Application of multiplex-ready PCR for fluorescence-based SSR genotyping in barley and wheat
- First Online:
- Cite this article as:
- Hayden, M.J., Nguyen, T.M., Waterman, A. et al. Mol Breeding (2008) 21: 271. doi:10.1007/s11032-007-9127-5
- 785 Views
Microsatellites (SSRs) are widely used in cereal research, and their use in marker assisted breeding has increased the speed and efficiency of germplasm improvement. Central to the application of SSRs for many purposes are methodologies enabling the low-cost acquisition of large quantities of genetic information for gene and genotype identification. In this study, multiplex-ready PCR was evaluated in barley and bread wheat as an approach for rapid and more automated SSR genotyping on a fluorescence-based DNA fragment analyzer. Multiplex-ready PCR is a method that allows SSR genotyping to be performed using a standardized protocol. The method enables flexible fluorescence labeling of SSRs, generates a relatively constant amount of PCR product for each marker, and has a high amenability to multiplex PCR (the simultaneous amplification of several SSRs in the same reaction). A high (92%) compatibility of published SSRs with multiplex-ready PCR is demonstrated, and the usefulness of the method for large scale genotyping is shown by its application for whole genome marker assisted breeding in barley. A database of more than 2,800 barley and wheat SSRs, and a suite of bio-informatic tools were developed to support the deployment of multiplex-ready PCR for various genetic applications, and are accessible at http://www.genica.net.au. Multiplex-ready PCR is broadly applicable to cereal genomics research and marker assisted breeding, and should be transferable to similar analyses of any animal or plant species.
KeywordsMarker assisted breedingMultiplex PCRMicrosatelliteSSRSemi-automated genotyping
Molecular markers are widely used in cereal breeding and research for phylogenetic studies, comparative genomics, and the mapping of genes and quantitative trait loci (QTLs) (Prasad et al. 2000; Stein and Graner 2004; Varshney et al. 2005). In breeding programs, molecular markers are used to improve the speed and efficiency of germplasm improvement through marker assisted selection (Crepieux et al. 2005; Koebner and Summers 2003; Langridge and Barr 2003). Microsatellites (SSRs) are currently the preferred type of marker due to their ease of assay, high level of polymorphism and codominant inheritance. The availability of dense SSR genetic maps for cereal crops such as barley, maize, rice, and wheat (Ramsay et al. 2000; Varshney et al. 2007; McCouch et al. 2002; Sharopova et al. 2002; Somers et al. 2004; Ramsay et al. 2000) provides marker choice and a high probability of finding polymorphic markers that tag a chromosomal region of interest.
Fluorescence-based SSR detection and allele sizing on an automated DNA fragment analyzer is one of the fastest and most accurate methods for SSR genotyping (Ziegle et al. 1992; Mansfield et al. 1994). This procedure is based on the separation of fluorescently labeled SSR amplicons by capillary or gel electrophoresis, and requires that one of the PCR primers used for SSR amplification is labeled with a fluorescent dye (Ziegle et al. 1992; Oetting et al. 1995). An advantage of fluorescence-based SSR genotyping is that several SSRs can be simultaneously separated in a single capillary or gel lane providing that the SSR fragments have non-overlapping sizes. In instances where SSR allele sizes are overlapping, coseparation can be achieved by labeling the SSR products with fluorescent dyes that have different emission wavelengths. Two approaches are commonly used to multiplex SSR genotyping. Post-PCR multiplexing (also called multi-pooling) involves the pooling of individual PCR assays for two or more SSRs prior to electrophoresis (Heller 2001). PCR multiplexing involves simultaneously amplifying two or more SSRs in a single PCR (Henegariu et al. 1997), and is typically used to repetitively genotype a small number of markers. The combined use of these approaches can be used to achieve highly paralleled, multiplexed SSR genotyping (Tang et al. 2003; Ponce et al. 1999).
Highly paralleled, fluorescence-based SSR genotyping can be difficult to achieve in cereal crops. Large genome size and polyploidy can limit the potential to perform multiplex PCR. For example, extensive experimental optimization is typically required for multiplex SSR amplification in bread wheat (Donini et al. 1998), an allohexaploid with a large and complex genome (Bennett and Smith 1976). The routine development of multiplex PCR assays in crop species with small diploid genomes is also challenging (Mitchell et al. 1997; Masi et al. 2003). One reason for this difficulty is the increased probability of undesirable primer–primer interactions in multiplex PCR that can lead to poor PCR sensitivity and specificity, and/or the preferential amplification of non-target loci (Edwards and Gibbs 1994). Multi-pooling strategies can be complicated by the amplification of vastly different amounts of PCR product for each SSR marker (Macaulay et al. 2001), which can lead to product overloading and genotyping inaccuracies. This problem arises from the inability of genotyping software to fully correct for background fluorescence resulting from the partial overlap of emission spectra for fluorescent dyes typically used on automated DNA fragment analyzers. In these instances, empirical optimization is required to determine the appropriate amount of PCR product to pool for each SSR marker.
