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Construction of a genetic linkage map of rootstock-used pumpkin using SSR markers and QTL analysis for cold tolerance
Scientia Horticulturae 220 (2017) 107–113
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Construction of a genetic linkage map of rootstock-used pumpkin using SSR markers and QTL analysis for cold tolerance Yang Xu, Shi-rong Guo, Sheng Shu, Yan Ren, Jin Sun ∗ College of Horticulture, Nanjing Agricultural University, Key Laboratory of Southern Vegetable Genetic Improvement, Ministry of Agriculture, Nanjing 210095, China
a r t i c l e
i n f o
Article history: Received 23 November 2016 Received in revised form 23 March 2017 Accepted 29 March 2017 Keywords: C. moschata Chilling tolerance SSR markers Linkage map QTL analysis
a b s t r a c t In China, cucumber plants suffer chilling stress during the winter and early spring, and grafting would help the plants adapt to chilling stress. Pumpkin (Cucurbita moschata; 2n = 40) is an important rootstock for cucumber (Cucumis sativus L.) grafting. However, there are no published molecular genetic studies of chilling tolerance in pumpkins used as rootstock. An F2 population with 166 individuals was derived from a cross between a chilling-stress-susceptible high-generation inbred line (‘5-5-6 ) and a chillingstress-tolerant high-generation inbred line (‘8-3-7 ). The chilling index (CI) was used to evaluate the cold tolerance of the parental lines and the F2 population. The continuous and normal distribution of the CI in the F2 population suggested that the trait of chilling tolerance was a typically quantitative trait controlled by multiple genes and was suitable for quantitative trait loci (QTL) mapping. A molecular linkage map was constructed using 95 simple-sequence repeat (SSR) markers, resulting in 15 linkage groups (LGs) covering a total distance of 830.7 cM with an average interval of 8.74 cM. Three QTLs were detected for CI, explaining 0.30%, 1.22% and 20.88% of the phenotypic variation. The genetic map and QTLs will serve as framework for future molecular breeding studies. © 2017 Published by Elsevier B.V.
1. Introduction Cucumber (Cucumis sativus L.) is a major protected cultivation vegetable that is susceptible to chilling stress (Liu et al., 2004). In China, cucumbers in protected areas are mainly cultivated from the winter to the next spring. Seedlings are grown in mid-to-late September, transplanted in mid-to-late October, and cultivated until early-to-mid June of the following year. The growth cycle lasts for more than eight months and three seasons (i.e., autumn, winter, and spring; Wang and Yu, 2013). Hence, cucumber plants would suffer from chilling stress during winter and early spring, which hinders physiological functions and results in decreased fruit yield and quality (Wang, 1990). Previous studies have concluded that grafting would help cucumber plants adapt to chilling stress (Bhatt et al., 2013; Colla et al., 2014; Gao et al., 2005; Yan et al., 2015). An appropriate rootstock could promote cucumber tolerance to low environmental or soil temperatures. The tolerance level of grafted plants to abiotic stresses is usually between that of the scion and the rootstock and is mainly affected by the tolerance level of the rootstock (Huang et al.,
∗ Corresponding author. E-mail address: [email protected] (J. Sun). http://dx.doi.org/10.1016/j.scienta.2017.03.051 0304-4238/© 2017 Published by Elsevier B.V.
2013; Schwarz et al., 2010; Yan et al., 2015). Therefore, a highly tolerant rootstock is crucial for the grafting of cucumber plants that are more tolerant to chilling stress. Pumpkins (Cucurbita; 2n = 40) are important rootstocks for watermelon and cucumber to increase disease resistance, stress tolerance and yield, and improve fruit quality (Muhie and Yassin, 2015; Wimer et al., 2015; Xing et al., 2014). The taxonomy of the Cucurbita genus is still under debate, as the number of species accepted by specialists varies from 13 to 30 (Burrows and Tyrl, 2013). The five domesticated common species in China are Cucurbita moschata, C. maxima, C. pepo, C. maxita and C. ficifolia (Esquinas-Alcazar and Gulick, 1983; Whitaker and Davis, 1962). There are very limited genomic and genetic resources available for Cucurbita compared to other cucurbits, such as cucumber, melon and watermelon. For the other cucurbits, microarrays (MascarellCreus et al., 2009; Wechter et al., 2008), saturated molecular genetic maps (Deleu et al., 2009; Ren et al., 2009; Ren et al., 2012), reverse genetic platforms (Fraenkel et al., 2012; González et al., 2011), transcriptomes (Ando et al., 2012; Blanca et al., 2011; Guo et al., 2010) and even whole genome sequences (Garcia-Mas and Puigdomènech, 2012; Guo et al., 2013; Huang et al., 2009) have been developed and completed. These powerful tools could facilitate the development of genetic and genomic analyses, promoting genetic linkage mapping and the breeding of new varieties (Bhawna
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et al., 2014; Hwang et al., 2014; Nimmakayala et al., 2013; Shang et al., 2014; Yang et al., 2012). Molecular linkage maps are important for QTL mapping, assembled genome scaffold anchoring and orientation, and markerassisted selection for efficient molecular breeding and function analysis (Argyris et al., 2015; Ren et al., 2012). However, genetic mapping of genus Cucurbita with different parental species is still in its infancy, with only a few maps reported. These maps include: one map that used interspecific crosses between C. maxima and C. ecuadorensis (Weeden and Robinson, 1986), two maps that used interspecific crosses between C. pepo and C. moschata (Brown and Myers, 2002; Lee et al., 1995), 4 intraspecific crosses of C. pepo (Esteras et al., 2012; Gong et al., 2008b; Zraidi et al., 2007), 2 intraspecific crosses of C. maxima (Singh et al., 2011; Zhang et al., 2015) and one intraspecific cross of C. moschata (Gong et al., 2008a). These published linkage maps used different types of markers and mapped populations derived from different parental lines for which some QTLs and loci have been identified. For example, QTLs for the depth of indentations between primary leaf veins and fruit shape (Brown and Myers, 2002), 12 QTLs for vine traits, immature fruit, mature fruit and flowering traits (Esteras et al., 2012), 10 QTLs for plant, flowering and fruit traits (Esteras et al., 2012), loci for seed coat character and resistance to Zucchini Yellow Mosaic Virus (ZYMV) (Zraidi et al., 2007) and loci for green rind (Gong et al., 2008a) have been established. However, there are no published molecular genetic studies regarding chilling tolerance in rootstockused pumpkins. The principal objective of the present study was to construct a linkage map for the pumpkin used for rootstock (Cucurbita moschata) using SSR markers and to detect QTLs for chilling-stress tolerance at the seedling stage. Subsequently, 95 pairs of polymorphic SSR primers were used to construct the linkage map in an F2 population with 166 individuals. Based on this new map, by investigating the chilling index (CI) in seedling-stage pumpkins, three QTLs related to chilling stress tolerance were detected using the composite interval mapping (CIM) method. The present data would facilitate genetic improvement of chilling tolerance and markerassisted selection (MAS) for rootstock-used pumpkin. 