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Putative paternal factors controlling chilling tolerance in Korean market-type cucumber (Cucumis sativus L.)
Scientia Horticulturae 167 (2014) 145–148
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Short communication
Putative paternal factors controlling chilling tolerance in Korean market-type cucumber (Cucumis sativus L.) Asjad Ali a,1 , Sun Woong Bang a,1 , Eun Mi Yang a , Sang-Min Chung a,∗ , Jack E. Staub b a b
Department of Life Science, Dongguk University-Seoul, Seoul 100-715, South Korea USDA, ARS, Forage and Range Research Laboratory, 696 N. 1100 E., Logan, UT 84322, USA
a r t i c l e
i n f o
Article history: Received 7 November 2013 Received in revised form 2 January 2014 Accepted 4 January 2014 Keywords: Abiotic stress Chilling injury Cytoplasmic effect Low temperature Maternal inheritance Paternal inheritance
a b s t r a c t Chilling temperatures (<10 ◦ C) may cause damages in cucumber plants (Cucumis sativus L.) during winter and early spring seasons. Inheritance of chilling injury in U.S. processing cucumber is controlled by cytoplasmic (maternally) and nuclear factors. To understand inheritance of chilling injury in Korean market-type cucumber, reciprocal crosses between chilling tolerant (CT1) and susceptible (CT4) lines produced F1 (CT1 × CT4) and F1 (CT4 × CT1) progenies. Reciprocal F2 (CT1 × CT4) and F2 (CT4 × CT1) populations were subsequently derived. Seedlings in the first true leaf stage were subjected to 4 ◦ C for 8 h (08:00 to 16:00) and damage level was assessed visually using 1 (no damage) to 5 (severe damage) rating scale. Means of damage rating for reciprocal F1 (CT1 × CT4) and F1 (CT4 × CT1) progenies were 1.1 and 1.1, respectively. This indicates that tolerance for chilling stress at 4 ◦ C in this germplasm is dominant. However, means of damage for F2 (CT1 × CT4) progenies and F2 (CT4 × CT1) progenies were 3.2 and 1.2, respectively. These data indicate that genetic control of chilling injury in these progenies is paternal. Based on the data, we hypothesize that line CT1 possesses a dominant nuclear factor that conditions chilling tolerance in both reciprocal F1 s and a paternal factor(s) that lead chilling tolerance only in F2 (CT4 × CT1). These putative nuclear and paternal genetic factors are designated as Ch-1 and Ch-p, respectively. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chilling injury can result in surface lesions in leaves and fruit, reduced leaf turgidity from water loss and cellular metabolite leakage, internal fruit discoloration, premature senescence, and increased ethylene production (Skog, 1998). Cucumber (Cucumis sativus L.) plants and fruit are sensitive to chilling temperatures between 1 and 12 ◦ C (Staub and Bacher, 1997; Smeets and Wehner, 1997). Chilling temperatures can decrease cucumber seedling germination and emergence (Nienhuis et al., 1983), which often results in lower yields depending on intensity and duration (Staub and Bacher, 1997). In 2012, cucumber crops were produced from open-field (966 ha) and greenhouse (3201 ha) (Korean Statistical Information Service, 2013). The relatively high heating requirement during the winter season for avoidance of chilling injury adds significantly to the cost of Korean cucumber production. Thus, the development of chilling tolerant germplasm would decrease production costs and increase managerial flexibility to cucumber greenhouse production operations.
Chilling injury is detectable in cucumber seedlings in controlled environments at 4 ◦ C (Chung et al., 2003; Kozik and Wehner, 2008; Smeets and Wehner, 1997). Chilling tolerance in U.S. processing cucumber has both nuclear (Kozik and Wehner, 2008) and maternal (Chung et al., 2003; Chung et al., 2007; Gordon and Staub, 2011) genetic components. U.S. processing cultivar Chipper (Chung et al., 2007, chilling tolerance conditioned by cytoplasmic factors) and line NC76 (Kozik and Wehner, 2008, chilling tolerance conditioned by nuclear gene Ch) are tolerant, and U.S. processing line GY-14 is susceptible. Likewise chilling tolerance (e.g., ‘Saeronchungjang’ and ‘Janghyungnachap’) and susceptibility (e.g., ‘Nacdongchungjang’) under these chilling regimes has been found in Korean market type cucumber (Ali et al., 2014). However, the inheritance of chilling tolerance in Korean cucumber is not known. Therefore, a study was designed to investigate the inheritance of chilling injury in two Korean market-type cucumber breeding lines, CT1 (tolerant) and CT4 (susceptible). This is the first step in the strategic integration of chilling tolerance genes into elite Korean cucumber germplasm through plant improvement. 2. Materials and methods 2.1. Germplasm
∗ Corresponding author. Tel.: +82 2 2260 8915. E-mail address: [email protected] (S.-M. Chung). 1 The authors equally contributed to this manuscript. 0304-4238/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2014.01.004
Based on phenotypic observations at the seedling stage, two highly inbred monoecious Korean market-type chilling tolerant
146
A. Ali et al. / Scientia Horticulturae 167 (2014) 145–148
Table 1 Damage rating means and standard deviations (Sd.) of chilling tolerant (CT1) and susceptible (CT4) parental cucumber (Cucumis sativus L.) lines and their derived reciprocal F1 , and F2 (CT1 × CT4) and (CT4 × CT1) progenies when challenged at 4 ◦ C for 8 h (immediate and constant) in two experiments (Exp. I and II). Parents and cross-progenies
CT1 CT4 F1 (CT1 × CT4) F1 (CT4 × CT1) F2 (CT1 × CT4) F2 (CT4 × CT1) a b c
Exp. I
Exp. II
Meana
Sd.
