000. We applied two different computational pipelines to analyze sequencing data; the reference-independent TASSEL pipeline and a reference-based pipeline using SAMtools. Sequence contigs positioned on the integrated physical and genetic map were used for read mapping and variant calling. We found high agreement in genotype calls between the different platforms and high concordance between genetic and reference-based marker order. There was, however, paucity in the number of SNP that were jointly discovered by the different pipelines indicating a strong effect of alignment and filtering parameters on SNP discovery. We show the utility of the current barley genome assembly as a framework for developing very low-cost genetic maps, facilitating high resolution genetic mapping and negating the need for developing <i>de novo</i> genetic maps for future studies in barley. Through demonstration of GBS on semiconductor sequencing platforms, we conclude that the GBS approach is amenable to a range of platforms and can easily be modified as new sequencing technologies, analysis tools and genomic resources develop.</p> </div

Genotyping-by-sequencing marker order based on the International Barley Sequencing Consortium reference framework (y-axis) compared to a <i>de novo</i> genetic order (x-axis) from 94 Morex x Barke recombinant inbred lines genotyped on either the Ion Torrent PGM or Proton platform.

<p>Each dot corresponds to one of 1,584 markers from the <i>de </i><i>novo</i> map that was positioned to the physical and genetic framework of barley.</p