The authors have declared that no competing interests exist.
Mechanized harvesting of cucumbers offers significant advantages compared to manual labor as both shortages and costs of labor increase. However the efficient use of machines depends on breeding plants with longer peduncles, but the genetic and molecular basis of fruit peduncle development in cucumber is not well understood. In this study, F2 populations were developed from a cross between two inbred lines, 1101 with a long peduncle and 1694 with a short peduncle. These were grown at two field sites, Hainan, with a tropical marine climate, in December 2014, and Beijing, with a warm temperate climate, in May 2015. Electron microscope examination of the pith cells in the peduncles of the two parental lines showed that line 1101 had significantly greater numbers of smaller cells compared to line 1694. The inheritance of cucumber fruit peduncle length (FPL) was investigated by the mixed major gene and polygene inheritance model. Genetic analysis indicated that FPL in cucumber is quantitatively inherited and controlled by one additive major gene and additive-dominant polygenes (D-2 model). A total of 1460 pairs of SSR (simple sequence repeat) primers were analyzed to identify quantitative trait loci (QTLs). Two similar genetic maps with 78 SSR markers which covered 720.6 cM in seven linkage groups were constructed based on two F2 populations. QTL analysis from the data collected at the two field sites showed that there are two minor QTLs on chromosome 1, named
Cucumber,
Many different traits relating to the quality of cucumber fruit have been reported, including the length, weight, and diameter of fruit [
Cui et al. [
At the molecular level, Yuan et al. [
In the present study, two inbred lines, one with a long fruit peduncle and the other with a short fruit peduncle, were crossed to construct genetic populations for inheritance analysis and chromosomal mapping of the related QTLs. The results from this study will promote the breeding of new cultivars of cucumbers that would be better adapted to mechanical harvesting and thus obviate the problems of both shortage of labor and higher costs.
Inbred line 1101 with long fruit peduncles and inbred line 1694 with short fruit peduncles were used as parental lines to develop a segregating population for inheritance analysis and QTL mapping. 1101, the female parent (P1), is from northern Europe with an average fruit peduncle length (FPL) of more than 5.5 cm whereas 1694, the pollen donor (P2), is from southern China with an average FPL of less than or equal to 2 cm. The F1 population was self-pollinated to generate the F2 population, and the F1 population was backcrossed with either 1101 to generate BC1P1 or with 1694 to generate BC1P2.
All plants were grown at two sites: the Sanya Science and Technology Academy for Crop Winter Multiplication at Sanya, Hainan in December, 2014 (18°15' N, 109°30' E; average day/night temperatures 22°C /19°C; daylength ~11 hr) and in the greenhouse of the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Science at Shunyi, Beijing in May, 2015 (40°10' N, 116°51' E; average day/night temperatures 26°C/14°C; daylength ~14 hr). Nineteen cucumber lines, including twelve with FPL > 5.0cm and seven with FPL < 1.2cm, were used to validate the molecular markers closest to the major QTL.
Fruit peduncles were collected from lines 1101 and 1694 14 days after anthesis. Samples for paraffin embedding were fixed in FAA (3.7% formaldehyde, 5% glacial acetic acid and 50% ethanol) and stored at room temperature for further embedding. The fixed samples were dehydrated in a graded series of ethanol (70%, 85%, 95%, and 100%), followed by a xylene/ethanol series (xylene:ethanol 1:3, 1:1, and 3:1) and finally 100% xylene. Xylene was replaced gradually with paraffin (Paraplast Plus, Sigma, P3683) at 60°C for two nights with four times replacement of paraffin. Ten μm sections were made using a HEISTION ERM3000 microtome and stained with Toluidine Blue O [
The peduncle lengths of three to five fruits of each plant were measured from the peduncle bottom to the joint with the stem with a precision of 0.1 cm when the fruits were commercially mature. The data were analyzed using Microsoft Excel 2013 using the mixed major gene and polygene inheritance model of Gai et al. [
The continuous phenotypic distribution of peduncle length was analyzed using the maximum likelihood estimation via the IECM algorithm [
Genomic DNA was extracted from young leaf tissue of the parents P1 and P2, and the F1 and F2 populations using a modified CTAB extraction procedure [
PCR amplification was carried out in a volume of 10 uL, containing 3 uL of DNA (15 ng/uL), 1 uL of both forward and reverse primers (50 ng/uL), and 5 uL of Go Taq Green Master Mix (Promega, USA). The PCR program was as follows: denaturation at 94°C for 4 min, 35 cycles of denaturation at 94°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 30 s, with a final extension at 72°C for 5 min. Amplified products were separated on 6.0% non-denaturing polyacrylamide gels at 150 V for 1 h, and the bands were visualized and photographed after silver staining.
