Mapping and Introgression of QTL Involved in Fruit Shape Transgressive Segregation into 'Piel de Sapo' Melon (Cucucumis melo L.)

Mapping and Introgression of QTL Involved in Fruit Shape Transgressive Segregation into ‘Piel de Sapo’ Melon ( Cucucumis melo L.) Aurora Dı ´az 1 *, Belkacem Zarouri 2 , Mohamed Fergany 3¤ , Iban Eduardo 3 , Jose ´M. A ´lvarez 4 , Bele ´n Pico ´ 5 , Antonio J. Monforte 1 * 1Instituto de Biologı ´a Molecular y Celular de Plantas (IBMCP). Universidad Polite ´cnica de Valencia (UPV)-Consejo Superior de Investigaciones Cientı ´ficas (CSIC), Valencia, Spain, 2Laboratorio de Biologı ´a Molecular. Dpto. Investigacio ´n Agroalimentaria. Instituto Madrilen ˜o de Investigacio ´n y Desarrollo Rural, Agrario y Alimentario (IMIDRA), Alcala ´de Henares, Madrid, Spain, 3Centre de Recerca en Agrigeno `mica (CRAG), IRTA-CSIC-UAB, Bellaterra, Cerdanyola del Valle `s, Barcelona, Spain, 4Centro de Investigacio ´n y Tecnologı ´a Agroalimentaria de Arago ´n (CITA), Zaragoza, Spain, 5COMAV-UPV, Institute for the Conservation and Breeding of Agricultural Biodiversity, Universitad Polite ´cnica de Valencia, Valencia, Spain Abstract A mapping F 2 population from the cross ‘Piel de Sapo’ 6PI124112 was selectively genotyped to study the genetic control of morphological fruit traits by QTL (Quantitative Trait Loci) analysis. Ten QTL were identified, five for FL (Fruit Length), two for FD (Fruit Diameter) and three for FS (Fruit Shape). At least one robust QTL per character was found, flqs8.1 (LOD = 16.85, R 2 = 34%), fdqs12.1 (LOD = 3.47, R 2 = 11%) and fsqs8.1 (LOD = 14.85, R 2 = 41%). flqs2.1 and fsqs2.1 cosegregate with gene a (andromonoecious), responsible for flower sex determination and with pleiotropic effects on FS. They display a positive additive effect (a) value, so the PI124112 allele causes an increase in FL and FS, producing more elongated fruits. Conversely, the negative avalue for flqs8.1 and fsqs8.1 indicates a decrease in FL and FS, what results in rounder fruits, even if PI124112 produces very elongated melons. This is explained by a significant epistatic interaction between fsqs2.1 and fsqs8.1, where the effects of the alleles at locus aare attenuated by the additive PI124112 allele at fsqs8.1. Roundest fruits are produced by homozygous for PI124112 at fsqs8.1 that do not carry any dominant A allele at locus a(PiPiaa). A significant interaction between fsqs8.1 and fsqs12.1 was also detected, with the alleles at fsqs12.1 producing more elongated fruits. fsqs8.1 seems to be allelic to QTL discovered in other populations where the exotic alleles produce elongated fruits. This model has been validated in assays with backcross lines along 3 years and ultimately obtaining a fsqs8.1-NIL (Near Isogenic Line) in ‘Piel de Sapo’ background which yields round melons. Citation: Dı ´az A, Zarouri B, Fergany M, Eduardo I, A ´lvarez JM, et al. (2014) Mapping and Introgression of QTL Involved in Fruit Shape Transgressive Segregation into ‘Piel de Sapo’ Melon (Cucucumis melo L.). PLoS ONE 9(8): e104188. doi:10.1371/journal.pone.0104188 Editor: Rongling Wu, Pennsylvania State University, United States of America Received February 4, 2014; Accepted July 10, 2014; Published August 15, 2014 Copyright: ß2014 Dı ´az et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants AGL2009-12698-C02-02 and AGL2012-40130-C02-02 from the Spanish Ministry of Economy and Competitiveness to AJM. AD was supported by a JAE-Doc contract from CSIC, MF by a Postdoctoral contract from GRAG, IE by a fellowship from the former Spanish Ministry of Education and BZ by a fellowship from Instituto Agrono ´mico Mediterra ´neo de Zaragoza (IAMZ), Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected]s (AD); amonfor[email protected] (AJM) ¤ Current address: Agronomy Department. Faculty of Agriculture Ain Shams University, Cairo, Egypt Introduction Melon (Cucumis melo L., 2n = 2x = 24) is one of the most important horticultural crops, ranking in the ninth position of the world production of vegetables, with more than 27 million tons in 2012 [1]. In the last two decades, melon production has experienced a 29% rise, what has been accompanied by an increase of 8% in the area harvested. It does not seem feasible to achieve the same gain in production in the next decades by increasing the land dedicated to its cultivation, so the efforts of plant breeders should be directed to improve traits having a great potential to increase yield and fruit quality. In this sense, fruit morphology can be considered a quality trait as it is one of the first features to be perceived. Although consumers associate particular sizes and forms to certain market classes (i.e. medium size and round cantaloupes or large and oval inodorus), a whole world of possibilities is being opened up before us with the development of products with original appearance, either by traditional breeding, like the ZESPRI GOLD kiwifruit cultivar [2], or by genetic engineering, like the purple tomato [3]. Modern market trends demand smaller melons, as the family size is progressively falling; and with rounder shapes, what minimizes costs and mechanical damages during storage and transport. Melon germplasm shows