2. Materials and methods 2.1. Plant materials Two varieties of pumpkin (Cucurbita moschata) with significantly different chilling injury indexes were crossed to generate F1 and F2 segregation populations. The female parent (‘8-3-7 ) was chilling stress tolerant (marked as C1 ) with a mean chilling index value of 2.00. The male parent (‘5-5-6 ) was chilling stress susceptible (marked as C2 ) with a mean chilling index value of 3.64. The two parents were high-generation inbred lines obtained in our laboratory that were rootstocks for cucumber grafts. The 166 F2 individuals, 20 F1 individuals, and 20 parent plants were grown and evaluated with regard to the chilling index. 2.2. The evaluation of cold tolerance for parent plants, the F1 and F2 population
the growth temperature was changed to 15/5 ◦ C (14-h day/10-h night) with no change in the other parameters described above. After 15 days, the chilling injuries of each individual, using chilling indexes (CI), were estimated in the C1 , C2 , F1 and F2 populations. The injuries of the individuals were visually scored on the true leaves and the heart leaves using a 0–5 scale criterion (Fig. 1). A score of 0 meant no injury on plants. A score of 1 meant the plant had few flaccid leaf margins. A score of 2 indicated that the plant had flaccid leaf margins and few dehydrated spots. A score of 3 meant that the plant had large areas of dehydrated spots and that parts of the true leaves were wilted. A score of 4 meant that the true leaves were wilted and only the heart leaves survived. A score of 5 meant that the plant completely died. A low CI value meant that the true leaves and heart leaves were less injured and more tolerant to the chilling stress. SPSS Statistics software version 20.0 (IBM Corp., Armonk, NY, USA) was used to analyze data variance and normal distribution. 2.3. DNA extraction and SSR marker analysis Genomic DNA from the parents and F2 population were extracted following the protocol of the plant genomic DNA kit (Tiangen, China). DNA quality and concentration were determined using 1% agarose gel electrophoresis and the Eppendorf Biophotometer plus (Eppendorf Inc., Hamburg, Germany). Subsequently, DNA was diluted to a concentration of 100 ng/L and stored at −20 ◦ C until use. A total of 1277 pairs of SSR primers with the prefixes ‘CMTm’, ‘CMTp’ (Gong et al., 2008b), ‘CUTC’ (Blanca et al., 2015) and ‘Unigene’ (Wu et al., 2014) were obtained from previously published studies and used to amplify regions of the two parent lines to select polymorphic markers. The polymerase chain reaction (PCR) amplification was performed in a 10 L solution containing 1 ng of genomic DNA, 1 M of each primer, 0.5 U of Premix taq DNA polymerase (TaKaRa, Japan) and 2 L double distilled water. The SSR amplification program was as follows: initial denaturation at 95 ◦ C for 5 min, 35 cycles of denaturation at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and elongation at 72 ◦ C for 30 s, with a final extension at 72 ◦ C for 10 min. The SSR products were added to 10 L loading buffer, then 2 L of each sample mixture was separated on an 8% non-denaturing polyacrylamide gel and electrophoresed in 0.5 × TBE electrophoresis buffer. The electrophoresis conditions were 240 V for 1 h at room temperature. The gels were then stained with rapid silver staining for detection. 2.4. Construction of the linkage map Marker segregation was tested against the expected ratios of 3:1 (dominant) or 1:2:1 (codominant) Mendelian segregation at P < 0.05 using the 2 test (Chi-square test). The 166 individuals from F2 populations and the polymorphic SSR markers selected as described above were used to construct a linkage map using JoinMap version 4.0 (Ooijen, 2006) with a minimum logarithm of odds (LOD) threshold of 3.0 and a recombination frequency value of 0.4. The Kosambi function was used to translate the recombinant ratios into map distances in centimorgans (Kosambi, 1943). The linkage map was generated using MapChart 2.3 software (Voorrips, 2001). 2.5. QTL analysis for seedling chilling index
Germinated seeds were sown in plastic nutrition pots (8 cm × 8 cm) filled with commercial organic substrates (vinegar waste compost:peat:vermiculite = 2:2:1, v/v/v; pH = 6.0, EC = 1.5 mS/cm; Beilei, Zhenjiang, China). One seed was planted per pot. Plants were grown in a growth chamber (RXZ-1000B, 1000 L; Ningbo Jiangnan Instrument Factory Inc.) with a 25/15 ◦ C (14-h day/10-h night) cycle under a light intensity of 400 mol m−2 s−1 and a relative humidity of 70%. At the stage of 2 true leaves,
Based on the map and raw data obtained from the Joinmap analysis, association analysis between markers and traits was performed using the composite interval mapping (CIM) method (Li and Ye, 2007) at 2.0 cM walk speed with Windows QTL Cartographer Version 2.5 (Basten et al., 2001). The threshold was determined by permutation analysis (Churchill and Doerge, 1994) of 1000 replications (P < 0.05). The position on a given linkage group with the
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Fig. 1. The chilling injuries in C. moschata were visually scored with the scale criterion 0–5. Score of 0 meant no injury on plants. Score of 1 meant the plant had few flaccid leaf margins. Score of 2 indicated that the plant had flaccid leaf margins and few dehydrated spot. Score of 3 represented that the plant had large area of dehydrated spot and parts of the true leaves were wilted. Score of 4 meant the true leaves were wilted and only the heart leaves survived. Score of 5 described that the plant completely died.
tion tests of the F2 individuals for chilling index were performed. The F2 population chilling index showed continuous phenotypic variations with the absolute values of kurtosis and skewness less than 1(0.117 and −0.387, respectively), reflecting a normal distribution of quantitative traits. The results are shown in the form of a histogram in Fig. 2. 3.2. Identification of polymorphic SSR markers Of the 1277 SSR primer pairs obtained from previous studies that were screened for polymorphisms between the parental lines ‘8-3-7 and ‘5-5-6 , 121 SSRs were found to be polymorphic. The 121 primer pairs were used to construct the linkage map. 3.3. Construction of the linkage map
Fig. 2. The frequency distribution of the value of chilling index (CI) in F2 population of C. moschata derived from ‘5-5-6 × ‘8-3-7 . Histograms were drawn using SPSS software.
maximum LOD score was considered to be the QTL position. The detected QTLs were denominated beginning with ‘q’, followed by the abbreviation of the trait name, the linkage name and finally the serial number (Mccouch et al., 1997).
