ORIGINAL PAPER

Summer Squash Identification by High-Resolution-Melting(HRM) Analysis Using Gene-Based EST–SSR MolecularMarkers

Aliki Xanthopoulou & Ioannis Ganopoulos & Aphrodite Tsaballa &

Irini Nianiou-Obeidat & Apostolos Kalivas & Athanasios Tsaftaris &Panagiotis Madesis

# Springer Science+Business Media New York 2013

Abstract Cucurbita pepo (squash, pumpkin, gourd), a world-wide cultivated vegetable of American origin, is extremelyvariable in fruit characteristics. Most of its widely grown com-mercial types are known as summer squashes and belong to theelongated forms of C. pepo ssp. pepo (Cocozelle, Vegetablemarrow and Zucchini groups). Here, we have integrated thehigh-resolution-melting (HRM) analysis method withexpressed sequence tags–simple sequence repeat (EST–SSR)marker genotyping, in order to facilitate the identification of 36summer squash landraces originated from Greece. The sixEST–SSR loci used were informative and generated a uniquemelting curve profile of EST-derived microsatellites for eachaccession allowing their comparison and classification.Moreover, HRMwas highly informative, as by using only fourmicrosatellite markers we were able to discriminate 36 summersquash landraces and by using six EST–SSRs. We were able toconstruct a highly informative and discriminative dendrogram

where the 36 genotypes were classified in six distinct clusters.Furthermore, we acquired information about the genes contain-ing the EST–SSRs using bioinformatics tools. We found thatthe EST–SSRs used in this study were hybridizing to genesinvolved in stress response to heavy metals and biotic stressesor the production of flavonoids or symporters of importantnitrogen sources, like xanthine and uric acid amongst others.The results presented here suggest that the panel of EST–SSRmarkers used in combination with HRM analysis could beuseful in a variety of applications, like squash biodiversityassessment but most importantly in managing squash germ-plasm to improve breeding programs.

Keywords Summer squash . Landraces . Genotyping .

EST–SSRmarkers . HRM

Introduction

Cucurbita pepo L. is the most economically important speciesof the genus Cucurbita L. (Cucurbitaceae). CultivatedC. pepo isconsidered to be consisted of two subspecies (Decker 1988;Formisano et al. 2012; Sanjur et al. 2002), each one encom-passing several cultivar groups:C. pepo ssp. pepo L. (includingPumpkin group, Vegetable marrow group, Cocozelle group andZucchini group) and C. pepo ssp. texana (Scheele) Filov (synssp. ovifera (L.) Decker) (includingAcorn group, Scallop group,Crookneck group and Straightneck group (Ferriol et al. 2003;Paris 1989; Paris et al. 2003). The primary economic value ofthis species is based on the culinary use of immature fruits, oftenreferred to collectively as "summer squash". Only cultivars ofthe Pumpkin and Acorn groups have a widespread use as"winter squashes", grown for the consumption of their maturefruits (Paris 2008).

Electronic supplementary material The online version of this article(doi:10.1007/s11105-013-0658-0) contains supplementary material,which is available to authorized users.

A. Xanthopoulou : I. Ganopoulos :A. Tsaballa :A. Tsaftaris (*) :P. Madesis (*)Institute of Applied Biosciences, CERTH, Thermi,Thessaloniki 570 01, Greecee-mail: tsaft@certh.gre-mail: pmadesis@certh.gr

A. Xanthopoulou : I. Ganopoulos :A. Tsaballa :I. Nianiou-Obeidat :A. TsaftarisDepartment of Genetics and Plant Breeding, School of Agriculture,Aristotle University of Thessaloniki, Thessaloniki 54 124, Greece

A. KalivasCotton and Industrial Plants Institute, National Agricultural ResearchFoundation, Thermi, Greece

Plant Mol Biol RepDOI 10.1007/s11105-013-0658-0

Countries of the Mediterranean region use different squashcultivars which meet their consumer's preferences, as squashis very popular in the region. Countries like Greece, Italy andSpain (Katsiotis et al. 2009; Paris 2008; Ferriol and Pico 2008)have unique and diverse landraces collections which havebeen used by the breeders in order to develop new or improveexisting varieties. National Genebank of Greece has a uniqueand invaluable collection of old Greek winter and summersquash cultigens of C. pepo ssp. pepo (Katsiotis et al. 2009),which are of great interest for breeders, thus we selected thiscollection to study.

