Diversity of cucumber fruit tip is attributed to the epistatic QTL pairs Ft4.1 and Ft6.1

doi:10.21203/rs.2.22509/v1

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Diversity of cucumber fruit tip is attributed to the epistatic QTL pairs Ft4.1 and Ft6.1

https://doi.org/10.21203/rs.2.22509/v1

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Background

The shape of fruit tip is critical to appearance quality and commodity value of cucumber (Cucumis sativus L.). Although diversity of cucumber fruit tip is rich, the genetic basis and molecular mechanism were poorly understood. In this study, the morphological and histological features of fruit tip were investigated to accurately evaluate the diversity in cucumber germplasm. QTLs of fruit tip were genetically mapped by using F₂ and RILs populations. Candidate genes of major-effect QTLs were predicted.

Results

The appearance of cucumber fruit tip was generally divided into sharp, oval-round and blunt-round categories, but morphological measurement showed that the angle of fruit tip was continuously distribute in range of 99~173° and the fruit tip index (the ratio of diameter and length of tips) was in range of 1.4~3.5. Histological analysis suggested that the different structures of fruit tips were owed to the different distribution of cell number in vertical and horizontal direction, rather than difference in cell size. In total, thirty fruit tip related QTLs were identified from two mapping populations including a F₂ population derived from EC1 (blunt round tip) × 8419s-1 (sharp tip) and a RILs population derived from IL52 (oval round tip) × CCMC (sharp tip). Two major-effect QTLs were detected, of which Ft4.1 was detected in both mapping population, while Ft6.1 was only identified in F₂ population. The phenotypes of Ft4.1 were epistatically suppressed by dominant Ft6.1 genotype, suggesting that Ft6.1 has epistatic dominance interaction to Ft4.1. The candidate genes of Ft4.1 were predicted based on genome re-sequencing of parental lines, including CsEXT (Extension-like family protein), CsPRK (leucine-rich repeat protein kinase protein) and CsRNApase (RNA polymerase beta subunit protein), of which CsEXT and CsPRK play important functions during cell division and expansion.

Conclusions

Different shapes of fruit tip were owed to the different frequency and orientation of cell division in tip structures. The fruit tip variation in cucumber maybe controlled by the major QTLs Ft4.1 and Ft6.1 through epistatic interaction.

Plant Physiology and Morphology

Plant Molecular Biology and Genetics

Cucumber

fruit tip

cell division

QTL

epistatic interaction

Cucumber (Cucumis sativus L.) is widely cultivated economic crop. Cucumber fruits shape exhibit extensive variation in cultivated cucumber. Fruit shape-related traits in cucumber such as fruit length, fruit diameter, stalk length, bending fruit, warty fruit, are considered as an important trait for cucumber breeding improvement and associate with the market value of cucumbers. Numerous studies have been performed to demonstrate that fruit shape relate-traits were a quantitative trait locus [1-5]. Several genes for fruit shape related traits, such as Tu (tuberculate fruit), csgl1 (cucumis sativus glabrous 1), csgl3 (cucumis sativus glabrous 3) and tril (trichome-less) for warty fruit [6-9], CsFUL (FRUITFULL-like) for fruit length [10], have been finely mapped or cloned in cucumber.

Fruit tip is a critical part of fruits shape and usually has a less desirable taste as compared with the rest of fruit in cucumber. The domestication and improvement of horticulture crops resulted in abundant variation in fruit tip, which usually was classified according to 3 categories including flat (angle of fruit tip is 180°), indented (angle of fruit tip is greater than 180°) and pointed (angle is less than 180°) shape [11-14]. These classiﬁcation have provided some insights into describing diversity fruit tip shape, while the classification cannot conduct precise quantitative measurements to identify change features of fruit tip shape in a reliable and systematic manner. In the previous study, Zhang et al. identified six QTLs for fruit tip shape in pepper using F₂ population derived from round fruit tip line ‘DaJiao’ and sharp fruit tip line ‘XiaoJiao’ [13]. Geng et al. detected three QTLs associated with fruit tip shape in eggplant using a F₂ population derived from round fruit tip line ‘283’ and sharp fruit tip line ‘284’ [14]. In cucumber, the female flower has a typical inferior ovary, hence the tip of cucumber fruit develops from the fusion part of ovary and receptacle. The complex structures lead to rich diversity of cucumber fruit tips. In East Asian ecotype, most of North-China ecotype cucumber cultivars produce thin and tapering fruit tip, while majority of South-China ecotype as well as Eurasian–ecotype cucumbers present thick and round fruit tip. Xishuangbanna and Indian ecotype usually bear round fruit tip. Diversity fruit tip shape occurs in cultivated cucumber, but very little progress has been made on clarifying morphological and genetic basis of cucumber fruit tip.