Among the 121 polymorphic primers, 59 pairs were regarded as distorted markers based on the 2 test for goodness-of-fit to the expected 1:2:1 or 3:1 Mendelian segregation ratios at P < 0.05 in the F2 population. Of the 59 pairs of markers, a total of 19 dominantly deviated from the segregation ratio of 3:1, and 40 deviated from the segregation ratio of 1:2:1. Subsequently, 7 dominant markers and 26 co-dominant markers were integrated into the map. Eventually, the linkage map was successfully constructed with 95 SSR markers, while 26 markers remained unlinked. A molecular linkage map was built based on 95 SSR markers with 15 linkage groups, spanning a total length of 830.7 cM with an average interval distance of 8.74 cM between adjacent markers. These 15 linkage groups were designated as LG1 to LG15, ranging in length from 9.0 cM in LG15 to 106.2 cM in LG1. The total number of markers in individual linkage groups varied from 2 to 15, and the marker density varied from 5.30 to 16.75 cM.
3. Results 3.4. QTL analysis for seedling chilling tolerance 3.1. Phenotype evaluation Segregation of the chilling index in the F2 family is shown in Fig. 2. The mean chilling index of the tolerant female parent ‘8-3-7 was 2.00 and the mean CI of the susceptible male parent ‘5-5-6 was 3.64. The mean chilling index of F1 plants (‘8-3-7 × ‘5-5-6 ) was 3.13, between those of ‘8-3-7 and ‘5-5-6 . Normal distribu-
The QTL analysis is shown in Table 1 and Fig. 3. The LOD score of 2.5 was determined according to the permutation test and the CIM method was performed to detect putative QTLs. Eventually, a total of 3 QTLs were mapped for chilling tolerance of the rootstock-used pumpkin at the seedling stage. They were named qCI-1-1, qCI-4-1, and qCI-10-1, respectively. The QTL qCI-1-1 was located on LG 1,
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Table 1 QTL analysis of chilling tolerance related to chilling index (CI) in C. moschata. QTL
Linkage groups
Flanking markersa
Peak Positions (cM) (support interval)b
LOD scorec
Additive effectd
R2 (%)e
qCI-1-1 qCI-4-1 qCI-10-1
LG1 LG4 LG10
CMTp163-CMTm140 CMTm113–Unigene0027389 CMTm214-Unigene0008127
65.5 (64.1–68.1) 58.9 (58.8–59.2) 34.9 (33.9–35.1)
2.60 2.70 2.56
−0.51 −0.04 0.52
0.30 1.22 20.88
a
Marker interval. QTL position of peak marker or interval in the LG and the support interval (cM). c Peak LOD value of the QTL flanking a peak LOD score (LOD ≥ 3.0). d Additive effect. A positive value indicates that the positive genes originating from male parent ‘5-5-6 increase the tolerance to chilling stress, and a negative value indicates that positive genes originating from female parent ‘8-3-7 increase the tolerance. e Percentage of the phenotypic variance explained by the putative QTL. b
flanked by the CMTp163 and CMTm140 markers. The distance of qCI-1-1 from the two markers was 0.97 cM and 22.95 cM, respectively. This QTL could only explain 0.3% of the phenotypic variance. The QTL qCI-4-1 was between the CMTm113 and Unigene0027389 markers in LG 4 and could account for 1.22% of the phenotypic variation. The distances between qCI-4-1 and its two flanking markers were 1.07 cM and 0.74 cM. The QTL qCI-10-1 was detected in LG 10, between the CMTm214 and Unigene0008127 markers, which explained 20.88% of the phenotypic variation. The distances between qCI-10-1 and the flanking markers were 1.99 cM and 6.71 cM. The positive genes of qCI-1-1and qCI-4-1 were donated from the female parent (8-3-7), while the positive genes of qCI-10-1 were from the male parent (5-5-6).
4. Discussion The construction of linkage maps is crucial for genetically dissecting the locations and organizations of genes or QTLs that are associated with complicated agronomic traits. Linkage mapping of interesting traits has been applied in many crops, such as rice (Ranawake et al., 2014), wheat (Anderson et al., 2001), Chinese cabbage (Yu et al., 2009) and tomato (Asins et al., 2013). Chilling stress tolerance is an important trait of pumpkins that are used as rootstock for cucumber grafts. To date, no QTL analyses of chilling stress tolerance have been reported, which has led to the study of the genetic basis of chilling stress tolerance in the pumpkin lagging behind such studies in other crops. Hence, the present linkage map and QTL analyses of chilling stress tolerance will provide a framework for further research and breeding. For the pumpkin (Cucurbita moschata), 20 LGs are expected to be mapped in accordance with the haploid chromosome number. Until now, the number of LGs in all published genetic maps of pumpkin (Cucurbita) ranged from 5 to 28 (Brown and Myers, 2002; Esteras et al., 2012; Gong et al., 2008a; Gong et al., 2008b; Lee et al., 1995; Singh et al., 2011; Weeden and Robinson, 1986; Zhang et al., 2015; Zraidi et al., 2007). However, only 3 maps (Gong et al., 2008a; Gong et al., 2008b; Zhang et al., 2015) contained the expected LG number corresponding to the number of chromosomes. In the present study, we obtained 15 LGs in the linkage map, which is less than the expected number of 20. These results indicated that the linkage map was not fully saturated and that some chromosome regions have not been covered in this map. Furthermore, the length of the map that we obtained was 830.7 cM, shorter than maps from the previous studies which ranged from 1195.2 cM to 2566.8 cM (Brown and Myers, 2002; Esteras et al., 2012; Gong et al., 2008a; Gong et al., 2008b; Singh et al., 2011; Zhang et al., 2015; Zraidi et al., 2007). Remarkably, map lengths have been shown not to be comparable between different software, as lengths computed by Joinmap are usually shorter than those obtained by MAPMAKER (Bradeen et al., 2001; Tani et al., 2003). The existence of unlinked markers also demonstrated that the saturated map could be larger than that generated in the present study. In further studies, it is
expected that additional markers may allow deeper connections between linkage groups. In this study, the SSR (Simple Sequence Repeats) marker system was used to analyze an F2 population with 166 individuals and construct a linkage map for Cucurbita moschata. SSRs contain 2–6 base pairs of DNA repeat sequences. They are widely used as molecular markers in genetic mapping, species classification and molecular-assisted selection in crops (Neeraja et al., 2007; Senior, 1998; Temnykh et al., 2001) because SSRs have good marker properties, such as co-dominant conditions, cost-efficient analysis, easy detection, stability and reliability (Quezada et al., 2014; Wu et al., 2014). Additionally, it has been indicated that SSRs show very low levels of polymorphism (Li et al., 2008). A total of 1277 pairs of SSR markers from previously published references (Blanca et al., 2015; Gong et al., 2008b; Wu et al., 2014) were used in the present study. The level of polymorphism detected in SSR markers in our study was 9.48%, which was low compared with other studies. There are two possible explanations for the low level of polymorphism. First, parts of the SSR markers used in this study were developed from the pumpkin transcriptome of Cucurbita