Different marker systems have been used before in order toassess the genetic variability within C. pepo like allozymesand different DNA markers (restriction fragment length poly-morphism [RFLP], random amplified polymorphic DNA[RAPDs], amplified fragment length polymorphism [AFLP]and inter simple sequence repeats [ISSRs]) reviewed byLebeda et al. (2006) and Esteras et al. (2011). Genetic andevolutionary relationships between the two C. pepo subspe-cies or between the cultivated groups and between the domes-tic and wild relatives of Cucurbitaceae have been the center ofprevious research efforts, unfortunately using only limitednumber of representatives of the European landraces (Ferriolet al. 2003; Paris et al. 2003).

Simple sequence repeats (SSRs) or microsatellite markersare powerful polymerase chain reaction (PCR)-based molecu-lar tools. They are characterized by their reproducibility, multi-allelic nature and co-dominant inheritance and thus they havebecome irreplaceable in plant breeding and genome studies.SSRs are highly abundant in the genome and they are distrib-uted relatively evenly in the genome, thus having wide genomecoverage. In order to design SSR markers, we could useinformation obtained from genomic DNA (gSSRs) (Panaudet al. 1995) or expressed sequence tags sequences (ESTs).EST–SSR markers have many advantages compared togSSRs, including their locus-specificity to the expressed genesand potential to be used as functional markers for associationstudies of candidate genes with phenotypic variations andcomparative studies for genetically related different species(Hwang et al. 2012; Park et al. 2010; Ritschel et al. 2004).EST–SSRs have been developed for many plant species withagronomic interest, including eggplants (Nunome et al. 2009),peppers (Yi et al. 2006), melons (Kong et al. 2007) and toma-toes (D'Agostino et al. 2007). Moreover, EST–SSR markershave been developed for Cucurbitaceae crops. DNA finger-printing and mapping of important traits have been developedvia EST–SSR markers in melon (Kong et al. 2006; Levi et al.2009). While recently, Formisano et al. (2012) have used gSSRand EST–SSR molecular markers in order to compare thenatural variation present in a collection of Spanish landraceswith commercial varieties and hybrids. Furthermore, Gonget al. (2008) used and enriched a genomic library from an oil-seed pumpkin variety for the development of almost 400 gSSR

markers. The advent of next-generation sequencing technolo-gies (Metzker 2009) has allowed (Blanca et al. 2011) the firstanalysis of the Cucurbita transcriptome, with 49,610 Cucurbitaunigenes assembled de novo from 512,751 high quality ESTs,sequenced using Roche GS/454. Analysis of these unigenes forSSR motifs identified a collection of 1,882 unigenes with SSRmotifs (EST–SSR), which could be considered to be the firstgenomic resource in this genus. Squash is a non-model cropwith narrow genetic base; thus, sequencing is still the methodof choice for EST–SSR detection.

The recent introduction of the high-resolution-melting(HRM) analysis, a sensitive detecting method able to detecteven single nucleotide polymorphisms in the field of genomicresearch, has extended the abilities for the analysis of DNAmelting curves which is a standard diagnostic feature in qPCR(Wilhelm and Pingoud 2003). HRM analysis is becoming themethod of choice for post PCR genotyping methods, as it is anefficient, cost-effective, quicker and alternative method to gelelectrophoresis. Genotype discrimination and genetic mappingcould be performed via HRM analysis through the detection ofa broad range of SNPs and indels (Lehmensiek et al. 2008;Chagné et al. 2008). Identification of cultivars using SSRs and/or SNPs has also been performed successfully via this method(Bosmali et al. 2012; Ganopoulos et al. 2011, 2013; Hwanget al. 2012; Mackay et al. 2008; Ricci et al. 2012).

In this study, we report the application of six gene-basedEST–SSR markers based on HRM analysis and the assess-ment of their utility in landraces identification and clarificationof the genetic relationship amongst them.

Materials and Methods

Thirty-six summer squash landraces collected from NationalGenebank of Greece were chosen for this study (Table 1).

Isolation of DNA from seeds was performed with theNucleoSpin Plant kit (Macherey-Nagel, Germany), accordingto the manufacturer's instructions. The DNA concentration wasestimated by standard spectrophotometric methods at 260- and280-nm UV lengths by an Eppendorf BioPhotometer and theintegrity by gel electrophoresis in a 0.8 % agarose gel. Sampleswere then diluted to 20 ng/μl work concentration.