an impressive array of fruit sizes and morphologies [4–6], ranging from a few tens of grams up to 10 Kg and from slightly flat to extremely elongated shapes. Most of this diversity is found in traditional Central Asian landraces and wild melons [7–8]. This gene pool is considered ‘‘exotic’’ when compared to the popular Occidental varieties (i. e. Galias, honeydews, western shippers, cantaloups, and inodorus like ‘Piel de Sapo’, from here on referred to as ‘PS’) although it represents a valuable source of variability with a huge potential in breeding programs. In this sense, the accession PI124112 (C. melo ssp. PLOS ONE | www.plosone.org 1 August 2014 | Volume 9 | Issue 8 | e104188 agrestis var. momordica) collected in 1937 in India is genetically very distant from commercial cultivars, as revealed by studies carried out with SSR (Simple Sequence Repeat) [5], [9], and AFPL (Amplified Fragment Length Polymorphism) and SNP (Single Nucleotide Polymorphism) [10] markers, and shows resistance to several fungal and viral diseases (reviewed in [7]). A few major genes with pleiotropic effects on melon fruit shape (FS) are currently known, like andromonoecious (a), that has been recently cloned [11] and is responsible for sex determination together with gynoecious (g), and like pentamerous (p), that controls carpel number. However, most of the phenotypic variation observed in FS seems to be caused by other Quantitative Trait Loci (QTL), as demonstrated by a number of QTL mapping studies that have been undertaken to genetically dissect its molecular basis [12–18]. Genotyping technologies have experienced an authentic revolution in the last few years [19–20], what has led to a renewed interest in QTL mapping. The availability of affordable highthroughput genotyping platforms will enable researchers to annotate more precisely the genomic regions of interest and speed up drastically the formerly time-consuming process of obtaining valuable plant material. All this will make possible to validate QTL and study putative interactions among them, at the same time that favorable alleles are transferred into elite varieties with a minimal drag of non-targeted fragments with potential detrimental effects. Consequently, it is expected that in a near future, new QTL will be added to those already cloned in important crops, like the QTL for fruit sugar content Brix9-2-5 [21], for fruit size fw2.2 [22], and those for fruit shape ovate [23], fasciated [24] and sun [25] in tomato; a QTL for grain shattering sh4 [26] and those for flowering date Hd1 [27], Hd6 [28], Hd3a [29], and Ehd1 [30] in rice; and the QTL for plant architecture Tb1 [31] and flowering time Dwarf8 [32] and Vgt1 [33] in maize, among others (reviewed in [34–35]). In most cases, the strategy followed was the positional cloning, for what the availability of Near-Isogenic Lines (NILs) was essential, highlighting the importance of generating the appropriate genetic material to be successful. In this sense, the development of a collection of NILs in melon between the Korean cultivar ‘Songwhan Charmi’ and ‘PS’ [36] has allowed to dissect in single Mendelian factors economically important traits as fruit morphology [14], [17], climacteric ripening [37] and virus resistance [38]. In the present work, we identify and localize several QTL associated with melon fruit morphological traits in a cross between the Spanish cultivar ‘PS’ and the Indian accession PI124112 and study the interaction between those with the strongest and most robust effects. Furthermore, we have validated the QTL for FS Figure 1. Fruit Shape (FS) polymorphism at each stage of the crossing program to obtain the fsqs8.1 -NIL. Fruits of (A) ‘PS’; (B) PI124112; (C) 2M158-3 (F 1 ); (D) different F 2 plants to show the transgressive segregation of FS; (E) 3M70-47 (F 2 ); (F) 5M113-1 6‘PS’ (BC1); (G) 6M59-13 6‘PS’ (BC2); (H) 7M36-1 6‘PS’ (BC3); (I) 8M42-24 6‘PS’ (BC4); (J) 9M7-15 : (BC4S1); (K) 10M2-30 6‘PS’ (BC5); (L) 11M27 : (BC5S1), (L.1) 11M27-20: heterozygous at fsqs8.1 and (L.2) 11M27-11: homozygous at fsqs8.1; (M) the fsqs8.1-NIL 12M57-3 OP (BC5S2). doi:10.1371/journal.pone.0104188.g001 Mapping and Introgression of Melon Fruit Shape QTL PLOS ONE | www.plosone.org 2 August 2014 | Volume 9 | Issue 8 | e104188 fsqs8.1 in a QTL-NIL developed in the genetic background of the cultivar ‘PS’, what produces totally round melons instead of the typical oval ones. Materials and Methods All experiments were conducted in compliance with the current Spanish laws. No specific permissions were required for the experiments carried out in these two locations, Cabrils and Zaragoza, as they are research institutes, Institut de Recerca i Tecnologia Agroalimentaria (IRTA) and Centro de Investigacio´n y Tecnologı ´a Agroalimentaria de Arago´n (CITA), respectively. Moreover, authors IE and JMA belong to IRTA and CITA, respectively. Plant material and phenotypic evaluation The F 2 plants of the mapping population were derived from a single cross between the monoecious Indian landrace accession PI124112 (momordica group) and the andromonoecious Spanish cultivar ‘PS’ (inodorus group) (Figure 1). PI124112 typically produces medium size, elongated and non-sweet