In this study, we characterized the physiological differences and cell morphology in diversity cucumber. With the aim of identify QTL associated with fruit tip in cucumber, three indexes (fruit tip diameter, fruit tip length and fruit tip index) were used for identifying fruit tip shape. Genetic mapping of fruit tip shape was performed based on phenotype data of F₂ population derived from the cross of EC1 (blunt round fruit tip) and 8419s-1 (sharp fruit tip) and RILs population derived from the cross of IL52 (oval round fruit tip) and CCMC (sharp round fruit tip). Integration of all QTL information, two major-effect QTL Ft4.1 and Ft6.1 were detected. Further, the interaction between Ft4.1 and Ft6.1 the was analyzed. Candidate regulatory genes of fruit tip formation were predicted by whole genome re-sequencing (WGS) technique. This study will provide important insights into genetic basis of fruit tip in cucumber.

Evaluation and classification of fruit tip shape in cucumber

In practice, fruit tip shape of cucumber cultivars was simply divided into sharp and round types. In present study, the commercial fruits at 10 days after pollination (DPA) of 186 cucumber inbred lines were classified into three categories including blunt-round fruit tip (BFT), oval-round fruit tip (OFT) and sharp fruit tip (SFT) separately based on the appearance of fruit tip (Fig. 1A, Additional file 1: Table S1). The fruit tip angles (α) and fruit tip index (a/b) as described in Fig.1B, were utilized to conduct precise the shape of fruit tip accurately. Among the cucumber inbred lines, fruit tip angle was in range of 99°~173° (Fig. 1C, Additional file 1: Table S1). The fruit tip index was in range of 1.4~3.5 (Fig. 1D, Additional file 1: Table S1). According to the data of fruit tip angle and fruit tip index, thresholds were set to classify BFT, OFT and SFT categories (Fig.1 C-D). The cucumber with range of fruit tip angle less than 125° or range of fruit tip index less than 2.1 were classified into SFT group. The OFT group were considered that had fruit tip angle range from 125° to 150°or fruit tip index range from 2.1 to 2.5. The BFT group were considered that had fruit tip angle range from 150° to 180° or range of fruit tip index greater than 2.5. Though correlation analysis suggested that fruit tip angle was significant correlated with fruit tip index (Fig. 1E), the mismatch rate of defined categories to tip appearance of fruit tip angle (7.6%) was greater than fruit tip index (4.3%) (Supplementary Table S1). Hence, fruit tip index is a flexible application that provides a good indicator to classification of fruit tip shape.

Phenotypic variation of fruit tip related traits

The phenotypic quantification methods of fruit tip shape (the FTD, FTL and FTI) were further to analyze the genetic basis. Phenotypic data of FTD, FTL and FTI were collected from four parents, their F₁, B/S (F₂ population) and O/S (RILs population) population (Table 1, Fig. 2, Additional file 1: Table S2-S5). Significant differences (P=0.05) for fruit tip related traits were detected between EC1 and 8419s-1, CCMC and IL52 (Table 1). All distribution of phenotypic data from B/S and O/S population (RIL population) showed a clear single distribution in violin and box plots (Fig. 2E-G), which suggested that multiple major-effect QTLs controlled fruit tip shape in B/S population and one major-effect QTL controlled fruit tip shape in O/S population. Significant difference of genotypes (G), environment (E), and G × E interactions were observed for all fruit tip related traits in two population. However, variance estimates of G × E interactions(σ²_GE) and error(σ²_E) were less important than the dominated effect of genotypes (σ²_G), which were led to relatively high estimates of heritability (h² >70%) (Additional file 1: Table S2, Table S3). High positive correlations of FTD, FTL, FTI in two environments were consistent with the dominated effect of genotypes (σ²_G) in the two population (Additional file 1: Table S5).

Histological section analysis and comparison of diversity fruit tips

Significant morphological differences (P=0.05) of FTD, FTL and FTI were observed in parental cucumber lines EC1, 8419s-1, IL52 and CCMC (Table 1). Furthermore, histological analysis of four parental cucumber lines were examined at 0 DPA (Fig. 3, Additional file 2: Fig. S1). The fruit tip of cucumber fruit develops from the fusion part of ovary and receptacle. Histological observation of fruit tips suggested that the morphological structure was varied in different cultivars (Fig. 3A). Cytological observation showed that cell number of sharp tip fruit (8419s-1, CCMC) were markedly greater than round tip fruit (EC1, IL52) in the vertical direction, and cell number of sharp tip fruit were fewer than round fruit tip fruit in the radial direction (Fig. 3C). But the cells size of fruit tips had no significant differences (Additional file 2: Fig. S1), which was indicated that the different structures of fruit tips were owed to the different distribution of cell number in vertical and horizontal direction, rather than different cell size. Furthermore, the significance difference of cell numbers ratio (T/H) as the same as FTI could be used for identifying fruit tip category.

QTL analysis

Mean phenotypic data of fruit tip related traits (FTD, FTL, FTI) was collected from B/S and O/S population in multiple environments. QTL analysis was performed in the WinQTLcart2.5 software package. For each fruit tip related traits QTL, the QTL peak location, LOD threshold, 1.5-LOD QTL interval, R² value, additive effects were illustrated in Table 2. QTLs for fruit tip related traits from O/S population were integrated in the genetic map of B/S population based on the physical position of flanking markers in the 9930 draft genomes (Additional file 1: Table S6). The interaction between the major-effect QTLs was analyzed (Table 3, Fig. 4 and Additional file 1: Table S7).