Nb
1.1 2.9 1.0 1.0 3.4 1.1
0.35 1.07 0 0 1.36 0.31
8 7 18 20 85 20
Mean
1.2 1.2 2.6 1.2
Exp. I + II Sd.
0.54 0.49 1.57 0.43
N
Mean
Sd.
N
Mean seperationc
16 20 37 18
1.1 2.9 1.1 1.1 3.2 1.2
0.35 1.07 0.38 0.35 1.47 0.37
8 7 34 40 122 38
A B A A B A
Visual damage rating from 1 to 5, where a damage rating (DR) of 1 = no damage, 2 = slight damage, 3 = moderated damage, 4 = advanced damage, and 5 = severe damage. Number of plants examined. Least significant grouping at p < 0.05.
(CT1) and susceptible (CT4) cucumber breeding lines were received from Dongbu Farm Hannong, Seoul, South Korea (Kihwan Song, 2011, personal communication). CT1 cucumber line has been derived with selection for chilling tolerance from mixed germplasm of native Korean market type of cucumber. Fruits of CT1 and CT4 have black spines and white spines, respectively. Reciprocal crosses were made by controlled pollination between lines CT1 and CT4 to produce reciprocal F1 (CT1 × CT4 and CT4 × CT1) and F2 progenies [F2 (CT1 × CT4), and F2 (CT4 × CT1)] via self-pollination. Subsequently, seedlings of these populations were evaluated for their response to chilling temperature stress under controlled conditions (growth chamber; pre- and post-chilling environment, and chilling environment) at Dongguk University at Seoul Korea. 2.2. Evaluation of plants in response to chilling temperature stress Parental lines (CT1 and CT4) and their reciprocal F1 and F2 progenies were sown in plug pots (50 cm × 50 cm) containing Sunshine Mix no. 4—aggregate plus (SunGro Horticulture, WA, U.S.A.). Seedling were grown under a 9-h photoperiod at a light level of 176 mol/m2 /s PPF supplied by cool-white fluorescent and incandescent lamps at 22 (light)/18 (dark) ◦ C, according to Chung et al. (2003). Relative humidity (RH) was held at (50%), and emerged seedlings were watered one time daily with water-soluble fertilizer (N:P:K = 20:10:20; Technigro: SunGro Horticulture) weekly. The chilling treatment was performed according to Chung et al. (2003) in two experiments separated in time (designated Exp. I and II). When first true leaves were fully opened with no remaining adaxial leaf curl, plants were subjected to a chilling treatment at 4 ◦ C for 8 (08:00–16:00, immediate and constant). This chilling temperature regime was chosen because of its historical application in several previous studies (Chung et al., 2003; Gordon and Staub, 2011; Smeets and Wehner, 1997). During chilling, light level was 149 mol/m2 /s PPF and RH was held constant at ∼80%. Water was supplied to seedlings during chilling treatment only to eliminate the dryness of soil, and then all plants were watered to soil saturation following chilling. After chilling treatment, plants were returned to pretreatment conditions, and leaf damage was quantified by visual ratings for 5 days after the chilling treatment. Chilling damage on the first true leaves was scored from 1 to 5, where a damage rating (DR) of 1 = no damage, 2 = slight damage, 3 = moderated damage, 4 = advanced damage, and 5 = severe damage according to Ali et al. (2014). This rating score scheme was modified from the 0–9 scale of Smeets and Wehner (1997) since the visual damage observed herein was comparatively less pronounced when contrasted to that study. 2.3. Data analysis Visual damage ratings data from two experiments was subjected to analysis of variance (ANOVA) using complete randomized design
(CRD). The Statistix 8.1 package (Analytical Software, Tallahassee, FL, USA) was used for data analysis. Entry means and standard deviations were calculated and mean difference tests were performed using least significant (LSD) tests to group germplasm responses (p < 0.05). 3. Results In Exp. I, the mean of chilling damage of line CT1 was low (DR = 1.1) when compared to line CT4 (DR = 2.9) (Table 1). Chilling response among reciprocal F1 progenies (CT1 × CT4 and CT4 × CT1) was identical (DR = 1), and demonstrated a tolerant reaction. In contrast, progeny response in F2 populations differed (p < 0.05), where F2 (CT1 × CT4) progenies were susceptible (DR = 3.4) while F2 (CT4 × CT1) individuals were tolerant (DR = 1.1). A similar chilling response pattern between reciprocal F2 populations was detected in Exp. II. While post-chilling evaluation of both reciprocal F1 populations in Exp. II indicated a similar reaction to chilling temperature stress (DR = 1.2), F2 (CT1 × CT4) progenies must be considered as susceptible (mean DR = 2.6) and F2 (CT4 × CT1) progenies as tolerant (mean DR = 1.2). Likewise, when taken collectively, mean value comparisons in Exp. I and II indicate that, although no differences were apparent between F1 reciprocal progenies, reciprocal F2 progenies differed in their response to chilling stress. Although both reciprocal F1 plants were chilling tolerant, reciprocal F2 (CT4 × CT1) progenies were, in the main, tolerant (DR = 1; 32 out of 38) and F2 (CT1 × CT4) progenies varied in their response (DR = 1–5) (Fig. 1). 4. Discussion In plants, nuclear, chloroplast, and mitochondria organelles possess genetic materials that can control trait expression. In cucumber, chloroplasts are maternally inherited (Chung et al., 2007; Corriveau and Coleman, 1988; Feierabend, 1977) and the mitochondrial (mt) genome is predominantly under paternal control (Havey, 1997). Differential transmission of organellar genomes was later documented in other cucurbits (Havey et al., 1998). Differential response to chilling temperatures in reciprocal cross-progenies may signify a non-nuclear genetic control to such challenge. Chung et al. (2003) demonstrated that such differences in chilling response (at 4 ◦ C) in reciprocal processing-type cucumber ‘Chipper’ × Gy-14 cross-progenies were controlled by cytoplasmic factors (designated as t for tolerance and s for susceptibility), which were subsequently mapped to three positions in the chloroplast genome (Chung et al., 2007). Kozik and Wehner (2008) identified a single dominant nuclear gene (Ch) that controls chilling tolerance in line NC-76 that was derived from PI 222783 (‘Khiar Sabz’; Bakhtaran, Iran) and PI 222985 (‘Khiar’; Tabriz, Iran). According to Gordon and Staub (2011), backcross progenies (tolerant plastid) derived from ‘Chipper’ (t/chch) × NC-76 (?/ChCh) F1 individuals responded similarly to chilling challenge regardless of their nuclear
A. Ali et al. / Scientia Horticulturae 167 (2014) 145–148
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Fig. 1. Frequency distributions of damage ratings (DR) of reciprocal F1 and F2 progenies derived from Korean market type chilling tolerant (CT1) and susceptible (CT4) exposed to chilling at 4 ◦ C under controlled environmental conditions.