For SSR analysis, PCR amplification, DNA melting, andend point fluorescence level acquiring, PCR amplificationswere performed in a total volume of 15 μl in a 36-wellcarousel of the Rotor-Gene 6,000 real-time 5P HRM PCRThermocycler (Corbett Research, Sydney, Australia)according to (Ganopoulos et al. 2011). PCR reaction mixtureconsisted of 20 ng genomic DNA, 1× PCR buffer, 2.5 mMMgCl2, 0.2 mM dNTP, 300 nM forward and reverse primers,1.5 mM Syto® 9 green fluorescent nucleic acid stain, and 1 UKapa Taq DNA polymerase (Kapa Biosystems, USA). ThePCR protocol used is as follows: an initial denaturing step of

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95 °C for 3 min followed by 35 cycles of 95 °C for 20 s, 55 °Cfor 20 s and 72 °C for 20 s, then a final extension step of 72 °Cfor 2 min. The fluorescent data was acquired at the end of eachextension step during PCR cycles. In order to perform the HRManalysis, the products were initially denatured at 95 °C for 5 s,and then annealed at 50 °C for 30 s to randomly form DNAduplexes. HRM protocol steps are the following: pre-melt at thefirst appropriate temperature for 90 s, and melt at a ramp of10 °C in an appropriate temperature range at 0.1 °C incrementsevery 2 s. The fluorescent data were acquired at the end of eachincrement step. End point fluorescence level was acquired fol-lowing the melting process by holding at 60 °C for 5 min.

The sequences of the EST–SSR primers used in the studyare shown in Table 1, and primers were designed as describedby Blanca et al. (2011). HRM was performed as describedpreviously (Ganopoulos et al. 2011).

For genotyping by HRM analysis, the genotype of eachDNA sample was determined based on the shape of curvesdepicted by temperature-shifted melting curves or differenceplots, and was scored for the binary data matrix. The matriceswere then analyzed by FreeTree v. 0.9.1.50 software (Hamplet al. 2001). Similarity of qualitative data was calculated usingthe Nei and Li/Dice similarity index (Nei and Li 1979), andsimilarity estimates were analyzed using UPGMA (Unweighted

Table 1 Summer squash (C. pe-po ssp. pepo) landraces used onEST–SSR-based genetic distanceanalysis

No Genebank accession number Origin Common Name Morphotype

1 GRC262/04 CHANIA Zucchini

2 GRC597/04 AHAIA Zucchini

3 GRC1025/04 AETOLOAKARNANIA Zucchini

4 GRC1369/04 ARTA Zucchini

5 ATS-030/06 ANDROS ISL. Zucchini

6 ATS-063/06 TINOS Zucchini

7 F-104/06 KASTORIA Zucchini

8 F-135/06 KASTORIA PITSIALA Zucchini

9 K-061/06 KOZANI Zucchini

10 K-161/06 GREBENA Zucchini

11 K-192/06 GREBENA Zucchini

12 M-047/06 LESVOS ISL. MAGIATIKO Zucchini

13 M-166/06 LESVOS ISL. Zucchini

14 P-054/06 ARKADIA Zucchini

15 P-085/06 MESSINIA Zucchini

16 P-170/06 LAKONIA Zucchini

17 P-199/06 ARKADIA KARDIA Zucchini

18 SK-067/06 SERRES Zucchini

19 T-024/06 KARDITSA Zucchini

20 T-121/06 KARDITSA Zucchini

21 T-223/06 KARDITSA Zucchini

22 T-373/06 TRIKALA Zucchini

23 T-528/06 TRIKALA RIZITES Zucchini

24 ANP-039/07 AMORGOS ISL. Zucchini

25 ANP-163/07 NAXOS ISL. Zucchini

26 AO-059/07 AG. OROS Zucchini

27 HL-018/07 IRAKLION FORMARES Zucchini

28 HL-195/07 LASITHI Zucchini

29 KD-014/07 KAVALA Zucchini

30 LKK-058/07 KALYMNOS ISL. Zucchini

31 RK-046/07 RODOS ISL SARAVANES Zucchini

32 ROX-091/07 XANTHI Zucchini

33 ROX-139/07 RODOPI Zucchini

34 SAS-010/07 ALONNISOS ISL. Zucchini

35 SAS-085/07 SKOPELOS ISL. Zucchini

36 XKA-010/07 CHALKIDIKI Zucchini

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Pair GroupMethod usingArithmetic Averages). Thematrices ofmutual coefficients of similarity calculated by FreeTree wereconverted to MEGA 5 software (Tamura et al. 2011) and theresulting clusters were expressed as dendrograms.