QTL analysis for B/S population

A total of 14 fruit tip related QTLs for fruit tip diameter (Bftd), fruit tip length (Bftl) and fruit tip index (Bfti), were detected in B/S population (Table 2). Three major-effect QTLs, Bfti4.1 (R² = 13.7-15.4% in 2016S and 2016F) and Bfti6.1 (R² = 21.0-35.9% in 2016S and 2016F) for fruit tip index, Bftd6.1 (R² = 32.6-33.6% in 2016S and 2016F) for fruit tip diameter, were detected in two environments. Location of major-effect QTL Bfti6.1 was consistent with QTL Bftd6.1. Four QTLs (Bftd2.1, Bftd3.1, Bftd4.1, Bftd6.1) for fruit tip diameter were detected accounting for 77.5% (2016S) and 33.6% (2016F) phenotypic variations observed. Four QTLs (Bftl1.1, Bftl1.2, Bftl1.3, Bftl4.1) for fruit tip length were detected accounting for 70.2% (2016S) and only 12.7% (2016F) phenotypic variations observed. Six QTLs (Bfti1.1, Bfti1.2, Bfti2.1, Bfti3.1, Bfti4.1, Bfti6.1) for fruit tip index were identified accounting for 43.45% (2016 S) and 76.28% (2016F) phenotypic variations observed.

QTL analysis for O/S population

Sixteen QTLs for fruit tip related traits were detected by analysis phenotypic data of the O/S population in spring and fall 2017 (Table 2). There are four QTLs for fruit tip diameter with one major-effect QTL, Oftd6.1 (R²=11.6-12.4% in 2017S and 2017F) and minor-effect QTL, Oftd3.1 (R²= 6.6% in 2017S) and Oftd3.2 (R²= 6.7% in 2017S). Three QTLs (Oftl3.1, Oftl3.2 and Oftl4.1) were consistent in positive additive effect for fruit tip length, of which Oftl4.1 was a major-effect QTL (R²=10.7-12.8% in 2017S and 2017F). Five QTLs were consistent in negative additive effect for fruit tip index, suggesting allele from IL52 can increase fruit tip index. Ofti4.1 was a major-effect QTL accounting for 11.0-15.5% phenotypic variation observed in two environments. The major-effect QTL Oftl4.1 and Ofti4.1 were also located in the same genomic region.

Two consensus interacting QTL, Ft4.1 and Ft6.1 control round fruit tip shape

Though QTL detection in different environment/population combinations in this study, the location of QTLs from B/S population was highly consistent with the region of QTL from O/S population (Additional file 1: Table S6). These QTLs at the same or near physical interval probably belong to the same QTL/gene for the fruit tip related trait. Four consensus QTLs, Ft3.1, Ft3.2, Ft4.1 and Ft6.1 were identified. Ft4.1 was a major-effect QTL harbored 5 QTL from B/S and O/S population, of which additive effect of Ofti4.1(allele from IL52) and Bfti4.1 (allele from EC1), were consistent in increasing fruit tip index. Ft6.1 was a major-effect QTL harbored two QTL (R²>10%) from B/S population, of which additive effect of Bfti6.1 (allele from EC1) can increase fruit tip index.

Possible epistasis interactions among major-effect QTL Ft4.1 and Ft6.1 were detected in B/S population by the methods of Yang et al [19] and Mackay et al [20]. Epistasis interactions in which the locus Ft6.1 is much greater in the oo of genetic background of Ft4.1 than that in the OO genetic background of Ft4.1 (Fig. 4). Epistasis interaction of Ft6.1/Ft4.1 was accounted for 12.9% of the phenotypic variance in two environments (Additional file 1: Table S7). Considering Ft4.1 and Ft6.1 at two chromosomes, the F₂ population would be expected to follow classical two-gene model in segregation. We examined phenotype data (fruit tip index, fruit tip shape) and genotype at locus Ft4.1 and Ft6.1. As expected, the segregation ratio of both genotype and phenotype did not deviate significantly from expected ratios of 12 blunt round fruit tip (_B_): 3 oval round fruit tip (O_bb): 1 sharp fruit tip (oobb) (Table 3), supporting epistatic dominance interaction between Ft6.1 and Ft4.1 control fruit tip formation. The phenotype of Ft4.1 was suppressed by epistatically suppressed by dominant Ft6.1 genotype (Table 3).

Candidate genes prediction of Ft4.1

Although the physical location of Ft4.1 has 680 genes in reference genome database of 9930 (data not shown), only three candidate genes, CsEXT (Csa4G291360), CsPRK (Csa4G385800), CsRNApase (Csa4G414910) were predicted based on genome resequencing of four parental cucumber line (Table 4). CsEXT encodes proline-rich extension-like family protein (PRP). PRP was an important call wall protein that was crucial for plant cell division and expansion [21-22]. CsPRK, an ortholog of AtPRK4, encodes leucine-rich repeat protein kinase family protein (PRK), which can regulate the actin cytoskeleton that was associate with cell division and expansion [23-25]. CsRNApase encodes RNA polymerase beta subunit protein. However, the RNA-seq data showed that only CsPRK (log2-fold change =1.87 and FDR-corrected P-value≤0.001) was differentially expressed during fruit development of EC1 and 8419s-1 [17].

