A High-Throughput Standard PCR-Based Genotyping Method for Determining Transgene Zygosity in Segregating Plant Populations

Abstract

In crop research programs that implement transgene-based strategies for trait improvement it is necessary to distinguish between transgene homozygous and hemizygous individuals in segregating populations. Direct methods for determining transgene zygosity are technically challenging, expensive, and require specialized equipment. In this report, we describe a standard PCR-based protocol coupled with capillary electrophoresis that can identify transgene homozygous and hemizygous individuals in a segregating population without knowledge of transgene insertion site. PCR primers were designed to amplify conserved T-DNA segments of the 35S promoter, OCS terminator, and NPTII kanamycin resistance gene in the pHellsgate-8 RNAi construct for the Gossypium hirsutum phytochrome A1 gene. Using an optimized multiplexed reaction mixture and an amplification program of only 10 cycles we could discriminate between transgene homozygous and hemizygous cotton control DNA samples based on PCR product peak characteristics gathered by capillary electrophoresis. The protocol was refined by evaluating segregating transgenic progeny from nine BC₁S₁ populations derived from crosses between the transgenic cotton parent 'E-1-7-6' and other cotton cultivars. OCS PCR product peak height and peak area, normalized by amplification of the native cotton gene GhUBC1, revealed clear bimodal distributions of OCS product characteristics for each BC₁S₁ population indicating the presence of homozygous and hemizygous clusters which was further confirmed via K-means clustering. BC₁S₁ plants identified as homozygous or hemizygous were self-fertilized to produce BC₁S₂ progeny. For the homozygous class, 19/20 BC₁S₂ families confirmed the homozygous BC₁S₁ prediction while 21/21 BC₁S₂ families confirmed the hemizygous prediction of the original parent. This relatively simple protocol provides a reliable, rapid, and high-throughput way of evaluating segregating transgenic populations using methods and equipment common to crop molecular breeding labs.

Keywords: PCR; bimodal; cotton; method; plant breeding; transgene.

FIGURE 1

GeneMapper^TM display of OCS_S PCR product peak heights for plants homozygous (A), hemizygous (B), or null (C) for the pHellsgate-8::PHYA1 RNAi construct. Note the change in scale between (A) and (B).

FIGURE 2

PCR product peak heights (A) and peak areas (B) for the pHellsgate-8-specific primer pairs 35S_S, NPTII-3, and OCS_S and the single copy control gene GhUBC1 (UBC1) using genomic DNA from plants homozygous (E-1-7-6) or hemizygous (F₁) for the pHellsgate-8::PHYA1 RNAi construct in an optimized 10 cycle PCR program.

FIGURE 3

Distributions of normalized OCS_S PCR product peak height values for nine BC₁S₁ populations derived from crosses between non-transgenic cultivars and the E-1-7-6 line homozygous for the pHellsgate-8::PHYA1 RNAi construct. The boxplot above each histogram shows selected quantiles of continuous distributions and outliers.

FIGURE 4

Significance test of sub-population means between homozygous (Group Genotype AA) and hemizygous (Group Genotype AB) entries identified by K-means clustering of normalized OCS_S PCR product peak height (A) and peak area (B) for nine BC₁S₁ populations.

FIGURE 5

Distribution of normalized OCS_S PCR product height values for BC₁S₂ populations derived from BC₁S₁ plants predicted to be hemizygous for the pHellsgate-8::PHYA1 RNAi construct. The red-dotted line in each histogram demarcates between hemizygous (AB) and homozygous (AA) progeny plants in each BC₁S₂ population.