Application of the High Resolution Melting Analysis for Genetic Mapping of Sequence Tagged Site Markers in Narrow-leafed Lupin (lupinus Angustifolius L.)

Sequence tagged site (STS) markers are valuable tools for genetic and physical mapping that can be successfully used in comparative analyses among related species. Current challenges for molecular markers genotyping in plants include the lack of fast, sensitive and inexpensive methods suitable for sequence variant detection. In contrast, high resolution melting (HRM) is a simple and high-throughput assay, which has been widely applied in sequence polymorphism identification as well as in the studies of genetic variability and genotyping. The present study is the first attempt to use the HRM analysis to genotype STS markers in narrow-leafed lupin (Lu-pinus angustifolius L.). The sensitivity and utility of this method was confirmed by the sequence polymorphism detection based on melting curve profiles in the parental genotypes and progeny of the narrow-leafed lupin mapping population. Application of different approaches, including amplicon size and a simulated heterozygote analysis, has allowed for successful genetic mapping of 16 new STS markers in the narrow-leafed lupin genome.


INTRODUCTION
Sequence tagged site markers represent single copy sequences in the genome and are commonly used in genetic and physical mapping.Moreover, these markers can be used as skeleton markers that allow comparison between genetic maps among one species or synteny analysis between different species (Croxford et al., 2008;Kroc et al., 2014).In the case of species with unsequenced genomes such an approach is still desirable in comparative studies.The gene-based STS legume anchor markers (Leg markers) used in this study were designed to facilitate the comparative genomics of less known legumes (Fredslund et al., 2006;Hougaard et al., 2008).To date these markers have been successfully applied in the synteny analyses of Phaseolus vulgaris, Lotus japonicas, Medicago truncatula and Arachis (Hougaard et al., 2008;Bertioli et al., 2009).Leg markers were also included within a larger set of markers used in the recent synteny analysis of Lupinus angustfolius and Medicago truncatula (Kroc et al., 2014).
High resolution melting (HRM) is a powerful technique used for genotyping and mutation scanning.This method takes advantage of special saturation dyes properties that fluoresce only in the presence of double stranded DNA.After PCR, amplicons bound to the fluorescent dye are denaturated and the fluorescence fades away.Since various genetic sequences melt at different temperatures, changes in the fluorescence registered during the analysis can be applied to single nucleotide polymorphism (SNP), simple sequence repeat (SSR), small insertion and/or deletion (InDel), as well as length polymorphism detection (Montgomery et al., 2007;Distefano et al., 2012).The HRM assay has been successfully applied in studies of genetic variability and SNP/ SSR marker genotyping of various plants, e.g.legumes, including alfalfa (Han et al., 2012), pea (Knopkiewicz et al., 2014), soybean (Monteros et al., 2010) and white lupin (Croxford et al., 2008).
Various molecular markers, such as amplified fragment length polymorphism (AFLP), microsatellite anchored fragment length polymorphism (MFLP), restriction fragments length polymorphism (RFLP), diversity arrays technology (DArT), InDel and STS have already been applied in various studies focused on the narrowleafed lupin genetic mapping (Nelson et al., 2006;Nelson et al., 2010;Yang et al., 2013;Kroc et al., 2014;Kamphuis et al., 2014).In the case of STS markers, DNA polymorphism has been detected with the aid of restriction enzymes such as either CAPS (cleaved amplified polymorphic sequence) or dCAPS (derived-CAPS) markers or the SNaPshot assay (Life Technologies Inc.) (Nelson et al., 2010;Kroc et al., 2014).However, these techniques have some limitations.Their protocols are multi-stage, time-consuming and labor-intensive.Moreover, in CAPS and dCAPS systems, SNP must create a restriction site, while polymorphism detection requires gel electrophoresis.On the other hand, the SnaPshot assay needs capillary electrophoresis to detect polymorphisms.In contrast to both these methods, HRM is a simple and fast analysis.Furthermore, it can be performed directly after PCR without additional sample processing (Wu et al., 2008).
The main aim of this study was to evaluate the effectiveness of the HRM method in genotyping STS markers in narrow-leafed lupin (Lupinus angustifolius L.) as well as HRM method optimization.