In order to acquire information about the genes containingthe EST–SSRs, the squash unigene contigs (Blanca et al. 2011)were introduced in BLASTx searches as queries against theGenBank proteins database at NCBI (http://www.ncbi.nlm.nih.gov/). Conserved domains of the predicted protein produced bythe squash unigene contigs translation, were identified by Pfamsearches (Punta et al. 2012). Furthermore, the BLASTn tool wasused in the Cucurbit Genomics Database (http://www.icugi.org/cgi-bin/ICuGI/index.cgi) for identifying the cucumber andwatermelon putative orthologs of the chosen squash unigenecontigs, since cucumber (Cucumis sativus), squash (C. pepo)and watermelon (Citrullus lanatus) share a high degree ofsimilarity as they belong in the Cucurbitaceae family. Theretrieved cucumber and watermelon putative unigeneorthologs were aligned with the nucleotide sequences of thechosen squash contigs using the ClustalW multiple alignmentmethod (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Results and Discussion

In order to genotype and distinguish the main Greek summersquash landraces, we evaluated six different gene-based EST–SSR markers. A total of 36 HRM profiles were revealed by thesix primer pairs used. For all the primer pairs, detection sensi-tivity and reproducibility tests have been confirmed by repli-cated DNA samples. Polymorphisms within the 36 summersquash landraces were detected based on the pattern oftemperature-shifted curves and difference plot (Figs. 1 and2a, b).

Analysis of conventional melting curves does not allow thesufficient discrimination of the different genotypes, as it usesonly the Tm values. On the contrary, the potential resolvingpower of HRM is much greater than conventional meltingcurve analysis. Figure 1 depicts the normalized HRMmeltingcurves of nine representative summer squash landraces, usingthe EST–SSR marker CUTC017708 (we only show theunique HRM genotypes). Using the shape of the meltingcurves, we could reveal the differences between the varietiesunder investigation and show that all varieties used could beeasily distinguished visually by their melting curves, for ex-ample in "K161/06" and "SAS10/07". The results with theother markers used were similar, showing a clear discrimina-tion of most of the landraces used (data not shown).

Figure 2b depicts the difference graph produced withCUTC002749 of a representative set of seven unique summersquash landraces as compared to "T373/06" genotype used asthe baseline. This closer examination of the HRM differencecurve presented in Fig. 2b, with the mean "T373/06" curve asthe baseline, revealed part of the curve sitting outside the 90 %CI curve, suggesting that all the examined varieties, via theHRM curves produced using the CUTC002749, are indeeddifferent. As shown in Fig. 3, we were able to estimate theconfidence value of similarity between "T373/06" and the othersummer squash landraces used in the study and show thatCUTC002749 was a sufficient EST–SSRmarker to distinguishmost of the 36 common squash landraces used in the study (forsimplicity reasons we show only seven landraces in the figure,HRM profile with all 36 landraces shown in Fig. S2). Thisestimation was further verified by assigning "T373/06" landra-ce as a reference genotype and by subtracting the area (differ-ence graph of "T373/06") from the rest of the produced meltingcurves by the other landraces. The resulting UPGMA dendro-gram, which combines the results produced by the six SSR

Fig. 1 HRM profiles of thegenotypes analyzed with themarker CUTC017708. HRMmelting profiles showing ninedistinct genotypes in normalizedmelting plot

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markers on the 36 C. pepo landraces, shows that the 36 Greeksummer squash landraces could be clearly discriminated usingthe six EST–SSR markers, when merged with the HRM anal-ysis (Fig. 3).

Assigning Possible Roles to the EST-Based SSRs,by BLASTx Analysis

A comprehensive description of the EST–SSRs and theirprimer sequences, as well as detailed information for theresults of PCR and polymorphism evaluation are given inTable 2.