fruits, whereas fruits coming from ‘PS’ are big, oval-shaped and very sweet [4–5]. A multi-location assay was conducted in summer of 2003 with a total of approximately 400 F 2 plants. On one hand, 200 of them were randomly distributed in a greenhouse at IRTA in Cabrils (Barcelona, Spain), grown in drip-irrigated peat bags and selfpollinated by hand. The remaining 200 plants were randomized in an open field without irrigation at CITA, located in Zaragoza (Spain) and allowed to open-pollinate. The fertilization method became irrelevant as all the fruit except the seed content is derived from the maternal genitor. Ten plants of every genitor plus the F 1 were included in both trials. Fruit length (FL) and maximum fruit diameter (FD) were measured on longitudinal sections of collected fruits and then, FS was calculated as the ratio of FL over FD. The absence of statistically significant genotype-by-environment (G6E) interactions was tested by calculating the least square means of FS in a two-way ANOVA with the data coming from six to nine replicates of F1, PI124112 and ‘PS’ in Cabrils and Zaragoza as a necessary condition to merge the data coming from the two locations. Pair-wise Pearson’s correlation coefficient was calculated between the three traits with the data coming from the whole F 2 population and also with those obtained in the two location assays (Cabrils and Zaragoza) independently. Percent mid-parental heterosis (MPH) was calculated using the data derived from a total of 15 replicates of each of the genotypes, F 1 and the both parents, as: MPH~2F1{P1{P2  =P1zP2  |100 where F1,P1and P2are the mean values of the F 1 , ‘PS’ and PI124112, respectively, for the trait under study. One-way ANOVA and t-Student mean comparison tests were carried out with the data coming from the whole F 2 population and also with those obtained in the two location assays independently. All statistical analyses were performed using JMP v5.1.2 software for Windows (SAS Institute Inc., Cary, NC). DNA marker analysis in the mapping population A selective genotyping strategy [39], which exploits maximum phenotypic differences with a minimal labor and time investment, was approached to study the FS trait. The plants selected to be genotyped were those with values more extreme than the mean of the F 2 population 61SD from each location, resulting in 84 individuals from both locations (File S1). DNA was isolated from young leaves according to [40], with slight modifications and genotyped with a total of 133 markers (66 SSRs, 65 SNPs and 2 Cleaved Amplified Polymorphic Sites, CAPS) chosen to cover all the genome among those available in the literature (File S2). SSR markers were amplified following the protocol with M13-tailed forward primers plus dye labeled M13 primers described by [41] and subsequently analyzed on a 3130 Genetic Analyzer (Applied Biosystems). Genotyping with SNP markers was carried out using the Sequenom MassArray iPLEX system (Illumina), followed by MALDI-TOF mass spectrometry at Servicio de Investigaciones Biome´dicas, Unidad Central de Investigacio´n (University of Valencia, Valencia, Spain). Amplifications of the CAPS markers, AluICAPS [11] and PSI_25-H03 [42], were performed in 25-ml volume solutions containing 20 ng of genomic DNA, 7.5 mM Tris HCl (pH 9.0), 5 mM KCl, 2 mM (NH 4 ) 2 SO 4 , 2.5 mM MgCl 2 , 0.2 mM of each dNTP, 0.2 mMof reverse and forward primers and 2.5 U of DNA polymerase (Biotools, Madrid, Spain). PCRs were carried out on a TC-512 thermocycler (Techne, UK) programmed with an initial denaturation step at 94uC for 5 min, followed by 35 cycles of 94uC for 30 s, 62uC for 45 s and 72uC for 1 min, plus a final elongation step at 72uC for 5 min. A 10-ml volume of the AluICAPS and PSI_25-H03 PCR products were digested with the restriction enzymes AluI and Bsh1236I (Fermentas, Fisher Scientific, Madrid, Spain), respectively, following the supplier’s recommendations and separated by agarose gel-electrophoresis. Linkage map construction and QTL analysis The linkage map was constructed using MAPMAKER 3.0 [43] and the distances were calculated with the Kosambi function [44]. Markers were associated with the group command with LOD. 3.0. When possible, they were put in order within each group using the order command with LOD.3.0, followed by a verification with the ripple command and the building of the framework map. If necessary, remaining markers in the group were included with the place command. If the previous approach did not render any map, the compare function was used to determine the best possible order for five starting markers, adding the remaining ones with the try command. In the case of groups with a low number of markers, the order and distances were estimated using the group command, followed by compare, what generated the framework map. Finally, maps were drawn with MapChart version 2.2 [45]. QTL analysis was performed for each location independently and for data from both locations merged together using Windows QTL Cartographer 2.5 [46] with the composite interval mapping (CIM, [47]) procedure. The LOD score threshold for a significant level p,0.05 was calculated by a permutation test with 1000 resamplings, giving a