In the present study, we describe a new method for semi-automated SSR genotyping on a fluorescence-based DNA fragment analyzer. The method, called multiplex-ready PCR, facilitates highly paralleled SSR genotyping by allowing flexible fluorescence labeling of SSRs and amplifying a similar amount of SSR product for each marker within a multiplex PCR assay and between independent reactions. These features simplify the development of multiplexed assays, and allow the use of a single multi-pooling procedure to prepare SSRs for electrophoretic analysis. The advantages of multiplex-ready PCR are discussed in the context of a laboratory that is deploying this technology to support genetic research and marker assisted breeding in barley and wheat.
Materials and methods
Sixteen barley and bread wheat lines were used to assess SSR amplification, uniformity of amplification yield and robustness of multiplex-ready PCR to variation in DNA quality and concentration. The barley lines were Alexis, Chebec, Clipper, Flagship, Harrington, Haruna Nijo, Sahara3771, and Sloop. The bread wheat lines were Barunga, VPM Cook, Chinese Spring, Gabo, Norin10, Olympic, Opata85, and WI7984 (a synthetic hexaploid wheat). A barley population consisting of 1961 F5 progeny derived from a four-way cross between doubled haploid breeding lines was used to demonstrate the utility of multiplex-ready PCR for marker assisted selection in the University of Adelaide barley breeding program. High-quality DNA was extracted from freeze-dried barley and wheat leaf material using the phenol–chloroform-based method described by Devos et al. (1992) with minor modifications. For high-throughput DNA extraction, DNA was extracted from fresh leaf material and seed using the sodium hydroxide-based method described by Paris and Carter (2000). The latter method produced DNA of quality suitable for immediate use in PCR but which underwent rapid degradation over a period of weeks.
PCR primers for SSRs obtained principally from the GrainGenes website (http://wheat.pw.usda.gov/GG2/index.shtml) were synthesized with generic non-complementary nucleotide sequences at their 5′-end. Specifically, the forward and reverse primer for each marker was synthesized with the nucleotide sequence 5′ ACGACGTTGTAAAA 3′ and 5′ CATTAAGTTCCCATTA 3′, respectively. Primer aliquots for each marker were prepared by mixing equimolar amounts of appropriate forward and reverse primer in 1 × TE (1 mM EDTA, 10 mM Tris–HCl, and pH 8.0), and are hereafter referred to as locus-specific primers. Two generic tag primers, tagF and tagR, with the sequences 5′ ACGACGTTGTAAAA 3′ and 5′ CATTAAGTTCCCATTA 3′, respectively, were also synthesized. The tagF primer was labeled at its 5′-end with one of the following fluorescent dyes: VIC, FAM, NED, and PET (Applied Biosystems, Warrington, UK).
The optimal concentration of locus-specific primer required for SSR amplification was determined empirically. Initially 20, 30, 40, and 80 nM of locus-specific primer were tested. PCR products were separated on a GelScan2000 instrument (see Uniplex and multiplex PCR) or on 2% agarose gels stained with ethidium bromide. The optimal primer concentration was determined by visual inspection as the strong amplification of a SSR fragment of the expected size with high-PCR specificity. In instances where it was desirable to improve PCR specificity and yield, additional locus-specific primer concentrations were tested.
Uniplex and multiplex PCR
The amplification of SSR markers by uniplex and multiplex PCR was performed under identical reaction conditions. PCR was performed in a 6 μl reaction mixture containing 0.2 mM dNTP, 1 × ImmoBuffer (Bioline, Luckenwalde, Germany) (16 mM (NH4)2SO2, 0.01% Tween-20, 100 mM Tris–HCl, and pH 8.3), 1.5 mM MgCl2, 100 ng/μl bovine serum albumin Fraction V (Sigma-Aldrich, Tanfkirchen, Germany), 75 nmol each of dye-labeled tagF and unlabeled tagR primer, 50 ng genomic DNA, 0.15 U Immolase DNA polymerase (Bioline), and an appropriate concentration of locus-specific primer (http://www.genica.net.au). For multiplex PCR, locus-specific primers for several markers were added to each reaction at the optimal concentration determined in uniplex assays. Following an initial denaturation step of 10 min at 95°C to heat activate the DNA polymerase, PCR was performed for a total of 55 cycles with the profile: 30 s at 92°C, 90 s at 50°C, and 60 s at 72°C for five cycles. The next 20 cycles were with 30 s at 92°C, 90 s at 63°C, and 60 s at 72°C, followed by 40 cycles with 15 s at 92°C, 30 s at 54°C, and 60 s at 72°C, and a final extension step of 10 min at 72°C.