Specifically, six EST-based SSRs were used in the presentstudy for identifying and distinguishing the squash landraces.Three of the SSRs (CUTC005800, CUTC011336 andCUTC046645) are found inside the corresponding coding

region of the gene, while the other three (those of the ESTsCUTC002749, CUTC009607 and CUTC017708) are 3′ UTRbased SSRs. CUTC002749 and CUTC017708 are ESTs of thesame squash transcript which code for a protein of thenucleobase-ascorbate transporter family (NAT), also callednucleobase cation symporter family (NCS2). The NCS2 familycontains proteins that belong to the symporter family, whichcould be subdivided to three groups according to the substratespecificity. Thus, substrates (and the respective groups) areoxidized purines — uric acid/xanthine — and nucleobase —uracil-H+. These symporters are found in bacteria, fungi andplants. A subfamily, of mammalian NATs that transfer L-ascor-bic acid is also included in NCS2 family (Diallinas andGournas 2008). Xanthine and uric acid are important nitrogensources for plants (Brychkova et al. 2008; Nakagawa et al.2007). Arabidopsis thaliana possesses 12 NAT genes, but for

Fig. 2 HRM analysis ofrepresentative summer squashgenotypes with EST–SSR markerCUTC002749. a NormalizedHRM melting curves for sixunique summer squash landracesthat are using HRM analysis. bRepresentative profiles of themelting curves (difference plotcurves) of EST-CUTC002749amplicons for summer squashlandraces. Difference graph of sixunique genotypes using "T373/06" as reference genotype

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none of these has there been assigned a role (Maurino et al.2006). CUTC002749 and CUTC017708 encode a 528-aa pro-tein that shares a high degree of similarity with NAT6 proteinfrom cucumber (92 % similarity) and NAT6 from A. thaliana(83 % similarity). The only plant NAT gene with knownfunction is the Leaf Permease 1 (Lpe1) gene of maize, whichis essential for the chloroplast structure development.Moreover, its expression in a purine transfer-defective fungusrescues the mutation thus restoring the uric acid/xanthine trans-fer (Argyrou et al. 2001; Schultes et al. 1996). The SSRs basedon the ESTs CUTC002749 and CUTC017708 are found on the3′ UTR. The SSR with the motif –TGC– (EST CUTC002749)

is common between cucumber, watermelon and squash(Fig. S1) meaning that this type of SSRs might be transferableto these species. A SSR that is included in the predicted codingregion, is SSR with the motif –ATC– based on the ESTCUTC046645. This EST encodes part of a protein that sharessimilarity with the acyl-CoA-binding domain-containing 2-likeprotein (ACBP) from cucumber and ACBP2 from A. thaliana(GenBank: AAG46057.1). The cucumber SSR motif codes fora series of nine aspartic acids (D) before the ACB domain(Fig. 4). AtACBP2 is one of the six ACBP proteins of A.thaliana which belongs to the Class I subfamily that containsendoplasmatic reticulum (ER) and plasma-membrane-based

Fig. 3 Dendrogram showing thegenetic relationships among 36summer squash landraces asrevealed by the EST–SSRmarkers

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proteins (Li and Chye 2003; Xiao and Chye 2011). AtACBP2and AtACBP1 proteins have been shown to be involved inseed/embryo growth of A. thaliana (Chen et al. 2010), in stressresponses to heavy metals and oxidative factors (Gao et al.2009; Xiao et al. 2008) as well as in biotic stress defense (Liand Chye 2004); thus, they could be very important in abreeding effort to develop stress tolerant plants.