constitution under the chilling conditions of Smeets and Wehner (1997) and Chung et al. (2003). In this study, we identified difference of chilling responses between reciprocal F2 , but not between reciprocal F1 populations in Korean market-type cucumber (Table 1). Given these results, it could be hypothesized that a dominant nuclear gene(s) controls chilling tolerance at 4 ◦ C in this germplasm. We provisionally designate the nuclear factor(s) controlling chilling injury at 4 ◦ C in this cucumber type as Ch-1. Allelism tests and identification of map location on the integrated cucumber map (Huang et al., 2009) would be prescriptive for determining putative genetic relationship(s) between dominant Ch (Kozik and Wehner, 2008) and Ch-1 as defined in this study. The presence of dominant nuclear factor(s) controlling chilling tolerance in Korean market-type cucumber, however, is equivocal given the segregation detected in F2 (CT1 × CT4) progenies (DR = 3.2) (Table 1; Fig. 1). The difference identified herein between reciprocal F2 populations (tolerant and segregating), but not between F1 reciprocal progenies (tolerant) suggests that paternal control of chilling tolerance (at 4 ◦ C) may be operating in CT4 × CT1 cross-progenies. This result is suggestive of the presence of a paternal factor that operates in conjunction with Ch-1 to control chilling injury at 4 ◦ C in these cucumber crosses. We provisionally designate this paternal factor(s) herein as Ch-p. However, if paternal control operates to control chilling at 4 ◦ C in this germplasm, then predictably F1 (CT1 × CT4) progenies would be susceptible given the hypothesized genetic constitution of line CT4 (s/ch-pch-p or s/ch-1ch-1). The paternal inheritance of of mtDNA is extremely rare in plants (Erickson et al., 1989; Kiang et al., 1994; Neale et al., 1991). In hexaploid wheat (Aegilops species) the differential amplification of the paternal mtDNA sequences varied depending on the plasmons of the maternal and the nuclear backgrounds of the paternal parents
(Tsukamoto et al., 2000). The relationship of Ch, and hypothesized Ch-1 and Ch-p described herein could be further investigated by the evaluation of F2 , F2 BC1 , and BC1 progenies derived from reciprocal matings between lines NC-76 × CT4 and NC-76 × CT1. Progeny segregation comparisons after chilling stress would allow for the test of organellar control and the elucidation of epistatic interactions between Ch, Ch-1, and Ch-p. Unequivocal confirmation of the mode of genetic control of chilling tolerance could be obtained using restriction enzyme marker techniques (Havey, 1997; Tsukamoto et al., 2000). If the mode of inheritance for chilling tolerance in Korean market cucumber is determined to be under organellar control, conventional breeding approaches for the development of chilling tolerant germplasm in this market-type would utilize elite cucumber germplasm (e.g., cultivars) as the maternal parent and line CT1 as paternal parent. The identification of molecular markers linked to chilling tolerance in this population would likely provide for greater flexibility during the recovery of tolerant genotypes (i.e., selection of unique tolerant individuals for phenotypic evaluation). Acknowledgements This work was supported by a grant from the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry, and Fisheries (Grant 109064-05-5-HD110), Republic of Korea. References Ali, A., Yang, E.M., Bang, S.W., Chung, S.M., Staub, J.E., 2014. Assessment of chilling injury and molecular marker analysis in cucumber cultivars (Cucumis sativus L.). Korean J. Hortic. Sci. Technol., In press.