JoinMap 4.0 software [
Longitudinal sections of peduncles in the parental lines showed that both the number and size of the cells differed in the pith tissue between the vascular bundles. There were significantly more cells per unit area in line 1101 compared to line 1694, and the cells were significantly smaller (
a-b Microscopic longitudinal sections of the fruit peduncle 14 days after anthesis in line 1101 (a) and line 1694 (b) (Vb, vascular bundles; Pi, pith). The dimensions of the white outlined box is 889 μm × 889 μm, and the black bar is 250 μm. c-d The cell number (c) and cell size (d) were approximately calculated. The bars show significance calculated by the unpaired t test, P<0.05.
Fruit peduncle lengths for line 1101 (P1) at Hainan in 2014 and at Beijing in 2015 varied from 4.50 to 7.50 cm and 5.10 to 6.70 cm, respectively whereas for line 1694 (P2) at the two sites the lengths varied from 0.90 to 3.50 cm and 0.50 to 1.50 cm, respectively. The mean fruit peduncle lengths of F1 at Hainan and Beijing were 4.60 cm and 2.90 cm, respectively (
a 1101 (P1, left), 1694 (P2, middle) and their F1 (right). 1101 had longer fruit peduncles than 1694, and F1 between them. b The frequency of fruit peduncle length of P1, P2, F1 and F2 populations at Hainan in December, 2014 and Beijing in May, 2015.
Values calculated for both AIC and the maximum likelihood function for the mixed major gene and polygene model suggested 23 kinds of inheritance (
Estimates of the first order parameters for the D-2 model indicated that the additive gene effect was higher than the dominant effect, and that parental line 1101 with long fruit peduncles contributed to the latter. However there were differences in heritability between the two sites. For example, the heritability of the major gene in the BC1P1 generation at Hainan was significantly higher than that for the polygenes, 53.30% compared to 5.80%, whereas it was slightly lower than polygenes at Beijing, 42.60% compared to 49.40%. On the other hand, the heritability of the major gene in BC1P2 and F2 generations was lower than that for the polygenes at Hainan but it was the opposite at Beijing (
Time | 1st order parameter | Estimation | 2nd order parameter | Estimation | ||
---|---|---|---|---|---|---|
B1 | B2 | F2 | ||||
m | 3.97 | σ |
1.53 | 0.78 | 3.09 | |
d | 1.04 | σ |
0.81 | 0.02 | 0.87 | |
2014-Hainan | [d] | 1.11 | σpg2 | 0.09 | 0.14 | 1.6 |
[h] | 0.51 | σ2 | 0.63 | 0.63 | 0.63 | |
h |
53.30% | 2.10% | 28.10% | |||
h |
5.80% | 17.80% | 51.70% | |||
m | 3.26 | σ |
3.12 | 0.34 | 2.56 | |
d | 1.49 | σ |
1.33 | 0.09 | 1.61 | |
2015-Beijing | [d] | 0.98 | σpg2 | 1.54 | 0 | 0.69 |
[h] | -0.6 | σ2 | 0.25 | 0.25 | 0.25 | |
h |
42.60% | 26.60% | 63.10% | |||
h |
49.40% | 0.00% | 27.10% |
m, the average of population; d, additive effect of major gene; [d], additive effect of polygene; [h], dominant effect of polygene; σ
After screening a total of 1,460 SSR markers, 236 (16.2%) were selected according to polymorphisms between the two parents. 78 markers scattered on cucumber chromosomes with proportional spacing and these were used to construct the linkage map (
Three QTLs were detected at the same location at both Hainan and Beijing, respectively.
