Multiplex-ready PCR: a new method for multiplexed SSR and SNP genotyping

Abstract

Background: Microsatellite (SSR) and single nucleotide polymorphism (SNP) markers are widely used in plant breeding and genomic research. Thus, methods to improve the speed and efficiency of SSR and SNP genotyping are highly desirable. Here we describe a new method for multiplex PCR that facilitates fluorescence-based SSR genotyping and the multiplexed preparation of DNA templates for SNP assays.

Results: We show that multiplex-ready PCR can achieve a high (92%) success rate for the amplification of published sequences under standardised reaction conditions, with a PCR specificity comparable to that of conventional PCR methods. We also demonstrate that multiplex-ready PCR supports an improved level of multiplexing in plant genomes of varying size and ploidy, without the need to carefully optimize assay conditions. Several advantages of multiplex-ready PCR for SSR and SNP genotyping are demonstrated and discussed. These include the uniform amplification of target sequences within multiplexed reactions and between independent assays, and the ability to label amplicons during PCR with specialised moieties such fluorescent dyes and biotin.

Conclusion: Multiplex-ready PCR provides several technological advantages that can facilitate fluorescence-based SSR genotyping and the multiplexed preparation of DNA templates for SNP assays. These advantages can be captured at several points in the genotyping process, and offer considerable cost and labour savings. Multiplex-ready PCR is broadly applicable to plant genomics and marker assisted breeding, and should be transferable to any animal or plant species.

Figure 1

Amplification of SSRs from six bread wheat varieties in uniplex reactions using conventional and multiplex-ready PCR. Markers from left to right are gwm301, gwm340, gwm389 and gwm513. Multiplex-ready PCR assays were performed using 80, 70, 40 and 40 nM of locus-specific primer, respectively. Conventional PCR was performed with T_a 55, 60, 60 and 60°C, respectively. PCR products were separated on a GelScan2000 instrument.

Figure 2

Distribution of ABI3730 fluorescence intensities for published SSRs amplified in four- and six-plex multiplex-ready PCR assays. A total of 8960 SSR loci were amplified in 32 six-plex PCRs from each of six apricot and cherry varieties, and 32 four-plex and 48 six-plex PCRs from each of eight barley and bread wheat varieties.

Figure 3

ABI3730 electrotraces showing six-plex PCRs amplified from eight bread wheat varieties. Multiplex-ready PCR was performed using the SSR markers (A) barc216, (B) barc64, (C) barc108, (D) gwm681, (E) barc273 and (F) cfa2028, and 100, 70, 120, 70, 120 and 30 nM of locus-specific primer, respectively.

Figure 4

Six-plex PCRs performed in duplicate for six apricot varieties. Multiplex-ready PCR for panel 1 was performed using SSR markers (A) UDP96-003, (B) UDP96-008, (C) BPPCT028, (D) BPPCT014, (E) pchgms21.2 and (F) pchgms17 with 50, 50, 40, 40, 70 and 50 nM of locus-specific primer, respectively; and for panel 2 using SSR markers (G) UDP96-010, (H) BPPCT039, (I) BPPCT004, (J) pchcms04, (K) pchgms11.2 and (L) pchgms23 with 60, 50, 50, 70, 70 and 70 nM of locus-specific primer, respectively. The SSR products were separated on a Gel Scan 2000 instrument.

Figure 5

Diagrammatic representation of multiplex-ready PCR.