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Mapping of a gene conferring alleviation of pollen–pistil incongruity found in an interspecific cross between Cucumis anguria L. and Cucumis melo L. (melon)
Scientia Horticulturae 146 (2012) 81–85
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Mapping of a gene conferring alleviation of pollen–pistil incongruity found in an interspecific cross between Cucumis anguria L. and Cucumis melo L. (melon) Yuichi Matsumoto a,∗ , Makoto Miyagi b a b
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Plant Biotechnology Institute, Ibaraki Agricultural Center, Kasama, Ibaraki 319-0292, Japan
a r t i c l e
i n f o
Article history: Received 20 June 2012 Received in revised form 15 August 2012 Accepted 16 August 2012 Keywords: Cucumis spp. Reproductive barrier Pollen tube Interspecific cross Mapping
a b s t r a c t In Cucumis, when melon (Cucumis melo L.) was crossed with Cucumis anguria L., PI 320052, at 24–30 ◦ C, pollen tube growth was arrested in the style. However, at 32 ◦ C and 34 ◦ C, the pollen–pistil incongruity was alleviated. Pollen tubes penetrated into ovules and fruit set was observed. However, these studies have not revealed the genetic and molecular mechanism of pollen–pistil incongruity alleviation. This study reveals the mode of inheritance and identifies molecular markers linked with the gene. We developed the F1 and F2 lines from the alleviate line and non-alleviate line, and evaluated the mode of inheritance of the gene. We constructed the linkage map by the phenotypes and conducted simple sequence repeat (SSR) analysis. We demonstrated that the pollen–pistil incongruity alleviation gene is single recessive and that it is located on LG I between CSN221 and SSR19844 at a distance of 3.7 cM and 4.5 cM from CSN221 and SSR19844, respectively. Therefore we assigned the gene as pollen–pistil incongruity alleviation (pia). This is the first report on the mapping of a gene conferring the pollen–pistil incongruity alleviation in interspecific crosses © 2012 Elsevier B.V. All rights reserved.
1. Introduction Pollen–pistil incongruity, a pre-zygotic barrier between selffertilizing species (Hogenboom, 1984), has been reported in many taxa such as Tulipa (Kho and Baër, 1971), Nicotiana (Kuboyama et al., 1994), Oryza (Suputtitada et al., 2000), and Cucumis (Kishi and Fujishita, 1970; Kho et al., 1980). In Cucumis, pollen–pistil incongruity is often observed in interspecific crosses between melon (Cucumis melo L.) and wild Cucumis spp. In these crosses, although pollens germinate and pollen tubes enter into the stigma, most pollen tubes are arrested in the style and fertilization is rarely observed. Wild Cucumis spp. reportedly has resistance for some melon pests. Some examples are downy mildew (Pan and More, 1996), powdery mildew (Lebeda, 1984; Pan and More, 1996), fusarium wilt (Pan and More, 1996; Matsumoto et al., 2011), and root-knot nematode (Sigüenza et al., 2005). Therefore, methods to bypass these reproductive barriers are available for expansion of usable genetic resources in melon breeding. Hedhly et al. (2005) reported temperature as an important factor affecting pollen performance during the progamic phase: from
∗ Corresponding author. Present address: Plant Biotechnology Institute, Ibaraki Agricultural Center, Kasama, Ibaraki 319-0292, Japan. Tel.: +81 299 458330; fax: +81 299 458351. E-mail address: [email protected] (Y. Matsumoto). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.08.021
pollination to fertilization. Regarding pollen–pistil incongruity, the alleviated effect by the temperature has been reported in interspecific crosses of Lilium (Ascher and Peloquin, 1968), Tulipa (Kho and Baër, 1971), Brassica (Matsuzawa, 1983), Oryza (Sitch and Romero, 1990), and Cucumis (Franken et al., 1988; Matsumoto et al., 2012). In Cucumis, we observed that when melon was crossed with Cucumis anguria L. at 24–30 ◦ C, pollen tube growth was arrested in the style. However, at 32 ◦ C and 34 ◦ C, the pollen–pistil incongruity was alleviated and pollen tubes penetrated into ovules and fruit set was observed (Matsumoto et al., 2012). However, the genetics of this pollen–pistil incongruity alleviation in interspecific cross was not studied. Such inheritance study is important to overcome reproductive barrier, and expansion of usable genetic resources. Hence we examined mode of inheritance of pollen–pistil incongruity alleviation and using molecular market made effort to locate gene for the pollen–pistil incongruity alleviation in Cucumis. For this study, we examined the mode of inheritance and the chromosome location of the gene conferring the pollen–pistil incongruity alleviation in C. anguria.