Electrophoresis and visualization of PCR products was performed on a GelScan2000 (Corbett Research, Sydney, NSW, Australia) and ABI3730 DNA analyzer (Applied Biosystems). For analysis on the GelScan2000, the PCR products were mixed with an equal volume of gel loading buffer (98% formamide, 10 mM EDTA, and 0.5% basic fuchsin as tracking dye), heated for 3 min at 95°C, chilled quickly on ice and separated on a 4% sequencing gel (Sambrook and Russell 2001). For ABI3730 analysis, a standardized multi-pooling procedure was used to prepare SSR products for electrophoresis. A detailed protocol is provided at http://www.genica.net.au. Briefly, diluted PCR products labeled with different fluorescent dyes were pooled at a ratio of 2:2:1:2 for VIC:FAM:NED:PET, and desalted by ultra-filtration using an AcroPrep 384 filter plate with 10 kDa Omega membrane according to the manufacturer’s instructions (PALL Life Sciences, Surry Hills, NSW, Australia). Three μl of desalted PCR product resuspended in water was added to 8 μl of deionized formamide containing 0.8 μl of GeneScan500 LIZ size standard (Applied Biosystems). The mixture was heated uncovered at 90°C for 5 min to evaporate the water and electrophoresed according to the manufacturer’s instructions. This multi-pooling procedure resulted in 0.03 μl of each PCR being electrophoresed. Semi-automated SSR allele sizing was performed using GeneMapper v3.7 software (Applied Biosystems). The pooling of PCR products with different dye-labels at the 2:2:1:2 ratio was to account for differences in the relative fluorescence of each fluorophore.
The SSR genotyping in the F5 progeny of the barley breeding population was performed using a Biomek3000 liquid handling robot (Beckman Coulter, Fullerton, CA, USA) to automate PCR setup and prepare samples for electrophoresis. Multiplex-ready PCR assays and post-PCR pooling of multiplexed assays were performed as described above. Marker panels comprised of SSRs with non-overlapping allele sizes were designed for multiplex PCR using custom-written software, called Binner, which is accessible at http://www.genica.net.au. Multi-pooling of multiplexed assays labeled with different fluorescent dyes allowed up to 24 SSRs to be simultaneously separated in each ABI capillary.
Converting published SSRs into multiplex-ready markers
Uniformity of amplification yield
Similarly, to determine if multiplex-ready PCR amplified a relatively constant amount of SSR product for each marker within a multiplexed assay, 48 six-plex PCR assays were performed in each of barley and bread wheat using the eight genetically diverse lines (Fig. 3). Marker panels for multiplex PCR were constructed using essentially single-locus SSRs with non-overlapping allele sizes. Of the 576 SSRs evaluated in the multiplex PCR assays, 408 (71%) were successfully amplified, as assessed by ABI3730 fluorescence intensities that fell within the range optimal for semi-automated allele sizing, i.e., 2,000–15,000 rfu. The remaining 168 (29%) SSRs did not produce fluorescence intensities within the optimal range for all eight lines, failed to generate PCR product of the expected size, or gave weak amplification. In barley and wheat, six SSRs were amplified in seven and 13 of the 6-plex PCRs, five SSRs were amplified in 13 and 18 of the 6-plex reactions, four SSRs were amplified in 18 and 8 of the 6-plex assays, and three or fewer SSRs were amplified in ten and nine of the multiplexed assays, respectively. This corresponded to an average success rate of 21% for the amplification of six SSRs, 32% for the amplification of five SSRs, 27% for the amplification of four SSRs, and 20% for the amplification of three or fewer SSRs in the six-plex PCRs performed in barley and bread wheat. Similar to uniplex assays, the main source of variation in fluorescence intensity between markers within the multiplex PCR assays was the presence of SSR stutter bands. As observed in conventional multiplex PCR in barley and bread wheat, non-specific fragments were often amplified but these did not generally interfere with marker scoring.