Another SSR is present in the coding region of ESTCUTC005800. This EST encodes a 280-aa protein that sharessimilarity with a C. sativus uncharacterized protein of 291-aaand Arabidopsis MYB12 protein (GenBank: ABB03913). Allproteins have a Myb DNA binding domain (Pfam: PF00249)in their N′ terminus. As shown by the alignment in Fig. 3, thededuced amino acid sequence of EST CUTC005800 has twoadjacent R repeats similar to AtMYB12 (R2R3 motifs) thatconstitute the Myb domain, a finding that shows that thecucurbita sequence is probably a R2R3-type MYB transcrip-tion factor. The SSR codes for a series of glutamic acids (E)after the Myb domain (Fig. 5). AtMYB12 (AT2G47460) ispredicted to be targeted by ath-miR858 of A. thaliana inpositions 291–310 of the mRNA. This area is highly similarbetween AtMYB12 gene and CUTC005800. However, amiRBase search for predicted miRNAs targets onCUTC005800 upon similarity shows that the cucurbita ESTcould be targeted by miR828s of species like Vitis vinifera ,Salvia sclarea , Glycine max , Populus trichocarpa , Malusdomestica , and Cucumis melo (Fig. 6), suggesting that possi-bly the MYB transcription factor encoded by CUTC005800can be a target of post-transcriptional regulation like manyplant MYB factors. AtMYB12 controls the expression of twofundamental genes involved in the production of plant flavo-noids, Chalcone synthase (CHS) and Flavonol synthase(FLS), by binding to promoter elements (Mehrtens et al.2005). Flavonoids serve not only for flower coloring but alsoas protectors against UV radiation and insects, as well as insignaling (Harborne and Williams 2000). When AtMYB12was over-expressed in tobacco, plants were producing moreflavonols, and at the same time many other genes linked toplant's stress protection were induced: tobacco plants wereable to defend against insects like Spodoptera litura andHelicoverpa armigera because of the high production ofrutin, a flavonoid glycosidase (Misra et al. 2010). Thus, ESTSSR CUTC005800 might contribute to the estimation ofbiotic or abiotic stress tolerant plant genotype which mightbe useful in a breeding effort.

EST CUTC011336 contains an SSR inside its predictedcoding region. The EST codes for a protein that resembles anuncharacterized protein from C. sativus . The cucurbita pre-dicted protein contains, according to Pfam, a RING fingerdomain of zing-finger type that binds zinc atoms. The zn-RING_2 (Pfam: PF13639) domain of cucurbita EST has aseries of cysteine (C) and histidine (H) amino acids that followthe C-X2-C-X[9–39]-C-X[1–3]-H-X[2–3]-C-X2-C-X[4–48]-T

able2

Characteristicsof

theEST–S

SRsselected

forgenotyping

thesummer

squash

collection

CucurbitaunigeneID

Expected

size

(bp)

HRM

genotypes

TopGenBankannotatedhit

Protein

C.sativus

hit

Watermelon

EST

hit

CUTC002749

183

7NCBI:XP_

004138122

Nucleobase-ascorbatetransporter6-lik

e[C.sativus]

Protein

xanthine-uracilp

ermease

family

(Pfam:00860)

CGDUnigene:C

U108113

CGDUnigene:W

MU39548

CUTC005800

a189

6NCBI:XP_

004150080

Uncharacterized

[C.sativus]

Myb-likeDNA-binding

(Pfam:

PF00249)

CGDUnigene:C

U104714

–

CUTC009607

146

5NCBI:XP_

004164933.1

Uncharacterized

[C.sativus]

Uknow

nfunctio

nCGDUnigene:C

U100137,

CU132453,C

U134113,

CU084595

–

CUTC011336*

149

5NCBI:XP_

004137212.1

Uncharacterized

[C.sativus]

ZingFinger,C

3HC4type

(ring

finger)(Pfam:P

F13639)

CGDUnigene:C

U115737,

CU111681,C

U14180

CGDUnigene:W

MU4570

CUTC017708

223

9NCBI:XP_

004138122

Nucleobase-ascorbatetransporter6-lik

e[C.sativus]

proteinXanthine-Uracilp

ermease

family

(Pfam:00860)

CGDUnigene:C

U108113

CGDUnigene:W

MU39548

CUTC046645

a194

4NCBI:XP_

004145936acyl-CoA

-binding

domain-containing

protein2-lik

e[C.sativus]

AcylC

oAbindingprotein(Pfam:

PF00887)

CGDUnigene:C

U122405,

CU099794,C

U087958

GDUnigene:W

MU73472

aSSRsin

thecoding

region

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C-X2-C motif (Borden and Freemont 1996). The domain isfound after the SSR coding glutamic acids. Finally, ESTCUTC009607 predicted amino acid sequence although highlysimilar to uncharacterized proteins fromC. sativus , V. vinifera(NCBI: XP_002285729), G. max (GenBank: ACU24472.1),does not possess a known Pfam domain.