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Corriveau, J., Coleman, A., 1988. Rapid screening method to detect biparental inheritance of plastid DNA and results from over 200 angiosperm species. Am. J. Bot. 75, 1433–1458. Chung, S.M., Staub, J.E., Fazio, G., 2003. Inheritance of chilling injury: a maternally inherited trait in cucumber. J. Am. Soc. Hortic. Sci. 128, 526–530. Chung, S.M., Gordon, V.S., Staub, J.E., 2007. Sequencing cucumber (Cucumis sativus L.) chloroplast genomes identifies differences between chilling-tolerant andsusceptible cucumber lines. Genome 50, 215–225. Erickson, L., Kemble, R., Swanson, E., 1989. The Brassica mitochondrial plasmid can be sexually transmitted: pollen transfer of a cytoplasmic genetic element. Mol. Gen. Genet. 218, 419–422. Feierabend, J., 1977. Capacity for chlorophyll synthesis in heat bleached 70S ribosome-deficient rye leaves. Planta 135, 83–88. Gordon, V.S., Staub, J.E., 2011. Comparative analysis of chilling response in cucumber (Cucumis sativus L.) through plastidic and nuclear genetic component analysis. J. Am. Soc. Hortic. Sci. 136, 256–264. Havey, M.J., 1997. Predominant paternal transmission of the mitochondrial genome in cucumber. J. Heredity 88, 232–235. Havey, M.J., McCreight, J.D., Rhodes, B., Taurick, G., 1998. Differential transmission of the Cucumis organellar genomes. Theor. Appl. Genet. 97, 122–128. Huang, S., Li, R., Zhang, Z., Li, L., Gu, X., Fan, W., Lucas, W.J., Wang, X., Xie, B., Ni, P., Ren, Y., Zhu, H., Li, J., Lin, K., Jin, W., Fei, Z., Li, G., Staub, J., Kilian, A., van der Vossen, E.A., Wu, Y., Guo, J., He, J., Jia, Z., Ren, Y., Tian, G., Lu, Y., Ruan, J., Qian, W., Wang, M., Huang, Q., Li, B., Xuan, Z., Cao, J., Asan, Wu, Z., Zhang, J., Cai, Q., Bai, Y., Zhao, B., Han, Y., Li, Y., Li, X., Wang, S., Shi, Q., Liu, S., Cho, W.K., Kim, J.Y., Xu, Y., Heller-Uszynska, K., Miao, H., Cheng, Z., Zhang, S., Wu, J., Yang, Y., Kang, H., Li, M., Liang, H., Ren, X., Shi, Z., Wen, M., Jian, M., Yang, H., Zhang, G., Yang, Z., Chen, R., Liu, S., Li, J., Ma, L., Liu, H., Zhou, Y., Zhao, J., Fang, X., Li, G., Fang, L., Li, Y., Liu, D., Zheng, H., Zhang, Y., Qin, N., Li, Z., Yang, G., Yang, S., Bolund, L., Kristiansen,
K., Zheng, H., Li, S., Zhang, X., Yang, H., Wang, J., Sun, R., Zhang, B., Jiang, S., Wang, J., Du, Y., Li, S., 2009. The genome of cucumber, Cucumis sativus L. Nat. Genet. 41, 1275–1281. Kiang, A.S., Connolly, V., McConnell, D.J., Kavanagh, T.A., 1994. Paternal inheritance of mitochrondria and chloroplasts in Festuca pratensis–Lolium perennne intergeneric hybrids. Theor. Appl. Genet. 87, 681–688. Korean Statistical Information Service, 2013. Statistics Korea. Korean Statistical Information Service, Daejeon, Republic of Korea, Retrievable at: www.kosis.kr/eng Last accessed: 6/2013. Kozik, E.U., Wehner, T.C., 2008. A single dominant gene Ch for chilling resistance in cucumber seedlings. J. Am. Soc. Hortic. Sci. 133, 225–227. Neale, D.B., Marshall, K.A., Harry, D.E., 1991. Inheritance of chloroplast and mitochrondrial DNA in incense-cedar (Calcedrus decurrens). Can. J. For. Res. 21, 717–720. Nienhuis, J., Lower, R.L., Staub, J.E., 1983. Selection for improved low temperature germination in cucumber (Cucumis sativus L.). J. Am. Soc. Hortic. Sci. 108, 1040–1043. Skog, L.J., 1998. Chilling Injury of Horticultural Crops. Ontario Ministry of Agriculture, Food and Rural Affairs, ON, Canada. Smeets, L., Wehner, T.C., 1997. Environmental effects on genetic variation of chilling resistance in cucumber. Euphytica 97, 217–225. Staub, J.E., Bacher, J., 1997. Cucumber as a processed vegetable. In: Processing Vegetables: Science and Technology IV. Technomic Publishing Co., Inc., Lancaster, PA. Tsukamoto, N., Asakura, N., Hattori, N., Takumi, S., Mori, N., Nakamura, C., 2000. Identification of paternal mitochondrial DNA sequences in the nucleus–cytoplasm hybrids of tetraploid and hexaploid wheat with D and D2 plasmons from Aegilops species. Curr. Genet. 38, 208–217.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Short communication