Robustness of multiplex-ready PCR
Highly paralleled SSR genotyping
To evaluate the suitability of multiplex-ready PCR for highly paralleled SSR genotyping, the method was applied for whole genome marker assisted selection in a barley breeding program. In this study, 117 SSRs amplifying 119 loci were assayed in 1961 F5 progeny derived from a four-way cross between doubled haploid breeding lines. Initially, 511 SSRs were screened on the parental lines and markers revealing polymorphism were selected for whole genome coverage and to tag chromosomal regions containing genes and QTL of interest. Multiplex PCR assays comprised of three to six markers were developed for the 117 selected SSRs using the parental allele size data, and tested on the parental lines and 20 random progeny. Successful multiplex PCR assays were used to genotype the remaining progeny, while SSRs (27) that initially failed to amplify in the multiplex PCR assays were assayed individually. On average, 17 SSR loci were assayed per ABI capillary. Overall, 220,896 genotypes were successfully scored using semi-automated genotyping performed by GeneMapper software, which corresponded to a 94.7% allele call rate. Frequency distribution analysis of the fluorescence intensities of the scored alleles revealed that 83% were within the range optimal for semi-automated allele calling, i.e., 2,000–15,000 rfu. The remaining 17% had fluorescence intensities >15,000 rfu. In general, SSR alleles with high-fluorescence intensities were amplified in uniplex PCR assays. SSR alleles not called by GeneMapper had fluorescence intensities below the allele detection threshold of 2,000 rfu.
The importance of SSRs as molecular markers in cereal breeding and genetic research is well documented (Gupta and Rustgi 2004; Gupta and Varshney 2000; Gupta et al. 1999). The availability of dense SSR maps for barley and wheat make it possible to implement marker assisted breeding strategies to select for multiple chromosomal regions in a single cross, and even attempt to manipulate entire genomes by simultaneously using a large number of markers to select and combine the most favorable regions of chromosomes. Numerous studies have shown that PCR multiplexing and multi-pooling strategies can significantly reduce the cost of fluorescence-based SSR genotyping and increase genotyping throughput (Narvel et al. 2000; Tang et al. 2003). Technical improvements to further improve PCR assay throughput and reduce genotyping costs have the potential to increase the efficiency and speed of marker assisted breeding and cereal genetic research in general.
An important prerequisite of any new method for SSR genotyping is compatibility with previously published markers, flexibility for marker deployment and assay tolerance to a wide range of reaction conditions. In the present study, a success rate of 92% was achieved for the amplification of publicly available barley and wheat SSRs in multiplex-ready PCR, with more than 2,800 markers now optimized (see http://www.genica.net.au). Similar to the M13-tailed primer method (Oetting et al. 1995), multiplex-ready PCR enables SSR products to be dye-labeled with a fluorophore of choice during PCR amplification by interchange of the dye-labeled tag primer used in each assay (see Fig. 3). This capacity provides maximum flexibility for the detection of SSRs on an automated DNA fragment analyzer, as SSRs with overlapping fragment sizes can be multiplexed by labeling the markers with different fluorophores. Multiplex-ready markers can also be deployed on DNA fragment analyzers detecting different fluorophores using the same set of locus-specific primers, since only appropriately dye-labeled tag primers need be resynthesized. Multiplex-ready PCR showed a high tolerance to variation in the amount of DNA template, indicating that quantification of DNA samples is unnecessary (Fig. 4). Similarly, high-PCR specificity and yield was achieved for SSRs amplified from DNA extracted from leaf and seed material using a sodium-hydroxide method, indicating general compatibility with high-throughput DNA extraction methods often used in marker assisted breeding (Fig. 5). This tolerance to variation in DNA quality and quantity results from the use of a low concentration of tag primers and an excess of PCR cycles for the second stage of amplification to ensure that all tag primer is incorporated into PCR product. This characteristic ensures similar SSR fluorescence intensities between independent assays despite variation in template concentration and quality.
A practical advantage of multiplex-ready PCR for high-throughput SSR genotyping is the ability to perform marker assays under standardized reaction conditions. Conventional PCR requires that marker assays are grouped according to their optimal annealing temperature, and often other factors such as Mg2+ requirements. Published barley and wheat SSRs are reported to have a wide range of optimal annealing temperatures ranging from 45 to 63°C, especially among older sets of markers (e.g., see Roder et al. 1998; Song et al. 2005). Differences in PCR requirements can limit the potential to perform multiplex PCR for a given set of SSR markers. In contrast, the ability to perform multiplex-ready PCR assays under standardized conditions can improve genotyping throughput by facilitating PCR assay automation, and enables any combination of markers to be deployed in multiplex PCR. In the present study, a 71% success rate was achieved for the amplification of barley and wheat SSRs in six-plex PCRs without assay optimization. On average, five or more SSRs were successfully amplified in more than half (54%) of the 96 six-plex assays performed. This amenability to multiplex PCR results from the use of relatively low concentrations of locus-specific primer, which helps to reduce undesirable primer–primer interactions, and exhaustive conditions to ensure that the locus-specific primer is fully incorporated into PCR product at the end of the first stage of amplification. The latter helps to ensure that a similar amount of SSR product is amplified for each locus before the second stage of amplification.