Both additivity and epistasis of Ft4.1 and Ft6.1 control the fruit tip variation

Many studies demonstrated that traits of fruit shape were complex that controlled by several QTLs. Fruit tip is a critical part of fruits that associate with the appearance quality of fruit crops. The six QTLs for pepper fruit tip shape were detected by Zhang et al. [13]. In eggplant, three QTLs for fruit tip shape were identified by Geng et al. [14]. In this study, phenotype data of fruit tip related traits showed that fruit tip shape of cucumber was also controlled by multiple genes (Fig. 2), of which QTL Ft4.1 and Ft6.1 (R²>10% in multiple environments) were detected in multiple environments suggesting the QTL pair are major-effect QTLs controlling variation of cucumber fruit tip (Table 3; Additional file 1: Table S6). Integration analysis of fruit shape related QTLs showed that the major QTL Ft4.1 and Ft6.1 co-located with many other fruit shape related QTLs such as QTLs of fruit stalk length [13, 26-27], fruit length [3, 4, 27] and fruit diameter [4] (Additional file 1: Table S6). These results implied that Ft4.1 and Ft6.1 maybe involved in pleiotropic regulation processes of cucumber fruit development.

Efforts to QTL mapping for quantitative traits have mainly focused on estimating additive effects of single loci, which was dominated narrow-sense heritability (h²) [20]. The additive effect of Ft4.1 and Ft6.1 from round fruit tip cucumber lines (EC1 and IL52) were consistent in increasing fruit tip index, which accounted for 12.9 to 35.9% of the phenotypic variance, respectively (Table 2). Epistasis interaction for quantitative traits refers to modification effect that the phenotype of one locus is either enhanced or suppressed by genotypes at the other locus [20,28-29]. Epistasis interaction of Ft6.1/Ft4.1 was accounted for 12.9% of the phenotypic variance. (Additional file 1: Table S7). Under epistatic dominance interaction between Ft6.1 and Ft4.1 in B/S population (Table 3), Ft4.1 and Ft6.1 were controlled formation of oval round fruit tip, blunt round fruit tip respectively. But the phenotype of Ft4.1 was suppressed by epistatically masked by a homozygous or heterozygote dominant Ft6.1 genotype.

Similarly, expected genotypic data of Ft4.1 among 155 RILs population were consistent with 1 OO: 1 oo ratio (AA: aa=69:80, data not shown). Although there were 19 RILs population with blunt round fruit tip, expected phenotypic data were consistent with 1 oval round fruit tip: 1 sharp fruit tip ratio (70: 66, data not shown), suggesting that major-effect QTL Ft4.1 controlled formation of oval round fruit tip in O/S population. The phenotypic data deviation from expected segregation in O/S population may also be related with environmental conditions. With two QTLs model of Ft4.1 and Ft6.1, it possible provides insight into fruit tip shape formation controlling by a pair epistasis gene.

Orientation of cell division is critical to fruit tip development in cucumber

Frequency of cell division is critical to fruit morphogenesis in a variety species such as Tomato [30-33], melon [37], pumpkin [35], cucumber [36-39]. In present study, we found that the fruit tip shape in 186 cucumbers inbred lines also were a complex trait and exhibit significant variations with angle range from 99 to 173° and fruit tip index range from 1.4 to 3.5. Histological sections showed that the cell size in fruit tips had no significant differences in cucumber cultivars (Additional file 2: Fig. S1). However, cell number in fruit tip had significant differences in the vertical and radial direction (Fig. 3). On the one hand, there was a markedly greater cell number of sharp fruit tip (SFT) in the vertical direction, but round fruit tip (BFT and OFT) had greater cell number in the radial direction (Fig. 3C), suggesting cell division of SFT was more active than round fruit tip in vertical direction, round fruit tip was more active than SFT in radial direction. On the other hand, although cell number of round fruit tip was not significantly different in vertical directions, BFT had a greater cells number than OFT in the radial direction (Fig. 3C), suggesting cell division of BFT was more active than OFT in radial direction. Three candidate genes for Ft4.1 were identified based on re-sequencing data in B/S and O/S parental lines (Table 4), of which CsERK and CsPRK were reported that involved in the processes of cell division and expansion [21-25]. Hence, the different structures of fruit tips were associated with the orientation of cell division.

Diversity of cucumber fruit tip maybe owed to selection and domestication of fruit size

Significant variations in fruit shape in cultivated cucumber owed to the continuous cucumber domestication and improvement [40]. The progenitor of the cultivated cucumber, cucumis sativus var. hardwickii [41], bear blunt-round fruit tip fruit with fruit tip angel of 166°±5° and fruit tip index of 3.231±0.118 (Additional file 1: Table S1). In European, breeders are interested in selecting gynoecious cucumber cultivars with limited fruit size to increase fruit load, thus improve yield per plant. So, most of Eurasian–ecotype cucumbers have relatively small size and round fruit tips that similar to Cucumis sativus var. hardwickii. In China, people used for improving fruit size of cucumber, including fruit diameter and fruit length, to increase yield. For instance, majority of South-China ecotype and Xishuangbanna ecotype cucumber present thick and oval-round or blunt round fruit tip with relatively larger fruit diameter. Most of North-China ecotype cucumber cultivars produce very long fruit with a thin and tapering fruit tip as well as an obvious fruit stalk. Hence, we speculated that diversity of cucumber fruit tip maybe owed to selection and domestication of fruit size.