Putative paternal factors controlling chilling tolerance in Korean market-type cucumber (Cucumis sativus L.) Asjad Ali a,1 , Sun Woong Bang a,1 , Eun Mi Yang a , Sang-Min Chung a,∗ , Jack E. Staub b a b
Department of Life Science, Dongguk University-Seoul, Seoul 100-715, South Korea USDA, ARS, Forage and Range Research Laboratory, 696 N. 1100 E., Logan, UT 84322, USA
a r t i c l e
i n f o
Article history: Received 7 November 2013 Received in revised form 2 January 2014 Accepted 4 January 2014 Keywords: Abiotic stress Chilling injury Cytoplasmic effect Low temperature Maternal inheritance Paternal inheritance
a b s t r a c t Chilling temperatures (<10 ◦ C) may cause damages in cucumber plants (Cucumis sativus L.) during winter and early spring seasons. Inheritance of chilling injury in U.S. processing cucumber is controlled by cytoplasmic (maternally) and nuclear factors. To understand inheritance of chilling injury in Korean market-type cucumber, reciprocal crosses between chilling tolerant (CT1) and susceptible (CT4) lines produced F1 (CT1 × CT4) and F1 (CT4 × CT1) progenies. Reciprocal F2 (CT1 × CT4) and F2 (CT4 × CT1) populations were subsequently derived. Seedlings in the first true leaf stage were subjected to 4 ◦ C for 8 h (08:00 to 16:00) and damage level was assessed visually using 1 (no damage) to 5 (severe damage) rating scale. Means of damage rating for reciprocal F1 (CT1 × CT4) and F1 (CT4 × CT1) progenies were 1.1 and 1.1, respectively. This indicates that tolerance for chilling stress at 4 ◦ C in this germplasm is dominant. However, means of damage for F2 (CT1 × CT4) progenies and F2 (CT4 × CT1) progenies were 3.2 and 1.2, respectively. These data indicate that genetic control of chilling injury in these progenies is paternal. Based on the data, we hypothesize that line CT1 possesses a dominant nuclear factor that conditions chilling tolerance in both reciprocal F1 s and a paternal factor(s) that lead chilling tolerance only in F2 (CT4 × CT1). These putative nuclear and paternal genetic factors are designated as Ch-1 and Ch-p, respectively. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Chilling injury can result in surface lesions in leaves and fruit, reduced leaf turgidity from water loss and cellular metabolite leakage, internal fruit discoloration, premature senescence, and increased ethylene production (Skog, 1998). Cucumber (Cucumis sativus L.) plants and fruit are sensitive to chilling temperatures between 1 and 12 ◦ C (Staub and Bacher, 1997; Smeets and Wehner, 1997). Chilling temperatures can decrease cucumber seedling germination and emergence (Nienhuis et al., 1983), which often results in lower yields depending on intensity and duration (Staub and Bacher, 1997). In 2012, cucumber crops were produced from open-field (966 ha) and greenhouse (3201 ha) (Korean Statistical Information Service, 2013). The relatively high heating requirement during the winter season for avoidance of chilling injury adds significantly to the cost of Korean cucumber production. Thus, the development of chilling tolerant germplasm would decrease production costs and increase managerial flexibility to cucumber greenhouse production operations.