pepo, which is different from the plant materials used in this study (Cucurbita moschata). Although the previous study showed that the genomic SSRs developed from C. pepo and C. moschata have a high inter-species transferability (Gong et al., 2008b), a low level of polymorphism was detected in our study. Second, we used intra-species pumpkins as parent lines to construct the genetic map. It is generally believed that the degree of polymorphism is lower in an intra-species population than in an inter-species population (Chen and Chen, 2010). The intra-species parents would have a similar genetic background and a lower level of genetic variability compared to the inter-species parents. As expected, the rate of polymorphism was indeed low. Out of the 121 analyzed markers, 59 (48.8%) were segregationratio distorted from the expected Mendelian segregation ratio at P < 0.05. Distorted segregation could be caused by competition among gametes during fertilization, which results from gametophyte genes expressed in the haploid genotype. Hybrid sterility genes cause the abortion of certain genotypes and could also lead to distorted segregation. Moreover, sublethal and lethal genes can also generate segregation distortion if they lead to embryos which are too feeble to germinate normally. Additionally, physiological and environmental factors could also affect the distorted segregation ratios (Brown and Myers, 2002; Faris et al., 1998; Ky et al., 2000). After grouping the SSR markers on the map, 26 markers could not be integrated. The resulting map covered 830.7 cM in 15 LGs comprising 95 SSR markers, with an average of 8.74 cM between intervals. In addition, small regions (less than 5 markers) in the maps (LG4, LG7, and LG15) showed that the markers are sparse in some regions of the genome and dense in other regions (Becker et al., 1995; Kesseli et al., 1994). The number of LGs was less than the haploid chromosomal number of C. moschata (n = 20). The relatively low polymorphism and the lack of markers would contribute
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Fig. 3. Genetic linkage map of C. moschata and QTLs for CI generated from a ‘5-5-6 × ‘8-3-7 F2 population. Genetic distances (cM) were shown on the left side and marker on the right. The red rectangles mean the support intervals for QTLs in LG1, LG4 and LG10. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
to this phenomenon. This map could become more saturated with additional markers in future research. As far as we know, no available QTL analyses of chilling tolerance in pumpkin have been published. Past studies have shown that
the cold tolerance of plants was a complex quantitative trait controlled by multiple genes (Hu et al., 2016; Liu et al., 2016; Ranawake et al., 2014; Thomashow, 1999). In the present study, we used the CI values of an F2 population derived from a cross of the tolerant
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inbred line ‘8-3-7 and the susceptible inbred line ‘5-5-6 . There was a significant difference between the parental lines, with a lower CI value for the tolerant inbred line ‘8-3-7 than for the susceptible inbred line ‘5-5-6 . The continuous and normal distribution observed in the CI values among the F2 population demonstrated that the trait of chilling stress tolerance was a typically quantitative trait controlled by multiple genes. Identification of potential QTLs is related to material difference, the type of markers, map saturation, LOD value and the length of the map (Tanksley, 1993). Considering that the length of the current map, 830.7 cM, was shorter than the expected whole distance, some underlying QTLs may not be identified in regions that the present map does not cover. Here, for the first time, we detected QTLs for chilling tolerance on different linkage groups by utilizing a CIM strategy. In total, three QTLs were found on three linkage groups with genetic contributions of 0.30%, 1.22% and 20.88%, respectively (Table 1, Fig. 3). Among the three QTLs, QTL qCI-10-1 has the largest effect (>20%), while the others have minor effects (<10%). The genetic distance between QTL qCI-10-1 and the closest flanking marker CMTm214 was 1.99 cM. The allele from the female parent ‘8-3-7 increased the tolerance to chilling stress via QTLs on LG 1 and LG 4, while the male parent ‘5-5-6 increased tolerance via the QTL on LG 10. These findings indicate that parental lines with phenotypically weak performance in the desired agronomic traits can also retain positive alleles in some QTLs. This phenomenon has been reported in previous studies in other crops (Devicente and Tanksley, 1993; Maheswaran et al., 2000). In the practical breeding program for chilling stress tolerance in C. moschata, it is not accurate to screen parental lines by phenotype alone, as the positive alleles may exist in susceptible parent lines as well. Identification of molecular markers linked with QTLs promotes detection of the underlying positive allele and the opportunity to integrate many of these alleles into one objective breeding cultivar with Marker-Assisted Selection (MAS). There are few reports on QTL identification for chilling stress tolerance in rootstock-used pumpkin and we considered it crucial to carry out further work on this genetic map, not only for its theoretical value but also for practical purposes. Acknowledgements This work was financially supported by the China Earmarked Fund for Modern Agro-industry Technology Research System (CARS-25-C-03) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Anderson, J.A., Stack, R.W., Liu, S., Waldron, B.L., Fjeld, A.D., Coyne, C., Moreno-Sevilla, B., Fetch, J.M., Song, Q.J., Cregan, P.B., 2001. DNA markers for Fusarium head blight resistance QTL in two wheat populations. Theor. Appl. Genet. 102, 1164–1168. Ando, K., Carr, K.M., Grumet, R., 2012. Transcriptome analyses of early cucumber fruit growth identifies distinct gene modules associated with phases of development. BMC Genom. 13, 518. Argyris, J.M., Ruizherrera, A., Madrizmasis, P., Sanseverino, W., Morata, J., Pujol, M., Ramosonsins, S.E., Garciamas, J., 2015. Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly. BMC Genom. 16, 4. Asins, M.J., Villalta, I., Aly, M.M., Olías, R., Alvarez De Morales, P., Huertas, R., Li, J., Jaime-Pérez, N., Haro, R., Raga, V., Carbonell, E.A., Belver, A., 2013. Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+ /K+ homeostasis. Plant Cell Environ. 36, 1171–1191. Basten, C.J., Weir, B.S., Zeng, Z.B., 2001. QTL Cartographer Version 1.15. Department of Statistics, North Carolina State University, Raleigh. Becker, J., Vos, P., Kuiper, M., Salamini, F., Heun, M., 1995. Combined mapping of AFLP and RFLP markers in barley. Mol. Gen. Genom. 249, 65–73. Bhatt, R.M., Harish, D.M., Srilakshmi, 2013. Significance of grafting in improving tolerance to abiotic stresses in vegetable crops under climate change scenario. In: Singh, H.C.P., Rao, N.K.S., Shivashankar, K.S., Pflanzen (Eds.), Climate-resilient Horticulture: Adaptation and Mitigation Strategies. Springer India, India, pp. 159–175.