value of LOD.2.5 for the whole population for all traits and larger numbers in the individual populations. Additionally, the threshold LOD.2.5 was also established for the single location analyses. Markers associated to the most robust QTL (6 out of the 10 QTL as several of them share the same marker as the strongest linked one, i.e. flqs8.1 and fsqs8.1) were genotyped in all F 2 plants in order to obtain unbiased estimates of the gene action (d/[a]). The QTL were named according to the nomenclature system adopted by [48], where the first letters Mapping and Introgression of Melon Fruit Shape QTL PLOS ONE | www.plosone.org 3 August 2014 | Volume 9 | Issue 8 | e104188 designate the trait abbreviation, followed by a ‘‘Q’’ that stands for QTL, then a letter referred to the mapping experiment (‘‘s’’, in this case), followed by a digit representing the linkage group (LG) to which the QTL maps, and then followed by a dot and a final digit that distinguishes different QTL from the same experiment on the same LG. In those cases in which two overlapping QTL were identified in a limited region and for the same trait, they were effectively considered as only one QTL. Study of QTL interactions Epistatic interactions between the FS QTL detected were investigated by two-way ANOVA with the JMP v5.1.2 software for Windows and using the genotypic data from the linked markers in all F 2 plants (not only the subset employed for the selective genotyping). These markers were AluICAPS in the case of fsqs2.1, CMAT141 for fsqs8.1, and ECM 67 for fsqs12.1. Interactions were considered to be statistically significant when p#0.05. Sum of squares values were used to calculate the percentage of the phenotypic variation explained by the interaction between two QTL (R 2 ). QTL validation by developing a QTL-NIL The strategy followed to validate one of the FS QTL detected and to introgress it into the ‘PS’ elite cultivar consisted in selecting a single F 2 plant from the trial located in Cabrils, that carried the PI124112 alleles at the QTL region and also showed round fruit, and backcross it to the parental ‘PS’ (Figure S1). Five rounds of backcrossing guided by a doubled selection, phenotypic (favoring round-shaped fruits) and assisted by markers linked to the target QTL (to monitor the introgressions harboring PI124112 alleles), were carried out. Simultaneously, background markers covering all the genome were also employed to gradually increase the percentage of the recurrent parent genome by selecting against the donor (PI124112) genome. An advanced backcross selection, 10M2-30, was genotyped with 548 SNPs using the Golden-Gate assay (Illumina, San Diego, CA) described in [10] in order to verify introgression size and integrity, and the composition of the genomic background. After two cycles of self-fertilization, the definitive introgression line in the BC5S2 generation was obtained (Figure 1M). Phenotypical evaluations of the fruits were conducted in the greenhouse at several stages of the backcross scheme. In 2008, 42 fruits collected from 37 plants belonging to three full-siblings BC4 families (8M42-37, 41 and 42) carrying the PI124112 allele for the FS QTL in LG VIII (homozygous and heterozygous for two linked markers placed 23.3 cM apart in the genetic map) were evaluated in Cabrils. In 2009, BC4S1 plants (Figure S1) were genotyped with the markers linked to the QTL and 30 of their fruits were phenotyped in a greenhouse at Polytechnic University of Valencia. The cosegregation of markers and FS phenotypes was assessed by ANOVA. Finally, in 2012, melon fruits from the BC5S2 family 12M57 and the parental cultivar ‘PS’ were analyzed in Paiporta (Valencia). 12M57, derived from a homozygous plant for the closest markers to the FS QTL fsqs8.1 (11M27-11, Figure S1), contained the introgression for the PI124112 allele at those same markers linked to the QTL. Means of the fruits produced by 10 12M57 plants were compared with means of the fruits collected from 15 ‘PS’ plants by a t-test. Results Phenotypic variation in morphological traits Cultivar ‘PS’ produces oval-shaped fruits (FS = 1.460.1; Table 1 and Figure 1A), while PI124112 displays elongated fruits (FS = 1.760.2; Table 1 and Figure 1B). The F 1 exhibited higher FS values (FS = 2.060.2, Table 1 and Figure 1C), in accordance with best-parent heterosis for this character previously observed in melon [16], particularly in hybrids between exotic accessions and ‘PS’ (inodorus group), caused mainly by an increase in FL [5]. No significant G6E interaction was observed for FS, therefore the parental mean values were calculated with the data coming from both locations. In Cabrils, the MPH values were 77%, 10% and 60% for FL, FD and FS, respectively; in the assay located in Zaragoza, 120%, 34% and 68%, respectively; and for the whole set of data, 100%, 24% and 64%, respectively. In all cases, heterosis was confirmed and, though there are slight differences in the MPH values for each character among assays, the ranking remains unaltered. A continuous distribution of values was observed in the F 2 (Figure 2), as expected in a quantitative trait like FS, with values ranging from 0.86 (oblate fruits) to 3.23 (extremely