Different shapes of fruit tip were owed to the different frequency and orientation of cell division in tip structures. Two novel epistatic interaction loci Ft4.1 and Ft6.1, which attribute to the fruit tip shape formation in cucumber, were detected using QTL mapping.

Plant materials and growth conditions

One-hundred and eighty-six cucumber inbred lines from different cucumber ecotype were preserved in State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, of which four cucumber inbred lines were used for genetic mapping of fruit tip shape. One hundred and forty-five F₂ populations (B/S population) were generated from the cross between ‘EC1’ and ‘8419s-1’ [15]. One hundred and fifty-five RILs population (O/S population) were derived from the cross of ‘CCMC’ and ‘IL52’ [16]. ‘EC1’ is an inbred line derived from a European type ‘Delta star’ [15], which bear blunt round fruit tip with fruit tip index in range of 2.873±0.110 and fruit tip angel in range of 151°±7° (Fig. 2A, Additional file 1: Table S1). 8419s-1 is an inbred line derived from European type ‘Thaminbeit alpha’ [15], which bear sharp round fruit tip with fruit tip index in range of 1.727±0.111 and fruit tip angel in range of 114°±5° (Fig. 2A, Additional file 1: Table S1). IL52 is an introgression line derived from interspecific hybridization between cucumber and the wild relative species Cucumis hystrix Chakr. [16,42-44], which bear oval round fruit tip with fruit tip index in range of 2.441±0.047 and fruit tip angel in range of 127°±4° (Fig. 2B, Additional file 1: Table S1). CCMC, a typical North China fresh market type cucumber-derived inbred line with high harvest index [16], bear sharp fruit tip with fruit tip index in range of 1.691±0.098 and fruit tip angel in range of 107°±2°. (Additional file 1: Table S1, Fig. 2B). All plants were grown 30/20°C (day/night), 12 hours photoperiod and a relative humidity of 85% at green house of Baima field base of Nanjing Agricultural University.

Paraffin section and microscope observation

Ovaries sample of ‘EC1’, ‘8419s-1’, ‘IL52’ and ‘CCMC’ at 0 DAP were collected from the 10-20 plant nodes. The fruit tip was isolated immediately from ovary and immersed in FAA solution (formalin: acetic acid:75% ethanol, 5:5:90, v/v/v) for 24 hours [17]. Fruit tip samples were dehydrated through a graded ethanol/xylene series and embedded in paraffin. The paraffin blocks were cut with a microtome into 5 µm thick sections along the vertical axis of ovaries and the sections stained with hematoxylin and eosin staining [18]. The paraffin sections were observed and photographed using an Olympus BX53 microscope equipped with SPOT digital camera under the magnification of 40´10.

Phenotypic data collection

Phenotypic data for fruit tip related traits, including fruit tip angle (FTA), fruit tip diameter (FTD), fruit tip length (FTL) and fruit tip index (FTI), were collected at 10 DPA. As showed in Fig 1B, the angle between two tangents that draw from the top point of fruit tip was measured as FTA (α), the diameter at the junction point between endocarp and mesocarp was measured as FTD (a), the shortest length between endocarp and top point of fruit tip was measured as FTL (b), FTI was the ratio of FTD to FTL.

In present study, FTI and FTA were a flexible application that provides a good indicator to classification of fruit tip shape. The FTI and FTA of 186 cucumber inbred-lines were investigated in spring 2016 with two repeat experiments. Phenotypic data of B/S population including FTD, FTL and FTI were performed in two experiments in spring and fall of 2016, which were designated as 2016S, 2016F, receptively. Phenotype data of O/S population including FTD, FTL and FTI were performed in two experiments in spring and fall of 2017, which were designated as 2017S, 2017F, receptively.

For each inbred line and individual of the populations, 5 plants were grown as biological repeats using randomized complete block experimental design (RCBD). Enlarged fruits were limited to 2 per plant and lateral branches were cut off to reduce the effect of ‘first-fruit inhibition’ which could lead to abnormal fruit set and malformed fruit.

QTL detection and refinement

The genetic linkage maps of the mapping populations were constructed by researchers in our lab. The genetic map of B/S population was constructed by Wu et al (2016), including 133 SSR and 9 InDels markers [15]. The genetic map of O/S population was constructed by Zhang et al, including 197 SSR and 19 Indel markers [16].