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Construction of a genetic linkage map of rootstock-used pumpkin using SSR markers and QTL analysis for cold tolerance Yang Xu, Shi-rong Guo, Sheng Shu, Yan Ren, Jin Sun ∗ College of Horticulture, Nanjing Agricultural University, Key Laboratory of Southern Vegetable Genetic Improvement, Ministry of Agriculture, Nanjing 210095, China
a r t i c l e
i n f o
Article history: Received 23 November 2016 Received in revised form 23 March 2017 Accepted 29 March 2017 Keywords: C. moschata Chilling tolerance SSR markers Linkage map QTL analysis
a b s t r a c t In China, cucumber plants suffer chilling stress during the winter and early spring, and grafting would help the plants adapt to chilling stress. Pumpkin (Cucurbita moschata; 2n = 40) is an important rootstock for cucumber (Cucumis sativus L.) grafting. However, there are no published molecular genetic studies of chilling tolerance in pumpkins used as rootstock. An F2 population with 166 individuals was derived from a cross between a chilling-stress-susceptible high-generation inbred line (‘5-5-6 ) and a chillingstress-tolerant high-generation inbred line (‘8-3-7 ). The chilling index (CI) was used to evaluate the cold tolerance of the parental lines and the F2 population. The continuous and normal distribution of the CI in the F2 population suggested that the trait of chilling tolerance was a typically quantitative trait controlled by multiple genes and was suitable for quantitative trait loci (QTL) mapping. A molecular linkage map was constructed using 95 simple-sequence repeat (SSR) markers, resulting in 15 linkage groups (LGs) covering a total distance of 830.7 cM with an average interval of 8.74 cM. Three QTLs were detected for CI, explaining 0.30%, 1.22% and 20.88% of the phenotypic variation. The genetic map and QTLs will serve as framework for future molecular breeding studies. © 2017 Published by Elsevier B.V.
1. Introduction Cucumber (Cucumis sativus L.) is a major protected cultivation vegetable that is susceptible to chilling stress (Liu et al., 2004). In China, cucumbers in protected areas are mainly cultivated from the winter to the next spring. Seedlings are grown in mid-to-late September, transplanted in mid-to-late October, and cultivated until early-to-mid June of the following year. The growth cycle lasts for more than eight months and three seasons (i.e., autumn, winter, and spring; Wang and Yu, 2013). Hence, cucumber plants would suffer from chilling stress during winter and early spring, which hinders physiological functions and results in decreased fruit yield and quality (Wang, 1990). Previous studies have concluded that grafting would help cucumber plants adapt to chilling stress (Bhatt et al., 2013; Colla et al., 2014; Gao et al., 2005; Yan et al., 2015). An appropriate rootstock could promote cucumber tolerance to low environmental or soil temperatures. The tolerance level of grafted plants to abiotic stresses is usually between that of the scion and the rootstock and is mainly affected by the tolerance level of the rootstock (Huang et al.,
∗ Corresponding author. E-mail address: [email protected] (J. Sun). http://dx.doi.org/10.1016/j.scienta.2017.03.051 0304-4238/© 2017 Published by Elsevier B.V.
2013; Schwarz et al., 2010; Yan et al., 2015). Therefore, a highly tolerant rootstock is crucial for the grafting of cucumber plants that are more tolerant to chilling stress. Pumpkins (Cucurbita; 2n = 40) are important rootstocks for watermelon and cucumber to increase disease resistance, stress tolerance and yield, and improve fruit quality (Muhie and Yassin, 2015; Wimer et al., 2015; Xing et al., 2014). The taxonomy of the Cucurbita genus is still under debate, as the number of species accepted by specialists varies from 13 to 30 (Burrows and Tyrl, 2013). The five domesticated common species in China are Cucurbita moschata, C. maxima, C. pepo, C. maxita and C. ficifolia (Esquinas-Alcazar and Gulick, 1983; Whitaker and Davis, 1962). There are very limited genomic and genetic resources available for Cucurbita compared to other cucurbits, such as cucumber, melon and watermelon. For the other cucurbits, microarrays (MascarellCreus et al., 2009; Wechter et al., 2008), saturated molecular genetic maps (Deleu et al., 2009; Ren et al., 2009; Ren et al., 2012), reverse genetic platforms (Fraenkel et al., 2012; González et al., 2011), transcriptomes (Ando et al., 2012; Blanca et al., 2011; Guo et al., 2010) and even whole genome sequences (Garcia-Mas and Puigdomènech, 2012; Guo et al., 2013; Huang et al., 2009) have been developed and completed. These powerful tools could facilitate the development of genetic and genomic analyses, promoting genetic linkage mapping and the breeding of new varieties (Bhawna
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et al., 2014; Hwang et al., 2014; Nimmakayala et al., 2013; Shang et al., 2014; Yang et al., 2012). Molecular linkage maps are important for QTL mapping, assembled genome scaffold anchoring and orientation, and markerassisted selection for efficient molecular breeding and function analysis (Argyris et al., 2015; Ren et al., 2012). However, genetic mapping of genus Cucurbita with different parental species is still in its infancy, with only a few maps reported. These maps include: one map that used interspecific crosses between C. maxima and C. ecuadorensis (Weeden and Robinson, 1986), two maps that used interspecific crosses between C. pepo and C. moschata (Brown and Myers, 2002; Lee et al., 1995), 4 intraspecific crosses of C. pepo (Esteras et al., 2012; Gong et al., 2008b; Zraidi et al., 2007), 2 intraspecific crosses of C. maxima (Singh et al., 2011; Zhang et al., 2015) and one intraspecific cross of C. moschata (Gong et al., 2008a). These published linkage maps used different types of markers and mapped populations derived from different parental lines for which some QTLs and loci have been identified. For example, QTLs for the depth of indentations between primary leaf veins and fruit shape (Brown and Myers, 2002), 12 QTLs for vine traits, immature fruit, mature fruit and flowering traits (Esteras et al., 2012), 10 QTLs for plant, flowering and fruit traits (Esteras et al., 2012), loci for seed coat character and resistance to Zucchini Yellow Mosaic Virus (ZYMV) (Zraidi et al., 2007) and loci for green rind (Gong et al., 2008a) have been established. However, there are no published molecular genetic studies regarding chilling tolerance in rootstockused pumpkins. The principal objective of the present study was to construct a linkage map for the pumpkin used for rootstock (Cucurbita moschata) using SSR markers and to detect QTLs for chilling-stress tolerance at the seedling stage. Subsequently, 95 pairs of polymorphic SSR primers were used to construct the linkage map in an F2 population with 166 individuals. Based on this new map, by investigating the chilling index (CI) in seedling-stage pumpkins, three QTLs related to chilling stress tolerance were detected using the composite interval mapping (CIM) method. The present data would facilitate genetic improvement of chilling tolerance and markerassisted selection (MAS) for rootstock-used pumpkin. 2. Materials and methods 2.1. Plant materials Two varieties of pumpkin (Cucurbita moschata) with significantly different chilling injury indexes were crossed to generate F1 and F2 segregation populations. The female parent (‘8-3-7 ) was chilling stress tolerant (marked as C1 ) with a mean chilling index value of 2.00. The male parent (‘5-5-6 ) was chilling stress susceptible (marked as C2 ) with a mean chilling index value of 3.64. The two parents were high-generation inbred lines obtained in our laboratory that were rootstocks for cucumber grafts. The 166 F2 individuals, 20 F1 individuals, and 20 parent plants were grown and evaluated with regard to the chilling index. 2.2. The evaluation of cold tolerance for parent plants, the F1 and F2 population