MATERIALS AND METHODS
Plant material.The mapping population of 96 F8 generation recombinant inbred lines (RILs) used in this study was established by the single seed descent method from a cross between a domesticated breeding line 83A:476 and a wild type P27255 (Boersma et al., 2005) and was provided within the framework of cooperation with Dr. Hua'an Yang (Western Australia Department of Agriculture and Food, Perth, Australia).
Young leaf samples were colleted from plants grown in the field and processed for DNA extraction using a DNeasy Plant Mini Kit (Qiagen) according to the manu-facturer's protocol with minor changes.These included: (1) an additional incubation step (30 min at room temperature) after the addition of Buffer AP1 and 3 µl of RNase A stock solution, (2) increased duration of the cell lysis step (30 min at 65°C) and (3) increased duration of the precipitation step (15 min on ice).
Polymorphic markers detection.Leg markers were designed with the aid of a bioinformatics pipeline on the basis of the coding sequences of model legumes (Lotus japonicus, Medicago truncatula) and legume crop species (Glycine max, Phaseolus vulgaris), with Arabidopsis thaliana genome sequence data used as a reference genome (Fredslund et al., 2005;2006).A list of all Leg primer pairs is available at http://cgi-www.daimi.au.dk/cgi-chili/GeneticMarkers/table and in Bertioli et al. (2009).
Temperature gradient PCR was used for the mapping population parental lines survey to set up the best annealing temperature for each Leg marker using Promega GoTaq ® Flexi Polymerase and the protocol provided by the manufacturer.Single PCR products of both parental lines were sequenced using a BigDye Terminator™ v.3.1 Cycle Sequencing Kit (Applied Biosystems) to confirm amplicon identity and further sequence polymorphism detection.
HRM primer pair design and HRM assay.Polymorphic markers of a length over 250 bp were re-designed into smaller amplicons not exceeding 160 bp covering SNP or InDel sites.The only exception was marker Leg435 kept in its original size of 278 bp as a test of the ability of HRM method to differentiate longer fragments.The HRM primers were designed to have a predicted annealing temperature of around 56-62°C using Primer3 (Koressaar & Remm, 2007;Untergrasser et al., 2012), taking care to exclude any possibility of secondary structure formation (Oligo Analyzer 3.1, https:// eu.idtdna.com/analyzer/Applications/OligoAnalyzer,Integrated DNA Technologies, Inc).
All PCR-HRM analyses were performed in 96-well plates using a LightCycler 480 (Roche) and either Light-Cycler 480 High Resolution Melting Master (Roche) or a combination of GoTaq ® Flexi Polymerase (Promega) with LightCycler ® 480 ResoLight Dye (Roche).All data were recorded and analyzed using LightCycler ® 480 Gene Scanning Software.During the optimization step, the quality and the specificity of the PCR products were verified by agarose gel electrophoresis and/or LightCycler ® 480 T m Calling Software.
LightCycler 480 High Resolution Melting Master.The HRM reactions were carried out in a final volume of 10 µl containing 1x LightCycler 480 High Resolution Melting Master (Roche), 0.2 µM of each primer, 1.5-3.5 mM MgCl 2 (Roche) and 25 ng of template DNA.After an initial denaturation step of 10 min at 95°C, 45-65 cycles were carried out with a repeated denaturation step at 95°C for 10 sec, an annealing step at 54-62°C for 15 sec and an extension step at 72°C for 10 sec.After the amplification, samples were heated to 95°C for 1 min and then cooled to 40°C for 1 min to encourage duplex formation.HRM curve data were obtained by melting over increasing temperatures from 65 to 95°C at a rate of 25 acquisitions per 1°C.This protocol applies to markers: Leg33MGm, Leg245, Leg256, Leg318, Leg325, Leg425, Leg435, Leg445, Leg713, Leg735, Leg736 and Leg871.
Linkage analysis.Integration of the Leg markers into the newest reference linkage map of narrow-leafed lupin (Kamphuis et al., 2014) was performed using Map-Manager software version QTXb20 (Manly et al., 2001).In the mapping analysis only the skeleton markers from Kamphuis et al. (2014) were incorporated.The new markers were added to the map with a P-value≤0.001.The Kosambi mapping function was applied for conversion of the recombination rate into genetic map distance (cM).Graphic illustration of linkage groups was performed with the aid of MapChart software (Voorrips et al., 2002).