The developed SSR molecular markers based on genesimplicated in plants stress responses, such as the ESTsencoding for acyl-CoA-binding domain-containing proteinand a Myb-domain protein, could be potentially useful foridentifying SSR alleles with mutations that could control dif-ferentially the expression of these genes. SSR markers like theSSRs based on cucurbita ESTs CUTC046645, CUTC005800and CUTC011336, since they are located inside coding regionswhich are conserved among species, could be used to identify

"real" genetic diversity as opposed to genetic diversity identi-fied by genetic DNA markers on non-coding genomic se-quences that are not conserved (Gupta et al. 2003). The SSRbased on EST CUTC005800 is cucurbita-specific and thusabsent from other Cucurbitaceae species, showing an exampleof how such a marker can be used for distinguishing speciesthat belong to the same family based on a polymorphism insidea functional gene. If the polymorphism alters the function of thegene is something that requires further experimentation.Supposing the alteration might give a desirable phenotype itcould have significant implications for marker-assisted selec-tion (MAS) and breeding in cucurbitaceae. Another SSR usedin the present study, based on EST CUTC002749, is commonamong cucurbita, watermelon and cucumber (Fig. S1). Theability of transferring SSRs across species, a more frequent

Fig. 4 Alignment of AtACBP2 protein from Arabidopsis thaliana ,cucumber ACBP2 (predicted protein) and the predicted protein sequencethat is encoded by the ESTCUTC046645. The nine aspartic acids that areencoded by the SSR motif based on the cucurbita EST are highlighted in

blue. A thick black line underlines the highly conserved ACB domain(Pfam: PF00887), one of the two domains that ACBP Class I proteinspossess, that bind long-chain acyl-CoA esters in vitro

Fig. 5 Part of the alignment of the AtMYB12 protein from A. thalianaand the predicted protein sequence encoded by the cucurbita ESTCUTC005800 sequence. The two adjacent R repeats (R2R3) of theMyb Domain of AtMYB12 are boxed and the conserved tryptophan

amino acids inside these repeats are colored yellow. The alignment showsthe high conservation of amino acids inside the Myb domain while theother protein part is highly unconserved something that was noticed in thepast (Dubos et al. 2010)

Plant Mol Biol Rep

characteristic of the EST-based SSRs in comparison to thetraditional gemonic SSRs, has been proven useful in plants(Pashley et al. 2006; Varshney et al. 2005).

The present study produced HRM profiles in a populationof 36 summer squash landraces, which covers a wide range ofgenotypes, using an array of six EST–SSR markers selectedfrom the study of (Blanca et al. 2011). The results presentedhere showed a clear separation of the landraces by thesemarkers. This suggests that this panel of EST–SSR markerscould be applied in order to identify different varieties andlandraces using theHRM approachwithout the requirement ofpost PCR procedure, as has been required in traditional mi-crosatellite analysis. Therefore, the information generatedhere could be used in other biodiversity and breeding pro-grams by the international summer squash community.Moreover, HRM analysis of EST–SSR markers adapted frompublications or developed de novo could have wider use in thefields of plant species biodiversity analysis, genetic mappingand breeding programs.

There are numerous advantages of the HRM method inscoring SNPs/microsatellites in comparison to existing sys-tems which are based on high-resolution gel or capillaryelectrophoresis (Reed et al. 2007). Any SNP or length poly-morphism giving rise to a melt polymorphism can be scoredwithout characterization, thus there is not a requirement nei-ther to identify the polymorphic position nor to identity theSNP/microsatellite. Moreover, there is no need for labeledprimers, and the method is a closed tube one, performed inthe same apparatus soon after the completion of the PCRreaction. The HRM analysis instrument collects more datapoints during the assays, which increases the overall through-put of the method. It is also important to mention that, becausethe melting curves shapes depend not only to amplified sizefragments but also to the base composition and SNP position,the method is more sensitive and precise in distinguishingclosely related genotypes, such as cultivars of the same spe-cies, compared to conventional methods (Madesis et al. 2013).

In this study, we showed that the combination of well-selected gene based EST–SSR markers combined with

HRM analysis could provide a fast, accurate and close-tubedway for summer squash identification with reduce genotypingcosts which could be used in current breeding programs.

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Fig. 6 Alignment of miR828s from diverse plant species and thecucurbita EST CUTC005800 sequence. The miR828s have only twomismatches with the EST sequence something that shows that possiblya similar or identical miR828 targets the EST for its post-transcriptional

control. The miRNA sequences have been reverse transcribed for clarity.The miR828 targets the EST from position 502 to position 523, 191 bpbefore the SSR position

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