the growth temperature was changed to 15/5 ◦ C (14-h day/10-h night) with no change in the other parameters described above. After 15 days, the chilling injuries of each individual, using chilling indexes (CI), were estimated in the C1 , C2 , F1 and F2 populations. The injuries of the individuals were visually scored on the true leaves and the heart leaves using a 0–5 scale criterion (Fig. 1). A score of 0 meant no injury on plants. A score of 1 meant the plant had few flaccid leaf margins. A score of 2 indicated that the plant had flaccid leaf margins and few dehydrated spots. A score of 3 meant that the plant had large areas of dehydrated spots and that parts of the true leaves were wilted. A score of 4 meant that the true leaves were wilted and only the heart leaves survived. A score of 5 meant that the plant completely died. A low CI value meant that the true leaves and heart leaves were less injured and more tolerant to the chilling stress. SPSS Statistics software version 20.0 (IBM Corp., Armonk, NY, USA) was used to analyze data variance and normal distribution. 2.3. DNA extraction and SSR marker analysis Genomic DNA from the parents and F2 population were extracted following the protocol of the plant genomic DNA kit (Tiangen, China). DNA quality and concentration were determined using 1% agarose gel electrophoresis and the Eppendorf Biophotometer plus (Eppendorf Inc., Hamburg, Germany). Subsequently, DNA was diluted to a concentration of 100 ng/L and stored at −20 ◦ C until use. A total of 1277 pairs of SSR primers with the prefixes ‘CMTm’, ‘CMTp’ (Gong et al., 2008b), ‘CUTC’ (Blanca et al., 2015) and ‘Unigene’ (Wu et al., 2014) were obtained from previously published studies and used to amplify regions of the two parent lines to select polymorphic markers. The polymerase chain reaction (PCR) amplification was performed in a 10 L solution containing 1 ng of genomic DNA, 1 M of each primer, 0.5 U of Premix taq DNA polymerase (TaKaRa, Japan) and 2 L double distilled water. The SSR amplification program was as follows: initial denaturation at 95 ◦ C for 5 min, 35 cycles of denaturation at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and elongation at 72 ◦ C for 30 s, with a final extension at 72 ◦ C for 10 min. The SSR products were added to 10 L loading buffer, then 2 L of each sample mixture was separated on an 8% non-denaturing polyacrylamide gel and electrophoresed in 0.5 × TBE electrophoresis buffer. The electrophoresis conditions were 240 V for 1 h at room temperature. The gels were then stained with rapid silver staining for detection. 2.4. Construction of the linkage map Marker segregation was tested against the expected ratios of 3:1 (dominant) or 1:2:1 (codominant) Mendelian segregation at P < 0.05 using the 2 test (Chi-square test). The 166 individuals from F2 populations and the polymorphic SSR markers selected as described above were used to construct a linkage map using JoinMap version 4.0 (Ooijen, 2006) with a minimum logarithm of odds (LOD) threshold of 3.0 and a recombination frequency value of 0.4. The Kosambi function was used to translate the recombinant ratios into map distances in centimorgans (Kosambi, 1943). The linkage map was generated using MapChart 2.3 software (Voorrips, 2001). 2.5. QTL analysis for seedling chilling index
Germinated seeds were sown in plastic nutrition pots (8 cm × 8 cm) filled with commercial organic substrates (vinegar waste compost:peat:vermiculite = 2:2:1, v/v/v; pH = 6.0, EC = 1.5 mS/cm; Beilei, Zhenjiang, China). One seed was planted per pot. Plants were grown in a growth chamber (RXZ-1000B, 1000 L; Ningbo Jiangnan Instrument Factory Inc.) with a 25/15 ◦ C (14-h day/10-h night) cycle under a light intensity of 400 mol m−2 s−1 and a relative humidity of 70%. At the stage of 2 true leaves,
Based on the map and raw data obtained from the Joinmap analysis, association analysis between markers and traits was performed using the composite interval mapping (CIM) method (Li and Ye, 2007) at 2.0 cM walk speed with Windows QTL Cartographer Version 2.5 (Basten et al., 2001). The threshold was determined by permutation analysis (Churchill and Doerge, 1994) of 1000 replications (P < 0.05). The position on a given linkage group with the
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Fig. 1. The chilling injuries in C. moschata were visually scored with the scale criterion 0–5. Score of 0 meant no injury on plants. Score of 1 meant the plant had few flaccid leaf margins. Score of 2 indicated that the plant had flaccid leaf margins and few dehydrated spot. Score of 3 represented that the plant had large area of dehydrated spot and parts of the true leaves were wilted. Score of 4 meant the true leaves were wilted and only the heart leaves survived. Score of 5 described that the plant completely died.
tion tests of the F2 individuals for chilling index were performed. The F2 population chilling index showed continuous phenotypic variations with the absolute values of kurtosis and skewness less than 1(0.117 and −0.387, respectively), reflecting a normal distribution of quantitative traits. The results are shown in the form of a histogram in Fig. 2. 3.2. Identification of polymorphic SSR markers Of the 1277 SSR primer pairs obtained from previous studies that were screened for polymorphisms between the parental lines ‘8-3-7 and ‘5-5-6 , 121 SSRs were found to be polymorphic. The 121 primer pairs were used to construct the linkage map. 3.3. Construction of the linkage map
Fig. 2. The frequency distribution of the value of chilling index (CI) in F2 population of C. moschata derived from ‘5-5-6 × ‘8-3-7 . Histograms were drawn using SPSS software.
maximum LOD score was considered to be the QTL position. The detected QTLs were denominated beginning with ‘q’, followed by the abbreviation of the trait name, the linkage name and finally the serial number (Mccouch et al., 1997).