The fruit tip related traits QTLs were detected by using WinQTLcart2.5 through the composite interval mapping (CIM) procedure based on phenotypic data of FTD, FTL and FTI from B/S and O/S populations. The presence of QTLs with a minimum LOD threshold of 2.5 were performed using permutation test with 1000 repetitions at 1.0 cM walk speed and threshold at P ≤ 0.05 [15-16]. The intervals for the genetic map locations of significant QTL were calculated using a 1.5 LOD interval. Nomenclature of fruit tip related QTLs were an abbreviation of the segregation population + fruit tip related trait + relevant chromosome number + QTL serial number on chromosome. A QTL, which can explain more than 10 % observed phenotypic variations (R²) and stable detected in multiple environment, was considered a major effect QTL.

Consensus QTLs for fruit tip related traits from B/S and O/S population was integrated based on the physical location of flanking markers. Consensus QTL was a synopsis of multiple QTLs, which were stable QTLs in at least one environment or population and shared the same or had overlapped physical location. Epistasis interactions of major-effect QTLs were performed by using the peak markers of the QTLs according to the methods of Yang et al. [19] and Mackay et al. [20]. The peak marker of Ft4.1 is SSR26165 and peak marker of Ft6.1 is SSR19174.

Identification of candidate genes for the Ft4.1

Genes located within the confidence interval of Ft4.1 were analyzed based on the whole genome parental resequencing data from Wu et al. [15] and Zhang et al. [16]. Only those common genes, which was filtrated on basis of polymorphic SNP/Indels causing non-synonymous mutations or frameshift deletion/ insertion, were considered as candidate genes in B/S and O/S population.

Statistical analysis

The software Statistical Analysis System (SAS, version 8.0) and R package was used for analyzing of phenotypic data including FTA, FTD, FTL and FTI. Violin and box plots were depicted phenotypic data distribution of fruit tip related traits by R package. Correlations between FTI and FTA of 186 cucumber inbred lines were analyzed by using PROCCORR function. Analysis of variance (ANOVA) of the phenotypic data including FTD, FTL and FTI from B/S and O/S population, were conducted with GLM (general linear models) program to the estimate the effect of gene and environment with a model of Y_ijk = mu + Genotype (G)_i+ Environment (E)_j+ G ´ E_ij+ Error_ijkin SAS. The broad sense heritability (h²) was calculated variance components and estimated with model of h²= σ²_G/ (σ²_G + σ²_GE /R_E + σ²_E/R_ERn). The segregation ratios in B/S and O/S population were analyzed with a χ2 goodness-of-ft test using FREQ (frequency) procedure in SAS.

ANOVA: Analysis of variance

BFT: Blunt round fruit tip

CIM: Composite interval mapping

DAP: Days after pollination

FTD: Fruit tip diameter

FTI: Fruit tip index

FTL: Fruit tip length

FTA: Fruit tip angel

OFT: Oval round fruit tip

QTL: Quantitative trait loci:

RILs: Recombinant inbred lines:

SD: Standard deviation:

SFT: Sharp round fruit tip:

SSR: Simple sequence repeat:

B/S population: F₂ population derived from the cross of EC1 (BFT) and 8419s-1(SFT)

O/S population: RILs population derived from the cross of IL52 (OFT) and 8419s-1(SFT)

WGS: Whole genome re-sequencing

Ethics approval and consent to participate

Not applicable.

Consent to publish

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by National Natural Science Foundation of China (No. 31672168), National Natural Science Foundation of JiangSu province (BK20191312) and National Natural Science Foundation of China (Key Program, 2016YFD0101705-5). The funding bodies had no role in the design of the study or collection, analysis, and interpretation of data and neither in writing the manuscript.

Authors’ contributions

JFC and JL conceived and supervised all of the study; PYZ and KL performed most of experiments and data analysis; KJZ, ZW participated in construction of QTL mapping; PYZ, JX, XYW, TTW and TLZ participated phenotypic data. PYZ drafted the manuscript; JFC and JL revised manuscript. All of the authors read and approved the manuscript.

Availability of data and materials

All relevant supporting data can be found within the Additional files accompanying this article.

Acknowledgements

We thank Dr. Liang Kun for technical assistance. We are very grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.

Author information

Affiliations

State Key Laboratory of Crop Genetics and Germplasm Enhancement, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China

Pinyu Zhu, Kaijing Zhang, Xueyan Wang, Xiaodong Qin,Tuantuan Wang, Tinglong Zhong, Jian Xu, Zhe Wu, Jinfeng Chen, Ji Li

Corresponding authors

Correspondence to Ji Li and Jinfeng Chen.

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Table1. Means, standard errors (SE) and least significant differences (LSD) for fruit tip-related traits in four cucumber parental inbred lines (EC1, 8419s-1, CCMC and IL52) and their F₁, B/S population (Spring and Fall 2016), O/S population (Spring and Fall 2017).