elongated fruits), making clear a transgressive segregation for this character in this population (Figure 1D and Figure 2C). Transgressive segregation was also observed for FL. In both cases, it was observed in both directions in the whole F 2 population (Table 1 and Figure 2), although the phenotypes where more extreme for the higher values, that means fruits with a more elongated shape. In all cases, individuals with fruits exceeding the mid-parent value 63.84SD were found. The trait showing a broader range of values was FL (from 7.1 to 38 cm), displaying extreme values corresponding to 26.56 and +13.52 times the SD of the mid-parental FL. Conversely, FD showed the lowest variation (from 7.30 to 17.70 cm), what represents the mid-parent FD value 23.84SD and +5.55SD, respectively. In between these two cases, the FS values ranged from 24.31SD and +11.23SD the mid-parent values, respectively. FL and FS showed a highly significant (p,0.001) and strong correlation with values for the Pearson’s correlation coefficient of 0.84 for the whole population, and 0.86 and 0.83 for the subpopulations of Cabrils and Zaragoza, respectively. In contrast, FD and FS were not significantly correlated in any case. Linkage map construction One hundred and twenty-two out of the 133 total markers (63 SNPs, 57 SSRs and 2 CAPS) used to genotype the F 2 mapping population could be incorporated to a genetic map consisting in 18 LGs that span 1437.3 cM (Figure 3), what renders a density of 1 marker every 11.8 cM. By comparing with the melon consensus map [48], each LG could be identified, finding that five of them were partial LGs of the integrated map. The map shows an average marker gap of 13.8 cM. QTL associated with fruit morphology traits Given that no G6E interaction was found among locations, data from both locations were merged for subsequent QTL analysis. A total of ten QTL for fruit morphology-related traits were positioned along the melon genetic map (Table 2 and Figure 3) with the selective genotyping strategy, five underlying FL (in LGs II, IIIb, VIa, VIII and Xb), two responsible for FD (in LGs IIIa and XII), and three controlling FS (in LGs II, VIII and XII). LOD score values ranged from 2.80 to 16.85, in the case of fdqs3a.1 and flqs8.1, respectively. The QTL with the highest effect for each of the three characters studied were flqs8.1 (LOD = 16.85, R 2 = 34%), fdqs12.1 (LOD = 3.47, R 2 = 11%) and fsqs8.1 (LOD = 14.85, R 2 = 41%). The remaining QTL accounted for 4 to 17% of the phenotypic variation. LOD peaks of the two major QTL located in LG II, flqs2.1 and fsqs2.1, and in LG VIII, flqs8.1 and fsqs8.1, overlapped completely (Figure S2). Mapping and Introgression of Melon Fruit Shape QTL PLOS ONE | www.plosone.org 4 August 2014 | Volume 9 | Issue 8 | e104188 The interval defined by the markers flanking the QTL ranged from 12.9 (flqs3b.1) to 69.1 (flqs6a.1) cM, being less than 26 cM for five out of the ten QTL (Table 2). The QTL on LG VIII (flqs8.1 and fsqs8.1) were consistently detected in the three analyses (that is, with the merged data and with those coming from individual locations, File S3). The QTL fsqs2.1 was also detected in the three analyses with a LOD = 2.5.The rests of the QTL were identified in two of the analyses, except for flqs10b.1. In all cases, the additive effects sign remained invariable across the different environments. Since fsqs2.1,fsqs8.1 and fsqs12.1 were the QTL most consistently detected among analyses, all plants were genotyped with markers AluICAPS, CMAT141, and ECM67, located in the respective QTL region, in order to obtain unbiased estimates of their effects (Table 2). Same direction effects of the exotic allele (PI124112) for FL and FS QTL with the same chromosomal location were observed. In this sense, flqs2.1 and fsqs2.1 displayed a positive value for the additive effect (a), 3.06 and 0.27, respectively, what means that the PI124112 allele causes an increase in FL and FS, producing more elongated fruits. Conversely, the negative value of afor flqs8.1 and fsqs8.1 (24.95 and 20.50, respectively) is indicative of a decrease in both characters, FL and FS, what results in rounder fruits, even if PI124112 itself produces very elongated melons. In all the remaining QTL, except for fdqs12.1, detected with the data coming from Cabrils and with the whole set (a=21.03 and a= 21.10, respectively), the PI124112 allele contributed to increase the value of the particular morphological trait (Table 2 and File S3). So, the PI124122 allele at fdqs12.1 will render elongated fruits by decreasing their diameters, whereas in the case of flqs2.1 and fsqs2.1, a similar phenotype will be obtained by increasing the length of the fruit. Gene action values ranged from 20.35 and 20.32 (flqs8.1 and fsqs8.1, respectively) to 0.75 (flqs2.1 and fsqs2.1; Table 2). In most cases, the alleles at the QTL loci showed an additive effect, except for flqs2.1 and fsqs2.1, where the inheritance mode is near complete dominance. In general, the gene action values obtained for the same QTL using the subsets of data coming from both locations (File S3) agree with the ones calculated when all the data were pooled together, with slight differences that can be