Among the 121 polymorphic primers, 59 pairs were regarded as distorted markers based on the 2 test for goodness-of-fit to the expected 1:2:1 or 3:1 Mendelian segregation ratios at P < 0.05 in the F2 population. Of the 59 pairs of markers, a total of 19 dominantly deviated from the segregation ratio of 3:1, and 40 deviated from the segregation ratio of 1:2:1. Subsequently, 7 dominant markers and 26 co-dominant markers were integrated into the map. Eventually, the linkage map was successfully constructed with 95 SSR markers, while 26 markers remained unlinked. A molecular linkage map was built based on 95 SSR markers with 15 linkage groups, spanning a total length of 830.7 cM with an average interval distance of 8.74 cM between adjacent markers. These 15 linkage groups were designated as LG1 to LG15, ranging in length from 9.0 cM in LG15 to 106.2 cM in LG1. The total number of markers in individual linkage groups varied from 2 to 15, and the marker density varied from 5.30 to 16.75 cM.
3. Results 3.4. QTL analysis for seedling chilling tolerance 3.1. Phenotype evaluation Segregation of the chilling index in the F2 family is shown in Fig. 2. The mean chilling index of the tolerant female parent ‘8-3-7 was 2.00 and the mean CI of the susceptible male parent ‘5-5-6 was 3.64. The mean chilling index of F1 plants (‘8-3-7 × ‘5-5-6 ) was 3.13, between those of ‘8-3-7 and ‘5-5-6 . Normal distribu-
The QTL analysis is shown in Table 1 and Fig. 3. The LOD score of 2.5 was determined according to the permutation test and the CIM method was performed to detect putative QTLs. Eventually, a total of 3 QTLs were mapped for chilling tolerance of the rootstock-used pumpkin at the seedling stage. They were named qCI-1-1, qCI-4-1, and qCI-10-1, respectively. The QTL qCI-1-1 was located on LG 1,
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Table 1 QTL analysis of chilling tolerance related to chilling index (CI) in C. moschata. QTL
Linkage groups
Flanking markersa
Peak Positions (cM) (support interval)b
LOD scorec
Additive effectd
R2 (%)e
qCI-1-1 qCI-4-1 qCI-10-1
LG1 LG4 LG10
CMTp163-CMTm140 CMTm113–Unigene0027389 CMTm214-Unigene0008127
65.5 (64.1–68.1) 58.9 (58.8–59.2) 34.9 (33.9–35.1)
2.60 2.70 2.56
−0.51 −0.04 0.52
0.30 1.22 20.88
a
Marker interval. QTL position of peak marker or interval in the LG and the support interval (cM). c Peak LOD value of the QTL flanking a peak LOD score (LOD ≥ 3.0). d Additive effect. A positive value indicates that the positive genes originating from male parent ‘5-5-6 increase the tolerance to chilling stress, and a negative value indicates that positive genes originating from female parent ‘8-3-7 increase the tolerance. e Percentage of the phenotypic variance explained by the putative QTL. b
flanked by the CMTp163 and CMTm140 markers. The distance of qCI-1-1 from the two markers was 0.97 cM and 22.95 cM, respectively. This QTL could only explain 0.3% of the phenotypic variance. The QTL qCI-4-1 was between the CMTm113 and Unigene0027389 markers in LG 4 and could account for 1.22% of the phenotypic variation. The distances between qCI-4-1 and its two flanking markers were 1.07 cM and 0.74 cM. The QTL qCI-10-1 was detected in LG 10, between the CMTm214 and Unigene0008127 markers, which explained 20.88% of the phenotypic variation. The distances between qCI-10-1 and the flanking markers were 1.99 cM and 6.71 cM. The positive genes of qCI-1-1and qCI-4-1 were donated from the female parent (8-3-7), while the positive genes of qCI-10-1 were from the male parent (5-5-6).
4. Discussion The construction of linkage maps is crucial for genetically dissecting the locations and organizations of genes or QTLs that are associated with complicated agronomic traits. Linkage mapping of interesting traits has been applied in many crops, such as rice (Ranawake et al., 2014), wheat (Anderson et al., 2001), Chinese cabbage (Yu et al., 2009) and tomato (Asins et al., 2013). Chilling stress tolerance is an important trait of pumpkins that are used as rootstock for cucumber grafts. To date, no QTL analyses of chilling stress tolerance have been reported, which has led to the study of the genetic basis of chilling stress tolerance in the pumpkin lagging behind such studies in other crops. Hence, the present linkage map and QTL analyses of chilling stress tolerance will provide a framework for further research and breeding. For the pumpkin (Cucurbita moschata), 20 LGs are expected to be mapped in accordance with the haploid chromosome number. Until now, the number of LGs in all published genetic maps of pumpkin (Cucurbita) ranged from 5 to 28 (Brown and Myers, 2002; Esteras et al., 2012; Gong et al., 2008a; Gong et al., 2008b; Lee et al., 1995; Singh et al., 2011; Weeden and Robinson, 1986; Zhang et al., 2015; Zraidi et al., 2007). However, only 3 maps (Gong et al., 2008a; Gong et al., 2008b; Zhang et al., 2015) contained the expected LG number corresponding to the number of chromosomes. In the present study, we obtained 15 LGs in the linkage map, which is less than the expected number of 20. These results indicated that the linkage map was not fully saturated and that some chromosome regions have not been covered in this map. Furthermore, the length of the map that we obtained was 830.7 cM, shorter than maps from the previous studies which ranged from 1195.2 cM to 2566.8 cM (Brown and Myers, 2002; Esteras et al., 2012; Gong et al., 2008a; Gong et al., 2008b; Singh et al., 2011; Zhang et al., 2015; Zraidi et al., 2007). Remarkably, map lengths have been shown not to be comparable between different software, as lengths computed by Joinmap are usually shorter than those obtained by MAPMAKER (Bradeen et al., 2001; Tani et al., 2003). The existence of unlinked markers also demonstrated that the saturated map could be larger than that generated in the present study. In further studies, it is
expected that additional markers may allow deeper connections between linkage groups. In this study, the SSR (Simple Sequence Repeats) marker system was used to analyze an F2 population with 166 individuals and construct a linkage map for Cucurbita moschata. SSRs contain 2–6 base pairs of DNA repeat sequences. They are widely used as molecular markers in genetic mapping, species classification and molecular-assisted selection in crops (Neeraja et al., 2007; Senior, 1998; Temnykh et al., 2001) because SSRs have good marker properties, such as co-dominant conditions, cost-efficient analysis, easy detection, stability and reliability (Quezada et al., 2014; Wu et al., 2014). Additionally, it has been indicated that SSRs show very low levels of polymorphism (Li et al., 2008). A total of 1277 pairs of SSR markers from previously published references (Blanca et al., 2015; Gong et al., 2008b; Wu et al., 2014) were used in the present study. The level of polymorphism detected in SSR markers in our study was 9.48%, which was low compared with other studies. There are two possible explanations for the low level of polymorphism. First, parts of the SSR markers used in this study were developed from the pumpkin transcriptome of Cucurbita pepo, which is different from the plant materials used in this study (Cucurbita