High-Throughput and Accurate Determination of Transgene Copy Number and Zygosity in Transgenic Maize: From DNA Extraction to Data Analysis

Abstract

It is vital to develop high-throughput methods to determine transgene copy numbers initially and zygosity during subsequent breeding. In this study, the target sequence of the previously reported endogenous reference gene hmg was analyzed using 633 maize inbred lines, and two SNPs were observed. These SNPs significantly increased the PCR efficiency, while the newly developed hmg gene assay (hmg-taq-F2/R2) excluding these SNPs reduced the efficiency into normal ranges. The TaqMan amplification efficiency of bar and hmg with newly developed primers was calculated as 0.993 and 1.000, respectively. The inter-assay coefficient of variation (CV) values for the bar and hmg genes varied from 1.18 to 2.94%. The copy numbers of the transgene bar using new TaqMan assays were identical to those using dPCR. Significantly, the precision of one repetition reached 96.7% of that of three repetitions of single-copy plants analyzed by simple random sampling, and the actual accuracy reached 95.8%, confirmed by T₁ and T₂ progeny. With the high-throughput DNA extraction and automated data analysis procedures developed in this study, nearly 2700 samples could be analyzed within eight hours by two persons. The combined results suggested that the new hmg gene assay developed here could be a universal maize reference gene system, and the new assay has high throughput and high accuracy for large-scale screening of maize varieties around the world.

Keywords: Southern blot; TaqMan assay; digital PCR; high throughput; maize endogenous reference gene; single-nucleotide polymorphism; transgene copy number; zygosity.

Figure 1

Development of universal TaqMan copy number assays in 384-well format for high-throughput screening. (A) Detected SNPs within amplified target DNA of hmg gene. Identical nucleotides are indicated by dots. White arrows and lines represent primers and probe attachment sites of hmg assay. Observed single-nucleotide polymorphism is shown in red boxes on white arrows. SNP of T to A was found at the first nucleotide from the 5′ end in the forward primer in maize line PH207, and another of G to T at the second nucleotide from the 5′ end in the reverse primer in maize line F7. No sequence variation was found in the probe. (B) Schematic diagram of pCAMBIA3301-ZmHMG. LB, left border; bar, phospinothricin acetyl transferase gene; hmg, fragment of maize endogenous gene hmg; gus, β-glucuronidase; RB, right border. Position of the EcoR I and HindIII restriction site is indicated above the T-DNA insert. (C) TaqMan assay efficiency for hmg and bar primer and probe sets. Five 10-fold serial dilutions of standard plasmid (1.0 × 10⁷ to 1.0 × 10³ copies/μL) were used in duplexed TaqMan copy number assays, as mentioned in Materials and Methods. Calculated Ct values were plotted against log of each concentration (copies/μL). Each sample was run in four replicates. Regression lines with their respective equations, correlation coefficient (R²), and amplification efficiency (E) are presented for each gene.

Figure 2

Correlation of TaqMan copy number assay data with Southern blot analysis. (A) Graphic representation of putative transgene insertion fragment (not to scale). pPBCXUN, binary vector construct in Agrobacterium strain EHA105 for Agrobacterium-mediated transformation; RB, right border; Pubi, ubiquitin promoter; Tnos, nopaline synthase terminator; P35S, 2 × CaMV 35S promoter; bar, phospinothricin acetyl transferase gene; T35S, CaMV 35S polyA; LB, left border. Position of HindIII restriction site is indicated above the T-DNA insert. Fragment used to generate bar-specific probes is indicated by the hatched box below bar gene. (B) Southern blot analysis of 45 independent T₀ transgenic maize plants. DNA samples from 45 T₀ events, as determined by TaqMan copy number assay, were digested with HindIII, fractionated by agarose gel electrophoresis, blotted to nylon, and probed with a bar-specific probe. Lines 1–45 correspond to 12141320, 12141321, 12151398, 12161497, 12161498, 12171579, 13181646, 13181668, 13191758, 13191764, 13191765, 13201829, 13201830, 12030065, 12050430, 12060607, 12060608, 12060612, 12060614, 12070775, 12080815, 12141331, 12161432, 12030070, 12040218, 12040220, 12050433, 12060625, 12060631, 12101014, 12111088, 12030068, 12030212, 12030213, 12030071, 12030210, 12030066, 12030067, 12010004, 12010005, 12030084, 12030085, 12030086, 12040234, and 12040236; WT represents untransformed ND101. (C) Correlation between copy numbers in transgenic maize T₀ lines determined by TaqMan assay and Southern blot analysis.

Figure 3

Development of TaqMan assay for high-throughput screening of transgene copy number and zygosity. (A) DNA extraction of samples in four 96-well plates and (B) TaqMan assay in 384-well plate are indicated by blue arrows. (C–H) Data analysis procedures are indicated by orange arrows: (C,D) sample corresponding to every well of 96-well plates; (E) sample corresponding to every well of 384-well plates; (F–H) automated data analysis results presented in three formats, (F) zygosity results of every line, (G) list of homozygous individual plants that need to be kept, and (H) copy number of individual T₁ plants.