Chilling injury is detectable in cucumber seedlings in controlled environments at 4 ◦ C (Chung et al., 2003; Kozik and Wehner, 2008; Smeets and Wehner, 1997). Chilling tolerance in U.S. processing cucumber has both nuclear (Kozik and Wehner, 2008) and maternal (Chung et al., 2003; Chung et al., 2007; Gordon and Staub, 2011) genetic components. U.S. processing cultivar Chipper (Chung et al., 2007, chilling tolerance conditioned by cytoplasmic factors) and line NC76 (Kozik and Wehner, 2008, chilling tolerance conditioned by nuclear gene Ch) are tolerant, and U.S. processing line GY-14 is susceptible. Likewise chilling tolerance (e.g., ‘Saeronchungjang’ and ‘Janghyungnachap’) and susceptibility (e.g., ‘Nacdongchungjang’) under these chilling regimes has been found in Korean market type cucumber (Ali et al., 2014). However, the inheritance of chilling tolerance in Korean cucumber is not known. Therefore, a study was designed to investigate the inheritance of chilling injury in two Korean market-type cucumber breeding lines, CT1 (tolerant) and CT4 (susceptible). This is the first step in the strategic integration of chilling tolerance genes into elite Korean cucumber germplasm through plant improvement. 2. Materials and methods 2.1. Germplasm
∗ Corresponding author. Tel.: +82 2 2260 8915. E-mail address: [email protected] (S.-M. Chung). 1 The authors equally contributed to this manuscript. 0304-4238/$ – see front matter © 2014 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2014.01.004
Based on phenotypic observations at the seedling stage, two highly inbred monoecious Korean market-type chilling tolerant
146
A. Ali et al. / Scientia Horticulturae 167 (2014) 145–148
Table 1 Damage rating means and standard deviations (Sd.) of chilling tolerant (CT1) and susceptible (CT4) parental cucumber (Cucumis sativus L.) lines and their derived reciprocal F1 , and F2 (CT1 × CT4) and (CT4 × CT1) progenies when challenged at 4 ◦ C for 8 h (immediate and constant) in two experiments (Exp. I and II). Parents and cross-progenies
CT1 CT4 F1 (CT1 × CT4) F1 (CT4 × CT1) F2 (CT1 × CT4) F2 (CT4 × CT1) a b c
Exp. I
Exp. II
Meana
Sd.
Nb
1.1 2.9 1.0 1.0 3.4 1.1
0.35 1.07 0 0 1.36 0.31
8 7 18 20 85 20
Mean
1.2 1.2 2.6 1.2
Exp. I + II Sd.
0.54 0.49 1.57 0.43
N
Mean
Sd.
N
Mean seperationc
16 20 37 18
1.1 2.9 1.1 1.1 3.2 1.2
0.35 1.07 0.38 0.35 1.47 0.37
8 7 34 40 122 38
A B A A B A
Visual damage rating from 1 to 5, where a damage rating (DR) of 1 = no damage, 2 = slight damage, 3 = moderated damage, 4 = advanced damage, and 5 = severe damage. Number of plants examined. Least significant grouping at p < 0.05.