moschata). Although the previous study showed that the genomic SSRs developed from C. pepo and C. moschata have a high inter-species transferability (Gong et al., 2008b), a low level of polymorphism was detected in our study. Second, we used intra-species pumpkins as parent lines to construct the genetic map. It is generally believed that the degree of polymorphism is lower in an intra-species population than in an inter-species population (Chen and Chen, 2010). The intra-species parents would have a similar genetic background and a lower level of genetic variability compared to the inter-species parents. As expected, the rate of polymorphism was indeed low. Out of the 121 analyzed markers, 59 (48.8%) were segregationratio distorted from the expected Mendelian segregation ratio at P < 0.05. Distorted segregation could be caused by competition among gametes during fertilization, which results from gametophyte genes expressed in the haploid genotype. Hybrid sterility genes cause the abortion of certain genotypes and could also lead to distorted segregation. Moreover, sublethal and lethal genes can also generate segregation distortion if they lead to embryos which are too feeble to germinate normally. Additionally, physiological and environmental factors could also affect the distorted segregation ratios (Brown and Myers, 2002; Faris et al., 1998; Ky et al., 2000). After grouping the SSR markers on the map, 26 markers could not be integrated. The resulting map covered 830.7 cM in 15 LGs comprising 95 SSR markers, with an average of 8.74 cM between intervals. In addition, small regions (less than 5 markers) in the maps (LG4, LG7, and LG15) showed that the markers are sparse in some regions of the genome and dense in other regions (Becker et al., 1995; Kesseli et al., 1994). The number of LGs was less than the haploid chromosomal number of C. moschata (n = 20). The relatively low polymorphism and the lack of markers would contribute
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Fig. 3. Genetic linkage map of C. moschata and QTLs for CI generated from a ‘5-5-6 × ‘8-3-7 F2 population. Genetic distances (cM) were shown on the left side and marker on the right. The red rectangles mean the support intervals for QTLs in LG1, LG4 and LG10. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
to this phenomenon. This map could become more saturated with additional markers in future research. As far as we know, no available QTL analyses of chilling tolerance in pumpkin have been published. Past studies have shown that
the cold tolerance of plants was a complex quantitative trait controlled by multiple genes (Hu et al., 2016; Liu et al., 2016; Ranawake et al., 2014; Thomashow, 1999). In the present study, we used the CI values of an F2 population derived from a cross of the tolerant
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inbred line ‘8-3-7 and the susceptible inbred line ‘5-5-6 . There was a significant difference between the parental lines, with a lower CI value for the tolerant inbred line ‘8-3-7 than for the susceptible inbred line ‘5-5-6 . The continuous and normal distribution observed in the CI values among the F2 population demonstrated that the trait of chilling stress tolerance was a typically quantitative trait controlled by multiple genes. Identification of potential QTLs is related to material difference, the type of markers, map saturation, LOD value and the length of the map (Tanksley, 1993). Considering that the length of the current map, 830.7 cM, was shorter than the expected whole distance, some underlying QTLs may not be identified in regions that the present map does not cover. Here, for the first time, we detected QTLs for chilling tolerance on different linkage groups by utilizing a CIM strategy. In total, three QTLs were found on three linkage groups with genetic contributions of 0.30%, 1.22% and 20.88%, respectively (Table 1, Fig. 3). Among the three QTLs, QTL qCI-10-1 has the largest effect (>20%), while the others have minor effects (<10%). The genetic distance between QTL qCI-10-1 and the closest flanking marker CMTm214 was 1.99 cM. The allele from the female parent ‘8-3-7 increased the tolerance to chilling stress via QTLs on LG 1 and LG 4, while the male parent ‘5-5-6 increased tolerance via the QTL on LG 10. These findings indicate that parental lines with phenotypically weak performance in the desired agronomic traits can also retain positive alleles in some QTLs. This phenomenon has been reported in previous studies in other crops (Devicente and Tanksley, 1993; Maheswaran et al., 2000). In the practical breeding program for chilling stress tolerance in C. moschata, it is not accurate to screen parental lines by phenotype alone, as the positive alleles may exist in susceptible parent lines as well. Identification of molecular markers linked with QTLs promotes detection of the underlying positive allele and the opportunity to integrate many of these alleles into one objective breeding cultivar with Marker-Assisted Selection (MAS). There are few reports on QTL identification for chilling stress tolerance in rootstock-used pumpkin and we considered it crucial to carry out further work on this genetic map, not only for its theoretical value but also for practical purposes. Acknowledgements This work was financially supported by the China Earmarked Fund for Modern Agro-industry Technology Research System (CARS-25-C-03) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). References Anderson, J.A., Stack, R.W., Liu, S., Waldron, B.L., Fjeld, A.D., Coyne, C., Moreno-Sevilla, B., Fetch, J.M., Song, Q.J., Cregan, P.B., 2001. DNA markers for Fusarium head blight resistance QTL in two wheat populations. Theor. Appl. Genet. 102, 1164–1168. Ando, K., Carr, K.M., Grumet, R., 2012. Transcriptome analyses of early cucumber fruit growth identifies distinct gene modules associated with phases of development. BMC Genom. 13, 518. Argyris, J.M., Ruizherrera, A., Madrizmasis, P., Sanseverino, W., Morata, J., Pujol, M., Ramosonsins, S.E., Garciamas, J., 2015. Use of targeted SNP selection for an improved anchoring of the melon (Cucumis melo L.) scaffold genome assembly. BMC Genom. 16, 4. Asins, M.J., Villalta, I., Aly, M.M., Olías, R., Alvarez De Morales, P., Huertas, R., Li, J., Jaime-Pérez, N., Haro, R., Raga, V., Carbonell, E.A., Belver, A., 2013. Two closely linked tomato HKT coding genes are positional candidates for the major tomato QTL involved in Na+ /K+ homeostasis. Plant Cell Environ. 36, 1171–1191. Basten, C.J., Weir, B.S., Zeng, Z.B., 2001. QTL Cartographer Version 1.15. Department of Statistics, North Carolina State University, Raleigh. Becker, J., Vos, P., Kuiper, M., Salamini, F., Heun, M., 1995. Combined mapping of AFLP and RFLP markers in barley. Mol. Gen. Genom. 249, 65–73. Bhatt, R.M., Harish, D.M., Srilakshmi, 2013. Significance of grafting in improving tolerance to abiotic stresses in vegetable crops under climate change scenario. In: Singh, H.C.P., Rao, N.K.S., Shivashankar, K.S., Pflanzen (Eds.), Climate-resilient Horticulture: Adaptation and Mitigation Strategies. Springer India, India, pp. 159–175.
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