Trait	EC1	8419s-1	F₁	B/S (2016S, n=145)		B/S (2016F, n=145)		Heritability (h²)
	Mean^a	Mean^a	Mean^a	Mean ± SD	Range	Mean ± SD	Range
FTD	2.73*	1.85*	2.28	2.30±0.28	1.44-3.09	2.39±0.27	1.45-3.17	0.762
FTL	0.97*	1.24*	1.04	1.01±0.13	0.79-1.25	1.05±0.14	0.76-1.38	0.719
FTI	2.87*	1.73*	2.68	2.58±0.34	1.72-3.46	2.70±0.35	1.79-3.65	0.742
Trait	CCMC	IL52	F₁	O/S (2017S, n=155)		O/S (2017F, n=155)		Heritability (h²)
	Mean^a	Mean^a	Mean^a	Mean ± SD	Range	Mean ± SD	Range
FTD	2.94*	1.96*	2.33	2.25±0.36	1.65-2.78	2.28±0.32	1.56-2.87	0.732
FTL	1.81*	0.96*	1.17	1.23±0.19	0.87-1.89	1.18±0.19	0.85-1.90	0.718
FTI	1.69*	2.44*	2.27	2.19±0.34	1.38-3.28	2.23±0.25	1.45-2.65	0.723

The unit of phenotypic data (FTD and FTL) is centimeters (cm).

^a Based on data across two environments.

* Significance difference (P＜0.05) between parental inbred lines.

Table 2 QTLs for fruit tip related-traits were detected in B/S and O/S population

QTL	Environments	Mapping population	Chr.	Position (cM)	LOD value	R2/%	Additive effect	1.5 LOD interval (cM)		Marker interval
QTL	Environments	Mapping population	Chr.	Position (cM)	LOD value	R2/%	Additive effect	Left	Right	Marker interval
Bfti1.1	2016F	B/S	1	0.01	3.0	10.6	0.166	0.0	2.1	SSR15108-SSR10018
Bftl1.1	2016S	B/S	1	31.5	3.7	22.5	-0.084	18.8	39.5	UW085396-UW084148
Bftl1.2	2016S	B/S	1	53.0	3.8	24.7	0.090	45.6	56.6	SSR12331-SSR04805
Bfti1.2	2016F	B/S	1	43.1	2.6	9.0	-0.157	31.3	56.2	SSR10134-SSR04805
Bftl1.3	2016S	B/S	1	60.0	3.4	23.0	0.085	56.6	74.0	SSR04805-UW37302
Bfti2.1	2016S	B/S	2	12.2	2.7	8.8	-0.123	0.0	19.2	SSR03070-SSR13532
Bftd2.1	2016S	B/S	2	33.8	2.7	11.5	-0.134	89.3	106.6	SSR20132-SSR1058
Oftd3.1	2017S	O/S	3	64.8	2.6	6.6	0.069	58.9	66.3	UW083972-SSR14669
Oftd3.2	2017S	O/S	3	71.8	2.7	6.7	0.073	66.3	81.9	SSR14669-SSR23725
Ofti3.1	2017S	O/S	3	74.8	2.5	7.0	-0.102	72.4	80.8	SSR04632-SSR20270
Oftl3.1	2017S	O/S	3	83.9	2.7	7.0	0.052	80.6	89.6	SSR10697-UW083999
	2017F	O/S	3	80.8	3.0	7.3	0.056	80.5	88.3	SSR10697-UW083999
Bftd3.1	2016S	B/S	3	83.5	3.1	14.0	1.980	77.6	93.6	SSR10367-SSR16667
Ofti3.3	2017F	O/S	3	96.3	3.1	9.5	-0.078	92.3	99.9	UW085097-SSR05678
Oftl3.2	2017F	O/S	3	105.9	3.0	8.0	0.059	99.6	115.2	SSR07131-SSR04628
Ofti3.2	2017S	O/S	3	112.2	4.5	14.6	-0.133	107.2	115.2	SSR07131-SSR06791
Bfti3.1	2016F	B/S	3	114.9	5.1	20.8	0.210	99.3	132.0	UW085395-SSR06791
Bftd4.1	2016S	B/S	4	13.9	3.7	19.4	2.400	0.0	29.8	UW019153-SSR17024
Oftl4.1	2017S	O/S	4	66.3	3.7	10.7	0.059	56.2	73.9	SSR20307-SSR26165
Oftl4.1	2017F	O/S	4	66.3	4.5	12.8	0.063	56.2	77.1	SSR20307- SSR22862
Ofti4.1	2017S	O/S	4	67.0	4.0	13.4	-0.115	56.2	75.5	SSR20307-SSR26165
Ofti4.1	2017F	O/S	4	67.0	2.9	15.5	-0.168	66.2	69.9	SSR15737-UW084951
Bftl4.1	2016F	B/S	4	75.1	2.6	12.7	0.519	71.6	88.3	UW029413-SSR21563
Ofti4.2	2017F	O/S	4	76.9	3.4	16.4	-0.064	69.9	83.1	UW08495-SSR10368
Bfti4.1	2016S	B/S	4	94.4	3.8	13.7	0.158	73.9	106.7	SSR05125-SSR21563
Bfti4.1	2016F	B/S	4	94.4	4.7	15.4	0.162	73.9	106.7	SSR05125- SSR21563
Oftd6.1	2017S	O/S	6	65.5	4.6	12.4	-0.095	55.5	72.5	SSR13884-SSR01566
	2017F	O/S	6	65.5	3.4	11.6	-0.067	59.3	84.9	SSR13884- UW085222
Bftd6.1	2016S	B/S	6	77.8	4.9	32.6	3.510	74.7	95.2	SSR19174-SSR12898
Bftd6.1	2016F	B/S	6	77.8	5.8	33.6	3.760	74.7	95.2	SSR19174-SSR12898
Bfti6.1	2016S	B/S	6	88.6	5.9	21.0	0.196	74.8	99.6	SSR19174-SSR12898
Bfti6.1	2016F	B/S	6	85.5	8.7	35.9	0.276	74.8	96.9	SSR19174-SSR12898