(CT1) and susceptible (CT4) cucumber breeding lines were received from Dongbu Farm Hannong, Seoul, South Korea (Kihwan Song, 2011, personal communication). CT1 cucumber line has been derived with selection for chilling tolerance from mixed germplasm of native Korean market type of cucumber. Fruits of CT1 and CT4 have black spines and white spines, respectively. Reciprocal crosses were made by controlled pollination between lines CT1 and CT4 to produce reciprocal F1 (CT1 × CT4 and CT4 × CT1) and F2 progenies [F2 (CT1 × CT4), and F2 (CT4 × CT1)] via self-pollination. Subsequently, seedlings of these populations were evaluated for their response to chilling temperature stress under controlled conditions (growth chamber; pre- and post-chilling environment, and chilling environment) at Dongguk University at Seoul Korea. 2.2. Evaluation of plants in response to chilling temperature stress Parental lines (CT1 and CT4) and their reciprocal F1 and F2 progenies were sown in plug pots (50 cm × 50 cm) containing Sunshine Mix no. 4—aggregate plus (SunGro Horticulture, WA, U.S.A.). Seedling were grown under a 9-h photoperiod at a light level of 176 mol/m2 /s PPF supplied by cool-white fluorescent and incandescent lamps at 22 (light)/18 (dark) ◦ C, according to Chung et al. (2003). Relative humidity (RH) was held at (50%), and emerged seedlings were watered one time daily with water-soluble fertilizer (N:P:K = 20:10:20; Technigro: SunGro Horticulture) weekly. The chilling treatment was performed according to Chung et al. (2003) in two experiments separated in time (designated Exp. I and II). When first true leaves were fully opened with no remaining adaxial leaf curl, plants were subjected to a chilling treatment at 4 ◦ C for 8 (08:00–16:00, immediate and constant). This chilling temperature regime was chosen because of its historical application in several previous studies (Chung et al., 2003; Gordon and Staub, 2011; Smeets and Wehner, 1997). During chilling, light level was 149 mol/m2 /s PPF and RH was held constant at ∼80%. Water was supplied to seedlings during chilling treatment only to eliminate the dryness of soil, and then all plants were watered to soil saturation following chilling. After chilling treatment, plants were returned to pretreatment conditions, and leaf damage was quantified by visual ratings for 5 days after the chilling treatment. Chilling damage on the first true leaves was scored from 1 to 5, where a damage rating (DR) of 1 = no damage, 2 = slight damage, 3 = moderated damage, 4 = advanced damage, and 5 = severe damage according to Ali et al. (2014). This rating score scheme was modified from the 0–9 scale of Smeets and Wehner (1997) since the visual damage observed herein was comparatively less pronounced when contrasted to that study. 2.3. Data analysis Visual damage ratings data from two experiments was subjected to analysis of variance (ANOVA) using complete randomized design
(CRD). The Statistix 8.1 package (Analytical Software, Tallahassee, FL, USA) was used for data analysis. Entry means and standard deviations were calculated and mean difference tests were performed using least significant (LSD) tests to group germplasm responses (p < 0.05). 3. Results In Exp. I, the mean of chilling damage of line CT1 was low (DR = 1.1) when compared to line CT4 (DR = 2.9) (Table 1). Chilling response among reciprocal F1 progenies (CT1 × CT4 and CT4 × CT1) was identical (DR = 1), and demonstrated a tolerant reaction. In contrast, progeny response in F2 populations differed (p < 0.05), where F2 (CT1 × CT4) progenies were susceptible (DR = 3.4) while F2 (CT4 × CT1) individuals were tolerant (DR = 1.1). A similar chilling response pattern between reciprocal F2 populations was detected in Exp. II. While post-chilling evaluation of both reciprocal F1 populations in Exp. II indicated a similar reaction to chilling temperature stress (DR = 1.2), F2 (CT1 × CT4) progenies must be considered as susceptible (mean DR = 2.6) and F2 (CT4 × CT1) progenies as tolerant (mean DR = 1.2). Likewise, when taken collectively, mean value comparisons in Exp. I and II indicate that, although no differences were apparent between F1 reciprocal progenies, reciprocal F2 progenies differed in their response to chilling stress. Although both reciprocal F1 plants were chilling tolerant, reciprocal F2 (CT4 × CT1) progenies were, in the main, tolerant (DR = 1; 32 out of 38) and F2 (CT1 × CT4) progenies varied in their response (DR = 1–5) (Fig. 1). 4. Discussion In plants, nuclear, chloroplast, and mitochondria organelles possess genetic materials that can control trait expression. In cucumber, chloroplasts are maternally inherited (Chung et al., 2007; Corriveau and Coleman, 1988; Feierabend, 1977) and the mitochondrial (mt) genome is predominantly under paternal control (Havey, 1997). Differential transmission of organellar genomes was later documented in other cucurbits (Havey et al., 1998). Differential response to chilling temperatures in reciprocal cross-progenies may signify a non-nuclear genetic control to such challenge. Chung et al. (2003) demonstrated that such differences in chilling response (at 4 ◦ C) in reciprocal processing-type cucumber ‘Chipper’ × Gy-14 cross-progenies were controlled by cytoplasmic factors (designated as t for tolerance and s for susceptibility), which were subsequently mapped to three positions in the chloroplast genome (Chung et al., 2007). Kozik and Wehner (2008) identified a single dominant nuclear gene (Ch) that controls chilling tolerance in line NC-76 that was derived from PI 222783 (‘Khiar Sabz’; Bakhtaran, Iran) and PI 222985 (‘Khiar’; Tabriz, Iran). According to Gordon and Staub (2011), backcross progenies (tolerant plastid) derived from ‘Chipper’ (t/chch) × NC-76 (?/ChCh) F1 individuals responded similarly to chilling challenge regardless of their nuclear
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Fig. 1. Frequency distributions of damage ratings (DR) of reciprocal F1 and F2 progenies derived from Korean market type chilling tolerant (CT1) and susceptible (CT4) exposed to chilling at 4 ◦ C under controlled environmental conditions.