Highly multiplexed SSR genotyping on an automated DNA fragment analyzer relies on the fluorescence intensities of markers falling within a range optimal for accurate allele detection and semi-automated scoring. Ideally, the fluorescence intensity for each SSR should be relatively uniform. This is especially important for the coseparation of SSR markers with overlapping fragment sizes but that are labeled with different fluorophores. Large differences in SSR fluorescence intensities can cause genotyping inaccuracies due to the inability of genotyping software to fully correct for background fluorescence, which results from the overlap of the emission spectra of different fluorophores. This phenomenon, known as “spectral bleed” and “pull-up,” results in the detection of a SSR allele where no dye-labeled amplicon actually exists. Multi-pooling strategies give similar SSR fluorescence intensities, although initial optimization is often required to determine the amount of product to pool. This optimization is required because SSRs are frequently amplified with different efficiencies in conventional PCR (Macaulay et al. 2001). In contrast, multiplex PCR often requires extensive optimization to achieve relatively uniform amplification for each SSR (Masi et al. 2003; Mitchell et al. 1997). In the present study, multiplex-ready PCR was observed to amplify a relatively consistent amount of SSR product for each marker within multiplexed assays and between independent reactions (Figs. 2, 3), as assessed by SSR fluorescence intensities that fell within an optimal range for semi-automated genotyping. The main source of variation in fluorescence intensities between markers appeared to be caused by SSR stuttering. Markers producing SSR stutter bands typically had lower fluorescence intensities than those with little, or no stuttering. This observation might be expected if multiplex-ready PCR amplified a relatively constant amount of SSR product, since a higher fluorescence signal would result if fewer dye-labeled fragments were amplified.
Numerous highly efficient multiplexed methods have been described for semi-automated fluorescence-based SSR genotyping in animals and plants. Methods based on multiplex PCR are most commonly described for DNA fingerprinting applications were common sets of marker loci are required or advantageous, such as in paternity testing, varietal identification and testing for seed purity (Tommasini et al. 2003; Blair et al. 2002; Perry 2004). The application of multiplex PCR primarily to DNA fingerprinting methods, rather than for genetic mapping and marker assisted breeding, is largely due to the effort required to develop multiplex PCR assays and that the combination of SSR markers required in genetic mapping and molecular breeding constantly changes. In practice, multi-pooling methods are more suited for these applications. Multiplex-ready PCR provides a new approach to highly paralleled SSR genotyping. The high amenability of SSR markers to multiplex PCR allows for the rapid development of multiplexed assays, while the ability to dye label SSR amplicons with a fluorophore of choice provides maximum flexibility for the separation of markers by multi-pooling. One disadvantage of multiplex-ready PCR, compared to other methods, is the initial requirement to determine the optimal concentration of locus-specific primer for SSR amplification. However, whilst this initial optimization is time-consuming, in our experience, this process is similar to the initial PCR optimization that is typically required for any new marker in conventional PCR.
Multiplex-ready PCR is currently deployed in several Australian cereal research and breeding programs, and has been used to generate more than one million SSR data points in the past 2 years. It has been used to construct several genetic maps in barley and bread wheat, perform whole genome scans for marker-trait associations using bulk segregant analysis and marker assisted breeding (Hearnden et al. 2007; Hayden et al., in preparation). To facilitate the use of multiplex-ready PCR for these applications, a marker database and suite of bio-informatic tools have been developed, and are accessible at http://www.genica.net.au. The database contains detailed marker information, allele size data, and electrotraces for each SSR amplified from genetically diverse germplasm.
In conclusion, multiplex-ready PCR is a new method for rapid and more automated fluorescence-based SSR genotyping. The advantages include significant cost savings for fluorescent primer labeling, ability to perform PCR assays and electrophoretic separation under standardized conditions, flexible dye labeling of SSR markers during amplification, and an inherent amenability to multiplex PCR. These advantages can be captured for SSR genotyping in any animal or plant species, and most types of applications ranging from highly repetitive genotyping, such as paternity testing, to projects that require the routine assemblage of new SSR combinations, such as genetic mapping and marker assisted breeding.
This research was supported by the Molecular Plant Breeding CRC and Grains Research and Development Corporation, Australia. The authors gratefully acknowledge Dr. J. Eglinton from the University of Adelaide barley breeding program for providing access to the F5 breeding material.