Application Guide - QIAxcel Advanced

QIAxcel Advanced Application Guide 10/2016 127 When no Wx gene products are present, the wheat line is referred to as “waxy wheat” which is not suitable for producing udon noodles. When only 1 or 2 gene products are missing (partially waxy wheat), the amylose content of the wheat is reduced and the wheat is more suitable for making udon noodles. Selection of a partial waxy line based on the actual amylose content is problematic because measuring amylose content is difficult and imprecise and measurements must be obtained from several generations of plants. Therefore, we developed the DNA markers listed in Table 1 for PCR analysis to select partial waxy wheat plants (1). An effective method for multiple selections requires a high-throughput system. Our laboratory used a simple DNA extraction method in conjunction with the QIAxcel system to efficiently select lines from many breeding sites throughout the country. Materials and methods Genomic DNA from Chinese Spring (CS, wild type), Mochi Otome (MO, mutations in all 3 Wx genes), and a heterozygous plant (H) was prepared for the detection of mutations. PCR amplification was performed using primers for the detection of mutations in Wx genes (Table 1), and the amplified products were analyzed on the QIAxcel system with the QIAxcel DNA Screening Kit and the AM420 method. Results and Discussion Representative results are shown in Figure 1. Clearly distinguishable separation was obtained for the PCR- amplified fragments, as seen with the 370 bp and 389 bp fragments in Figure 1A. The PCR products for Wx-A1 and Wx-D1 alleles (Figures 1A and 1C, respectively) are co-dominant, and the assessment of heterozygosity using the gel image generated by the QIAxcel system is easy and straightforward. BDFL and BRD primers (Wx-B gene) amplify a 425 bp fragment that is present in the wild type but not the mutant (Figure 1B). In the electropherograms, the height of the Wx-B peak from the heterozygote (Figure 1D) is half the height of the peak from the homozygous wild type (Figure 1E), clearly demonstrating differences in gene dosage. The throughput capacity of the QIAxcel system allowed simultaneous analysis of up to 96 samples to be performed (Figure 2). Table 1. Sizes of fragments that are specific PCR markers for mutants of Wx alleles Amplification product (bp) Gene Primer Wild type Mutant Wx-A AFC 389 370 AR2 (408, 410) (408, 410) Wx-B BDFL 425 none BRD (455, 497) (455, 497) Wx-D BDFL 2307 1731 DRSL — — Fragments amplified from other Wx genes are indicated in parentheses. Wx-A AFC 389370 Wx-D BDFL 23071731

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