RESULTS AND DISCUSSION
Sixteen Leg primer pairs were incorporated in the evaluation of HRM method effectiveness in markers genotyping for the purpose of their genetic mapping in the narrow-leafed lupin genome (Table 1).The genetic map constructed on the basis of skeleton markers from Kamphuis et al. (2014) was longer than originally as a result of different mapping software application (2 290.1 cM versus 2 263.9 cM and average spacing between skeleton loci of 2.92 cM versus 2.9 cM).Newly mapped Leg markers were distributed in ten linkage groups (Fig. 1, Table 2) In two cases map intervals between adjacent markers exceeded 10 cM, whereas all the remaining newly mapped markers were tightly linked (Table 2).The new linkage map length slightly increased and was 2 362.6 cM with an average of 2.96 cM between adjacent loci.The linkage groups varied in length from 74.2 cM (NLL-19) to 173.8 cM (NLL-11) (Fig. 1).
One of the main criterion of our HRM assay was to keep the amplicon size under 160 bp.Melting of smaller amplicons results in more significant differences of melting temperature (T m ) among genotypes, which greatly simplifies sample differentiation (Gundry et al., 2003;Liew et al., 2004).Secondary structures were avoided at the primer pairs design level since its formation influence the reaction kinetics, efficiency and specificity.The optimization of the PCR profile mainly involved MgCl 2 concentration (1.5 to 3.5 mM in 0.5 mM steps), since an optimal Mg 2+ concentration is es- sential to ensure the best specificity and yield of the PCR products (Montgomery et al., 2007).The annealing temperature for the primer pairs tested mostly oscillated around 56°C.The analysis of the actual HRM data was always preceded by the examination of amplification fluorescence data.The crossing point (C p ) which corresponds to the cycle number at which the fluorescence signal of the PCR product rises above the background should remain below 30 cycles for each reaction.This indicates an adequate amount of template DNA and a suitable amplification efficiency (LightCycler ® 480 Real-Time PCR System -Technical Note No. 1 High Resolution Melting: Optimization Strategies).In the case of four analyzed markers (Leg050, Leg055, Leg074, Leg156) the amplification carried out with the aid of LightCycler ® 480 High Resolution Melting Master (Roche) was not effective enough and required more than 30 cycles to achieve C p and even more than 60 cycles to achieve the plateau phase.Application of a previously optimized GoTaq ® Flexi Polymerase (Promega) and LightCycler ® 480 ResoLight Dye (Roche) combination instead of the HRM commercial reagent kit allowed the required C p and amplification efficiency to be reached.
High resolution melting of the PCR products can detect most homozygous mutations, however, some homozygous SNP have melting curves identical to those of the wild-type.This might be caused by overlong amplicons that influence the reaction sensitivity.Moreover, A>T and C>G mutation types result in only a slight T m difference between amplicons (usually less than 0.4°C), which makes their detection more difficult (Liew et al., 2004).In this case, addition of DNA of a known genotype to each unknown sample before PCR, results in heteroduplex formation which enables differentiation of homozygous and wild-type genotypes (Palais et al., 2005).This approach was successfully utilized by Croxford et al. (2008) to detect SNP markers in Lupinus albus.In our studies, the parental genotypes of six markers (Leg-33MGm, Leg074, Leg156, Leg713, Leg736 and Leg871) were difficult to distinguish, therefore, the comparison of melting profiles generated by each parent separately and  also by a mixed template containing DNA from both parental genotypes (a simulated heterozygote 1:1 mixed sample) was applied.On the basis of this approach, the distinction of both genotypes for these markers was feasible (Fig. 2).
Amplicon size has a significant impact on HRM method resolution.Han et al. (2012) reported successful employment of the HRM assay in SNP genotyping of 51-149 bp amplicons in alfalfa; Wu et al. (2008) analyzed 68-198 bp amplicons in almond; while De Koeyer et al. (2010) were able to detect SNP with amplicon sizes between 50-230 bp in potato.As mentioned above, the size of the HRM markers designed in our studies was kept under 160 bp, but one marker (Leg435) was used with its original, longer sequence size of 278 bp to test the possibility of SNP detection in longer amplicons (Fig. 3).According to Reed & Wittwer (2004), the melting of amplicons longer than 300 bp induces a depletion of sensitivity and specificity in single SNP detection.On the other hand, the increased number of polymorphic sites within the amplicon results in an improvement of HRM analysis resolution, thus enabling larger PCR product utilization (Hofinger et al., 2009).Most of the re-designed HRM markers in our studies covered one polymorphic site, but in the case of three markers, two (Leg074), three (Leg33MGm) or even five (Leg435) SNP and/or InDel were incorporated in the amplicon sequence.The increased number of polymorphic sites of marker Leg435 is probably the explanation of its successful HRM analysis.Knopkiewicz et al. (2014) also used the HRM method to successfully analyze 400 bp and 600 bp amplicons covering both three and seven SNPs, respectively.
The HRM technique offers a very sensitive and rapid method for SNP genotyping that after optimization does not require DNA restriction or electrophoresis to detect sequence polymorphisms.In our studies, melting curve analyses using LightCycler ® 480 T m Calling Software (Roche) proved to be completely sufficient and in some cases even more sensitive than standard agarose electrophoresis.Thus, in comparison with other popular genotyping methods, i. e. CAPS/dCAPS, the HRM technique is less time-consuming and labor-intensive.When the most appropriate chemistry set is selected, the important advantage of the HRM method is its flexibility in new markers examination.New primer set is the only requirement for each newly analyzed marker, while the chemistry remains unchanged in each analysis.No additional reagents are needed to detect any existing polymorphic site.HRM assay is therefore more profitable than other methods, i.e.CAPS/dCAPS requiring specific restriction endonucleases.The main obstacle in the HRM technique application is the requirement of a specialized and expensive equipment to conduct analyses.Fortunately, as qPCR thermocyclers adapted to HRM assay are becoming more and more popular, their cost gradually decreases.
The new Leg markers with its associated DNA sequence could potentially be used in the synteny analyses between the genome of Lupinus angustifolius and model legumes.Synteny between Lupinus angustifolius and Medicago truncatula as well as Lotus japonicus were previously undertaken, showing examples of marker colinearity between their genomes (Nelson et al., 2010;Nelson et al., 2006;Kroc et al., 2013).The current reference map of Kamphuis et al. (2014) was not involved in any synteny analysis and as it incorporated a significant number of new markers, their positions/order have changed compared to the previous map versions.It is therefore difficult to assess if the newly mapped Leg markers are involved and extend any previously reported synteny blocks.
In the course of our studies the optimized HRM markers were analyzed in the mapping population of narrow-leafed lupin.As a result, all of the 16 analyzed markers were successfully mapped in lupin genome.The incorporation of new Leg markers into the narrow-leafed lupin genetic map opens the possibility of synteny analyses with other legume species.Furthermore, the different approaches we applied in the HRM marker optimization process, might serve as a good starting point in overcoming difficulties when implementing HRM assays in other species.We conclude that the HRM assay proved to be an effective method for genotyping of STS markers in narrow-leafed lupin despite optimization problems encountered.It is therefore a good alternative to other popular genotyping methods.