The genetic map was used to detect QTLs for fruit peduncle length. After a first round of interval mapping (IM), markers with the highest LOD value were selected as cofactors for MQM mapping with the MapQTL4.0 software. Coincidently, both IM and MOM mapping approaches detected the same QTLs for the two sites. Two QTLs detected on chromosome 1 were named
Seasons | QTL | Chromosome (chr.) | Marker Interval | LOD | R2/% | Additive Effects |
---|---|---|---|---|---|---|
2014-Hainan | 1 | SSR03462-CMBR40(97) | 4.65 | 9.80% | 0.77 | |
1 | SSR04992-1CS6 | 4.67 | 9.80% | 0.76 | ||
6 | SSRFPL-1-UW021226 | 10.84 | 21.50% | 1.13 | ||
2015-Beijing | 1 | SSR10018-CMBR40(97) | 5.15 | 7.70% | 0.53 | |
1 | SSR04992-SSR10134 | 6.9 | 10.50% | 0.82 | ||
6 | SSRFPL-1-UW021226 | 12.95 | 27.50% | 1.22 |
Marker UW021226, the closest to
Material code | Fruit peduncle length/cm | UW021226 | Material code | Fruit peduncle length/cm | UW021226 |
---|---|---|---|---|---|
1101 | 5.10 ~6.70 | a | CG45 | 0.4 | b |
1694 | 0.50 ~1.50 | b | CG50 | 5.25 | b |
F1 | 2.10 ~4.10 | h | CG57 | 5.07 | a |
CG11 | 0.95 | b | CG90 | 0.97 | a |
CG15 | 1.1 | b | CG91 | 5.67 | a |
CG26 | 5.72 | a | CG94 | 5.86 | a |
CG30 | 5.41 | a | CG99 | 5.45 | a |
CG33 | 5.74 | a | CG104 | 5.74 | a |
CG37 | 1.07 | b | CG106 | 6.84 | h |
CG39 | 5.38 | b | CG111 | 0.82 | b |
CG40 | 5.69 | a | CG116 | 0.85 | b |
The phenotypic measurement was conducted in May, 2015 at Beijing.
The inheritance of fruit peduncle length (FPL) in cucumber was studied with six generations based on a cross between parents with long, 1101, and short, 1694, peduncles. These were P1, P2, F1, F2, BC1P1, and BC1P2. Plants were grown at two sites: Hainan in December (18°15' N, 109°30' E; average day/night temperatures 22°C /19°C; daylength ~11 hr) and Beijing in May (40°10' N, 116°51' E; average day/night temperatures 26°C/14°C; daylength ~14 hr). FPL showed a continuous distribution that was found to fit a quantitative inheritance model, although there were differences in average FPL between the two sites in each generation that were attributed to the different environments. This was particularly evident for the BC1P1 generation which showed an environmental variance as high as 80.1%. Lin et al [
FPL was found to be controlled by one additive major gene and several additive-dominant polygenes, and agrees with the finding by Cui et al [
For QTL analysis, two F2 populations were constructed from the cross between 1101 and 1694 and planted at Hainan in 2014 and Beijing in 2015. Two minor QTLs,
The validation of the link between marker UW021226 and QTL
The major QTL,
Comparison of the number and size of the cells in the pith of the parental lines shown by longitudinal sections of the peduncle (
This research will lead to the development of a marker for FPL in hybrid cucumber production and facilitate marker-assisted selection (MAS) of the long fruit peduncle trait in cucumber breeding. It will also lead to an understanding of the way in which the fruit peduncle develops and the future fine mapping and cloning of the
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Letters indicate significant differences among trait values at P = 0.05 level.
(DOCX)
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Loci with asterisks show segregation distortion.
(DOCX)
The authors would like to thank Dr. Graham Collins, formerly of the University of Adelaide, South Australia for proofreading. This study was supported by the earmarked fund for Modern Agro-industry Technology Research System (CARS-25), the Science and Technology Innovation Program of the Chinese Academy of Agricultural Science (CAAS-ASTIP-IVFCAAS), and the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, the People’s Republic of China.