attributed to the smaller sample size of the first analysis. The additive effect at fsqs8.1 is supported by the phenotypic data, as the heterozygous NIL-fsqs8.1 displays a FS value (i.e. 1.24) intermediate between the two homozygotes, the homozygous line for the PI124112 allele (i.e. 1.06) and the ‘PS’ parent (average FS = 1.40). QTL 6QTL interaction In order to detect the putative effects of other genomic regions on each of the FS QTL, all the possible digenic interactions between them were studied although only those that were statistically significant when p#0.05 are shown (Table 3 and Figure 4). Table 1. Mean and Standard Deviation (SD) values of the traits for the parents, the F 1 and the whole F 2 . FL FD FS Mean SD Mean SD Mean SD ‘PS’ 19.5 1.3 14.0 1.1 1.4 0.1 PI124112 14.8 1.8 9.2 1.1 1.6 0.2 F 1 25.1 3.5 12.7 1.8 2.0 0.2 F 2 19.5 5.7 11.7 2.0 1.7 0.5 FL: Fruit Length; FD: Fruit Diameter; FS: Fruit Shape. doi:10.1371/journal.pone.0104188.t001 Figure 2. Frequency distribution of the traits across the F 2 population derived from the cross ‘PS’ 6PI124112. (A) FL (Fruit Length), (B) FD (Fruit Diameter) and (C) FS (Fruit Shape). Both parents and F 1 values are marked. In the upper box, 25 th ,50 th and 75 th quartiles are displayed; the sample mean and the 95% confidence interval are represented by a diamond, and the outlier values as dots. The bracket along the edge of the box stands for the part of the graph in which 50% of the observations are gathered together. Mean and standard deviation estimates for a normal distribution are also shown. doi:10.1371/journal.pone.0104188.g002 Mapping and Introgression of Melon Fruit Shape QTL PLOS ONE | www.plosone.org 5 August 2014 | Volume 9 | Issue 8 | e104188 fsqs2.1 6fsqs8.1 A significant interaction was identified between fsqs2.1 and fsqs8.1 (p = 0.013, Figure 4A, Table 3), that explains a 4% of the genetic variance of FS. flqs2.1 has been previously identified as gene a[12], [49] and the marker linked to it (AluICAPS) is actually the causal mutation of andromonoecy [11]. For that reason and from now on, the alleles at this locus will be referred to as A and a. It is well known that the dominant allele A causes elongated shapes in some genetic backgrounds [12], as becomes clear in the cases of homozygous for PS allele at locus fsqs8.1 (PsPs). For each of the three possible genotypes at locus a (AA, Aa, aa), the lowest Figure 3. Location of 10 QTL responsible for morphological fruit traits mapped in a F 2 population derived from the cross ‘PS’ 6 PI124112. FL: Fruit length, open bars; FD: Fruit Diameter, hatched bars; and FS: Fruit Shape, solid bars. Length of QTL bars corresponds to the twoLOD support intervals (from peak marker) based on the results obtained by CIM using Windows QTL Cartographer 2.5. Numbers on the left side correspond to the distance in cM (according to [44]) from the top of each chromosome. doi:10.1371/journal.pone.0104188.g003 Mapping and Introgression of Melon Fruit Shape QTL PLOS ONE | www.plosone.org 6 August 2014 | Volume 9 | Issue 8 | e104188 Table 2. QTL detected for FL, FD and FS using the merged data from two locations of the F 2 population from the cross ‘PS’ 6PI124112. Trait LG QTL name a Flanking markers Interval size (cM) LOD score a b d b d/[a] b R 2c Homologue QTL ID a Homologue published QTL d Reference e FL II flqs2.1 CMPSNP431-ECM61 38.4 8.44 3.06 2.29 0.75 0.16 flqn2.1 fl2.1 [54] FL IIIb flqs3b.1 ECM205-CMPSNP998 12.9 2.92 +0.05 flqc3.5 3.5 [14] FL VIa flqs6a.1 ECM52-CMTCN41 69.1 9.56 +0.11 flqc6.4 6.4 [14] FL VIII flqs8.1 GCM241-PSI_25-H03 23.3 16.85 24.95 21.71 20.35 0.34 flqc8.3 8.3 [14] flqn8.1 fl8.1 [54] FL Xb flqs10b.1 CMPSNP671-CMPSNP665 31.6 3.02 – 0.04 – – – FD IIIa fdqs3a.1 CMBR100-AI_18-E05 25.6 2.80 – 0.11 – – – FD XII fdqs12.1 ECM67-CMTCN14 52 3.47 21.03 20.17 20.17 0.11 fdqi12.1 fw6.10 [65] FS II fsqs2.1 CMPSNP431-AluICAPS 16.9 6.74 0.27 0.20 0.75 0.17 fsqj2.1 fs2.3 [15] fsqq2.1 fs2.2 [12] fsqn2.1 fsh2.1 [54] FS VIII fsqs8.1 GCM241-PSI_25-H03 23.3 14.85 20.50 20.16 20.32 0.41 fsqc8.3 8.1 in [14] fsqn8.1 fsh8.1 [54] FS XII fsqs12.1 AI_35-A08-CMPSNP361 53.1 3.61 0.15 0.03 0.21 0.03 fsqa12.1 fs11.1 [13] fsqp12.1 fs12.1 [12] fsqc12.1 12.1 [14] FL: Fruit Length; FD: Fruit Diameter; FS: Fruit Shape. a Nomenclature according to [48]. b Only calculated for QTL in which the whole population was genotyped with the strongest linked marker(s); for the rest, only the sign is shown. c When possible, calculated using the data coming the whole population genotyped with the strongest linked marker(s); for the rest, the value obtained by the QTL analysis using the selective genotyping subset of samples is shown. d Nomenclature according to the authors that originally identified the QTL. e Reviewed in [48] and, the FS QTL, in [53]. doi:10.1371/journal.pone.0104188.t002 Mapping and Introgression of Melon Fruit Shape QTL PLOS ONE | www.plosone.org 7 August 2014 | Volume 9 | Issue 8 | e104188 values of FS index (rounder fruits) were always observed in the homozygous for the PI124112 allele at locus fsqs8.1 (PiPi) (Figure 4A). So, when the PI124112 allele is present at fsqs8.1 and the dominant A allele is absent at locus a(PsPiaa and PiPiaa), there is a considerable