Figure 2 .
Figure 2. High resolution melting curves of Leg736 (116 bp) containing a SNP substitution (C>G).(A) Verification of the PCR amplicon specificity by T m Calling analysis.Melting curves profile of the parental genotypes (83A:476 and P 27255) and the simulated heterozygote (83A:476/P 27255).Single peaks for each analyzed genotype indicate a proper specificity of the conducted analysis.(B) Normalized and shifted melting curves of the parental genotypes (83A:476 and P 27255) and the simulated heterozygote (83A:476/P 27255) amplicons.Parental genotypes of Leg736 were indistinguishable due to existing C>G mutation type causing only a slight T m difference between both amplicons.To overcome this problem melting profile generated by a mixed template of both parental DNA (a simulated heterozygote) was analyzed apart from individual parental genotypes.(C) Normalized and shifted melting curves of Leg736 amplicons after addition of 83A:476 parental line to each mapping population progeny sample.Figure presents the results for 88 RILs analyzed simultaneously on 96-well plate together with the mapping population parental lines as standards).Arrows represent: mapping population parental line 83A:476 and RILs with same genotype as well as simulated heterozygote 83A:476/P27255 and corresponding RILs.Application of this approach resulted in successful mapping of Leg736 marker in the NLL-09 linkage group.

Figure 3 .
Figure 3. HRM assay of Leg435 in narrow-leafed lupin mapping population.(A) Verification of the PCR amplicon specificity by T m Calling analysis.Arrows represent the mapping population parental lines 83A:476 and P 27255.Single peaks of the analyzed genotypes indicate a proper specificity of the conducted analysis.(B) Normalized and shifted melting curves of the 278-bp Leg435 amplicons.Parental genotypes contain five SNP substitution (C>T; G>A; G>A; G>A, A>C). Figure presents the results for 88 RILs analyzed simultaneously on 96-well plate together with the mapping population parental lines as standards).Arrows represent genotypes of the mapping population parental lines 83A:476, P 27255 and also their corresponding RILs.All of the RILs analyzed have been assigned to one of the parental genotypes and Leg435 marker was successfully mapped in the NLL-18 linkage group.

Table 1 . A list of markers selected for HRM analyses and incorporated into the narrow-leafed lupin genetic map.
CATCTCTGCATCATGAAGATG Genotyping of STS markers in narrow-leafed lupin by HRM method Figure 1.Linkage map of the narrow-leafed lupin genome comprising newly mapped HRM markers.

Table 2 . HRM markers map position and linkage details.
a HRM marker and preceding and succeeding marker in the linkage group.b linkage with preceding and succeeding marker