constitution under the chilling conditions of Smeets and Wehner (1997) and Chung et al. (2003). In this study, we identified difference of chilling responses between reciprocal F2 , but not between reciprocal F1 populations in Korean market-type cucumber (Table 1). Given these results, it could be hypothesized that a dominant nuclear gene(s) controls chilling tolerance at 4 ◦ C in this germplasm. We provisionally designate the nuclear factor(s) controlling chilling injury at 4 ◦ C in this cucumber type as Ch-1. Allelism tests and identification of map location on the integrated cucumber map (Huang et al., 2009) would be prescriptive for determining putative genetic relationship(s) between dominant Ch (Kozik and Wehner, 2008) and Ch-1 as defined in this study. The presence of dominant nuclear factor(s) controlling chilling tolerance in Korean market-type cucumber, however, is equivocal given the segregation detected in F2 (CT1 × CT4) progenies (DR = 3.2) (Table 1; Fig. 1). The difference identified herein between reciprocal F2 populations (tolerant and segregating), but not between F1 reciprocal progenies (tolerant) suggests that paternal control of chilling tolerance (at 4 ◦ C) may be operating in CT4 × CT1 cross-progenies. This result is suggestive of the presence of a paternal factor that operates in conjunction with Ch-1 to control chilling injury at 4 ◦ C in these cucumber crosses. We provisionally designate this paternal factor(s) herein as Ch-p. However, if paternal control operates to control chilling at 4 ◦ C in this germplasm, then predictably F1 (CT1 × CT4) progenies would be susceptible given the hypothesized genetic constitution of line CT4 (s/ch-pch-p or s/ch-1ch-1). The paternal inheritance of of mtDNA is extremely rare in plants (Erickson et al., 1989; Kiang et al., 1994; Neale et al., 1991). In hexaploid wheat (Aegilops species) the differential amplification of the paternal mtDNA sequences varied depending on the plasmons of the maternal and the nuclear backgrounds of the paternal parents
(Tsukamoto et al., 2000). The relationship of Ch, and hypothesized Ch-1 and Ch-p described herein could be further investigated by the evaluation of F2 , F2 BC1 , and BC1 progenies derived from reciprocal matings between lines NC-76 × CT4 and NC-76 × CT1. Progeny segregation comparisons after chilling stress would allow for the test of organellar control and the elucidation of epistatic interactions between Ch, Ch-1, and Ch-p. Unequivocal confirmation of the mode of genetic control of chilling tolerance could be obtained using restriction enzyme marker techniques (Havey, 1997; Tsukamoto et al., 2000). If the mode of inheritance for chilling tolerance in Korean market cucumber is determined to be under organellar control, conventional breeding approaches for the development of chilling tolerant germplasm in this market-type would utilize elite cucumber germplasm (e.g., cultivars) as the maternal parent and line CT1 as paternal parent. The identification of molecular markers linked to chilling tolerance in this population would likely provide for greater flexibility during the recovery of tolerant genotypes (i.e., selection of unique tolerant individuals for phenotypic evaluation). Acknowledgements This work was supported by a grant from the Technology Development Program for Agriculture and Forestry, Ministry for Food, Agriculture, Forestry, and Fisheries (Grant 109064-05-5-HD110), Republic of Korea. References Ali, A., Yang, E.M., Bang, S.W., Chung, S.M., Staub, J.E., 2014. Assessment of chilling injury and molecular marker analysis in cucumber cultivars (Cucumis sativus L.). Korean J. Hortic. Sci. Technol., In press.
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