decrease of FS. In the homozygous PiPi, the differences among the three different genotypes at locus aare the slightest. It seems that homozygous PiPi at fsqs8.1 diminish or, in some way, attenuates the effects of the dominant A allele of gene aon fruit elongation. In the case of heterozygous genotypes at fsqs8.1 (PsPi), some differences can be observed depending on the alleles present at locus a(A_ are expected to produce moderately elongated fruits while aa will rendered more rounded ones). So, we can only see marked differences in the three possible genotypes at gene awhen the PI124112 allele is not present at QTL fsqs8.1 (no masking effect) or it is in heterozygosis, PsPs or PsPi (moderate attenuation of the phenotype caused by A), with FS showing nearly parallel increments when these two scenarios are compared. In both cases, the elongated phenotype caused by the aforementioned dominant A allele becomes clear, as homozygous AA produce the most elongated fruits, followed by the heterozygous (Aa). fsqs8.1 6fsqs12.1 A significant interaction was identified between fsqs8.1 and fsqs12.1 (p = 0.014, Figure 4B, table 3), explaining 7% of the genetic variance of FS. Similarly to what can be observed in the interaction fsqs2.1 6 fsqs8.1, the lowest values of FS (rounder fruits) are obtained when the PI124112 allele is in homozygosis at fsqs8.1 QTL (PiPi) (Figure 4B). In such a case, the phenotypic variations due to the different allelic composition at fsqs12.1 locus are practically imperceptible. Those differences in the FS value become more evident with the presence of the PS allele at fsqs8.1, specifically when it is in homozygosis (PsPs). Interestingly, the highest value of FS (the most elongated fruits) are reached for the PS homozygous at fsqs8.1 locus that are homozygous for the PI124112 at fsqs12.1 (PsPsPiPi), what is totally in line with the positive additive value of fsqs12.1 QTL (the Pi allele causes an increase in the FS index). Here, unlike what happens in the previous interaction, within the same genotype for QTL fsqs8.1, the increase in FS due to the PI124112 allele at fsqs12.1 becomes clear when the PI124112 allele is absent at locus fsqs8.1. QTL validation in the fsqs8.1-NIL The identification and mapping of the fsqs8.1 QTL in the F 2 population has allowed us to use marker-assisted selection to precisely introgress the PI124112 allele responsible for the round shape into the elite cultivar ‘PS’ as the result of a backcross program (Figure S1). To ensure the integrity and maintenance of the QTL effects, two rounds of QTL verification were carried out in intermediate generations. Figure 4. Digenic interactions studied by two-way ANOVA between the FS QTL using the markers significantly linked to them. (A) fsqs8.1 and fsqs2.1 (fsqs2.1 6fsqs8.1); (B) fsqs12.1 (fsqs8.1 6fsqs12.1). Alleles at locus flqs2.1 are named A and a since this QTL has been previously identified as gene a[12], [49]. PsPs: homozygous for the allele PS (solid line); PsPi: heterozygous (dashed line); PiPi: homozygous for the allele PI124112 (dotted line). doi:10.1371/journal.pone.0104188.g004 Table 3. Significant digenic interactions between all the FS QTL. QTL pair Source DF Sum of Squares F Ratio Prob . F fsqs2.1-fsqs8.1 fsqs2.1 (AluICAPS) 2 7.83 35.72 ,0.0001 fsqs8.1 (CMAT141) 2 26.79 122.21 ,0.0001 fsqs2.1 (AluICAPS) 6fsqs8.1 (CMAT141) 4 1.42 3.24 0.0132 * fsqs8.1-fsqs12.1 fsqs8.1 (CMAT141) 2 23.02 82.76 ,0.0001 fsqs12.1 (ECM67) 2 1.59 5.71 0.0038 fsqs8.1 (CMAT141) 6fsqs12.1 (ECM67) 4 1.78 3.19 0.0142 * The interactions were explored by two-way ANOVA using the genotypes for the closest markers in the whole F 2 population derived from the cross ‘PS’ 6PI124112. * : Interactions were considered to be statistically significant when p#0.05. doi:10.1371/journal.pone.0104188.t003 Mapping and Introgression of Melon Fruit Shape QTL PLOS ONE | www.plosone.org 8 August 2014 | Volume 9 | Issue 8 | e104188 In 2008, the effect of the fsqs8.1 was monitored with markers CMAT141 and PSI_25-H03 in BC4 plants (Table 4, 8M-codes in Figure S1). Plants carrying the PI124112 allele in heterozygosis at fsqs8.1 produced significantly rounder melons than plants carrying the PS allele in homozygosis (F = 21.10 and F = 25.94 for CMAT141 and PS_25-H03, respectively, p,0.001). In 2009, the three genotypes at fsqs8.1 (monitored with marker CMAT141 and PSI_25-H03) segregated in a BC4S1 population (9M7, Figure S1), re-confirming the cosegregation of PI124112 alleles at fsqs8.1 with the round fruit phenotype (Table 4; F = 13.75 and p,0.001 for CMAT141; F = 5.71 and p,0.01 for PSI_25-H03). At the next stage, whole-genome genotyping was performed in a selected BC5 plant (10M2-30) in order to check the integrity of the introgressed region and the composition of the genetic background. The target introgression harbored 8.5 Mb in the LG VIII, and there were only seven additional introgressions ranging from 250 kb to 86.8 Mb. Finally, the definitive introgression line was fixed in a BC5S2 family (12M57, Figure S1). The family 12M57 showed highly significant shorter and rounder fruits, that is, lower values of FL and FS, respectively (Table 4; F = 10.27 and F = 105.71, for FL and FS, respectively, and