Table 3 The genotype and phenotype data conditioned by epistatic dominance interaction model QTL between Ft4.1 and Ft6.1 in B/S population

Genotype data of B/S population			Exception phenotype data			Observations phenotype data			χ2 value
Epistatic interaction model	Genotype	Observations	SFT	OFT	BFT	SFT	OFT	BFT	χ2 value
1 aabb	aabb	6	6	0	0	5	1	0	0.33< χ2(2,0.05)
3 A_bb	Aabb	22	0	22	0	2	15	5	2.55< χ2(2,0.05)
3 A_bb	AAbb	11	0	11	0	1	8	2	1.28< χ2(2,0.05)
12 B_	aaBb	13	0	0	13	1	0	12	0.154< χ2(2,0.05)
	aaBB	7	0	0	7	0	0	7	0< χ2(2,0.05)
	AaBb	40	0	0	40	1	7	32	2.85< χ2(2,0.05)
	AaBB	23	0	0	23	0	1	22	0.087< χ2(2,0.05)
	AABb	15	0	0	15	1	3	11	1.73< χ2(2,0.05)
	AABB	8	0	0	8	0	0	8	0< χ2(2,0.05)

χ2(2,0.05) =5.991

Table 4 Candidate genes of Ft4.1 based on re-sequencing analysis of parental line 8419s-1, EC1, IL52 and CCMC. The common mutant sites were symbolled in grey column.

Gene ID	Mutation (8419s-1→ EC1/position)	Amino acid change	Gene ID	Mutation (CCMC→ IL52/position)	Amino acid change	Annotation
CsEXT			CsEXT	C→G/11391101	Pro→Arg	Proline-rich extension-like family protein
	A→G/11391121	Ile→Val
	C→G/11391182	Pro→Arg
				G→A/11391346	Val→Ile
				C→A/11391620	Ser→Tyr
				G→A/11391623	Ser→Asn
	C→G/11391624	Ser→Arg		C→G/11391624	Ser→Arg
	G→A/11391719	Ser→Asn		G→A/11391719	Ser→Asn
	C→G/11391720	Ser→Arg		C→G/11391720	Ser→Arg
				G→A/11391886	Val→Met
				C→T/11391958	Pro→Ser
CsPRK			CsPRK	T→C/15310393	Asn→Asp	PRK4 (Pollen Receptor protein kinase) like protein
	A→T/15310395	Phe→Tyr		A→T/15310395	Phe→Tyr
	A→G/15310396	Phe→Leu		A→G/15310396	Phe→Leu
				A→T/15310397	Asp→Glu
CsRNApase	C→T/15525734	Ala→Thr	CsRNApase			RNA polymerase beta subunit
	G→T/15525761	Arg→Ser		G→T/15525761	Arg→Ser
	G→A/15525785	Arg→Cys

Additional files 1:

Table S1 Fruit tip phenotypic features of commodity fruits in 185 cucumber inbred lines. * means the mismatch between FTI or FTA defined categories to tip appearance defined categories (The fruits with fruit tip angel and fruit tip index that less than 125° and 2.1 were classified into SFT group. The OFT fruits were considered that had fruit tip angel ranged between 125° and 150°or fruit tip index ranged 2.1 to 2.5. The BFT fruits were considered that had fruit tip angel ranged between 150° and175° or fruit tip index ranged 2.5 to 3.5).

Table S2 Analysis of variance, variance component estimates and heritability of fruit tip related traits for B/S population of EC1×8419s-1 and O/S population of IL52 × CCMC.

Table S3 Restricted maximum likelihood variance components and heritability estimates based on B/S population of EC1× 8419s-1 and O/S population of CCMC × IL52.

Table S4 Correlation analysis of phenotype data of B/S population of EC1× 8419s-1 gathered from two individual experiments in 2016 spring and 2016 fall separately.

Table S5 Correlation analysis of phenotypic data of O/S population of CCMC × IL52 gathered from two individual experiments in 2017 spring and 2017 fall separately.

Table S6 Fruit shape related QTLs were integrated in the genetic map of B/S population based on the physical position of linkage markers. QTLs detected in different environment were marked by dark grey color ( B/S population) and light grey color(O/S population ); *, The major-effect QTLs(R2 >10%) were marked by stars(*); The major-effect and stable detected QTLs between B/S and O/S populations were considered as the consensus QTLs and marked in blue color; Fruit shape related QTLs that detected in others studies were also integrated in genetic map of B/S population.

Additional files 2:

Fig. S1 Fruit tip tissue sections of EC1(A),8419s-1(B), IL52(C), CCMC(D). The average size of cells has no significant differences in in the four inbred.

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Diversity of cucumber fruit tip is attributed to the epistatic QTL pairs Ft4.1 and Ft6.1

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