p,0.001). Additionally, significant effects on FD were also observed (F = 10.90, p,0.001, Table 4). Discussion The F 2 population between ‘PS’ and PI124112 showed an impressive variability for fruit morphology. Transgressive segregation in both directions was found for FL and FS, indicating that at least one of the parents contributed alleles with opposite effects and making possible to obtain genotypes that could render round fruits. In fact, the existence of an allele inducing round-shaped melons was identified in the genitor PI124112 and verified with an introgression line in the ‘PS’ background. The effects of this allele seem to be masked by epistatic interactions with other gene(s) in the PI124112 background, and those effects were unveiled in the ‘PS’ background. A similar discovery has been reported recently with the obtaining of a climacteric line derived from the cross between two non-climateric melon accessions, ‘PS’ and ‘Shongwan Charmi’ [37]. This is a further indication of the richness of the melon gene pool that remains still largely unexplored. The segregating population coming from parents genetically distant and with morphological differences used in this work has revealed itself as suitable for the genetic dissection of morphological traits and mapping QTL. Furthermore, the most robust FS (and the highly correlated FL) QTL detected (flqs2.1,fsqs2.1, flqs8.1 and fsqs8.1) were consistent across the locations tested, in agreement with other results published [16], in which QTL affecting FS displayed a high heritability, what explained their reproducibility in assays carried out in different locations and along several years. The availability of a melon consensus map including the position of QTL controlling a panoply of important traits [48] made easier the comparison of the QTL identified here with those previously described. In this sense, most QTL controlling the same characters studied in this work have been detected previously and mapped to similar genomic positions (Table 2). In fact, homologous QTL obtained in different populations can be assigned to all the QTL described here, except in the case of flqs10b.1 (only detected with the full set of data) and fdqs3a.1 (not found in Cabrils). Only a QTL associated to FL was recently reported in the LG II of the related cucurbit Zucchini (Cucurbita pepo L.) in a region syntenic to the LG III of melon at which fdqs3a.1 maps [50]. However, this QTL could not be confirmed in further backcross generations. The QTL in LG II (flqs2.1 and/or fsqs2.1) was previously identified as a pleiotropic effect of the gene a[12], [49], whose alleles are responsible for sex determination in melon flowers [51] as it has been reported in other plant species like Vitis ssp. [52]. The dominant inheritance mode observed for the alleles at flqs2.1 and fsqs2.1 loci also supports it. The cloning of the gene has unveiled that the causal mutation of the andromonoecious melon types is in the active site of 1-aminocyclopropane-1-carboxylic acid synthase [11]. The map position of fsqs8.1 (and flqs8.1, as they overlap) happened to be similar to those reported for other FS QTL (Table 2; reviewed in [48], [53]), fsqc8.3 (8.1 in [14]) and fsqn8.1 (fsh8.1 in [54]). Those QTL were detected in a NIL population derived from the cross ‘PS’ (C. melo var. inodurus)6PI161375 ‘Shongwan Charmi’ (C. melo var. chinensis), and a Recombinant Inbred Line (RIL) population coming from the cross PI414723 (C. melo var. momordica)6‘Dulce’ (C. melo var. reticulatus), respectively. So, fsqs8.1 seems to be allelic to those major QTL underlying fruit morphology in a broad array of melon germplasm. The PI161375 and PI414723 alleles produced elongated fruits. On the contrary, PI124112 allele (in a certain allelic combination in trans with gene avariants) induced round fruits, what may be showing the existence of an allelic series at this QTL when different germplasm sources are investigated. Three more QTL have been reported in the surrounding region (fsqa8.1, fsqn8.1 and fsqc8.1, compiled in [48]) and two additional ones further away in the same chromosome (fsqc8.1 and fsqp8.2, reviewed in [48]). These results suggest that an important part of the loci controlling FS in melon could be concentrated in the LG VIII. It is also possible that some of them are the same QTL that the one described here (harboring common and/or new alleles). To discern between these two cases, higher resolution in the mapping and QTL studies is required. Similar QTL clusters have Table 4. F-values obtained from ANOVA to validate the effects of QTL fsqs8.1 on FS. 2008 2009 2012 CMAT141 PSI_25-H03 CMAT141 PSI_25-H03 CMAT141 FL 5.58 * 8.76 ** 10.10 *** 4.82 * 10.27 *** FD 3.46 ns 2.49 ns 0.53 ns 0.38 ns 10.90 ** FS 21.10 *** 25.94 *** 13.75 *** 5.71 ** 105.71 *** Data coming from the assays carried out along 3 years were used. Plants were genotyped with linked markers (CMAT141 and/or PSI_25-H03). FL: Fruit Length; FD: Fruit Diameter; FS: Fruit Shape. *** P,0.001; ** P,0.01; * P,0.05; ns P.0.05. doi:10.1371/journal.pone.0104188.t004 Mapping and Introgression of Melon Fruit Shape QTL PLOS ONE | www.plosone.org 9 August 2014 | Volume 9 | Issue 8 | e104188