2. Materials and methods 2.1. Plant materials Melon line ‘MR-1 was obtained from the Institut National de la Recherche Agronomique (INRA), France, and used as the pollen
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Y. Matsumoto, M. Miyagi / Scientia Horticulturae 146 (2012) 81–85
parent. The C. anguria L. lines PI 320052 and PI 364475 were obtained from Germplasm Resources Laboratory, USDA, ARS, Beltsville, USA, and used. Eighty-four F2 plants from the cross between PI 320052 and PI 364475 were developed for mapping the population. 2.2. Pollen tube observation Pollination was conducted using mature flowers emasculated one day before anthesis. The flowering pistils of PI 320052 and PI 364475 were collected, put on a wetted paper towel, pollinated with ‘MR-1 pollen, and then incubated at controlled temperatures of 28 ◦ C (normal temperature) or 32 ◦ C (high temperature). The incubated pistils were collected 8 h after pollination and macerated by autoclaving in 50 g L−1 aqueous sodium sulfite and stained with aniline blue before squashing under a cover glass (Jefferies and Belcher, 1974). Pollen tubes were observed under a fluorescence microscope (blue–violet excitation filter cube, UMWBV, BX50; Olympus Corp., Tokyo, Japan). Photographs of pollen tubes were taken using a digital camera (DS-Fi1; Nikon Corp., Tokyo, Japan) and acquired with imaging software (NIS-Elements D; Nikon Corp., Tokyo, Japan). The images were registered on image tiling software (e-Tiling; Mitani Corp., Fukui, Japan). The distance from the stigma to the longest pollen tube was regarded as the approximate pollen tube length. 2.3. Segregation of expression types Pistils that pollinated under the high temperature condition were observed for pollen tube length. For these observations, 20 flowers of two parental line (PI 320052 and PI 364475) and F1 line, and 10 flowers of 84 F2 lines were examined. Pollen tube length was log-transformed and calculated using analysis of variance (ANOVA) via a least-squares fitting model. A Tukey–Kramer test was performed as a post hoc test for ANOVA to compare lines. For these analyses, statistical software (JMP ver. 9.0.0; SAS Institute Inc., Cary, NC, USA) was used. In these analyses, a line which showed a non-significant difference from PI 320052 (alleviated parent) was regarded as an alleviated line. A line which showed a non-significant difference from PI 364475 (non-alleviated parent) was regarded as a non-alleviated line. The observed ratios in the segregated F2 populations were tested for deviations from the expected ratios using the Chi-square goodness of fit test, with significance inferred for P < 0.05. 2.4. DNA extraction Total DNA was extracted according to Mukai and Yamamoto (1997) with some modifications. Briefly, each 2-cm-long young leaf tip from a single plant was put in a 1.5 ml tube. Then 290 L of extraction buffer (100 mM Tris–HCl (pH 8.0), 500 mM NaCl, 50 mM EDTA (pH 8.0), and 10 mM 2-mercapto ethanol) was added. The leaf was chopped with sharp plastic chips and a tube was incubated at 65 ◦ C for 10 min. After incubation, 100 L of 5 M potassium acetate were added and incubated at 4 ◦ C 15 min. After centrifuging, the supernatant was recovered and 300 L of 2-propanol was added. After centrifuging, the supernatant was discarded and the DNA pellet was rinsed with 300 L of 70% ethanol. The DNA pellet was dried and dissolved in 100 L of 1/10 TE buffer. 2.5. SSR analysis Sixty-two simple sequence repeat (SSR) markers developed by cucumber (Cucumis sativus L.) (Fukino et al., 2008; Watcharawongpaiboon and Chunwongse, 2008; Ren et al., 2009) and melon (Danin-Poleg et al., 2001; Chiba et al., 2003; Ritschel
et al., 2004; Gonzalo et al., 2005; Fukino et al., 2007; Cucurbit Genomics Database, http://www.icugi.org/), and which showed polymorphism between PI 320052 and PI 364475 were used to construct a genetic linkage map. PCR reaction was performed using the modified post labeled method (Schuelke, 2000) as follows: SSR forward primers were modified using 5 -concatenation with the T7 promoter sequence (aatacgactcactatagg), with the M13 forward sequence (tgtaaaacgacggccagt), or with the M13 reverse promoter (caggaaacagctatgacc) based on Moriya et al. (2010). For these three sequences, we also created tailed sequences labeled with fluorescent chemicals: FAM with the T7 promoter sequence (T7p-FAM), VIC with the M13 forward sequence (M13f-VIC), and NED with the M13 reverse sequence (M13r-NED). The PCR reaction mix contained 5.0 L of GoTaq Colorless Master Mix (Promega Corp., Madison, WI, USA), 0.4 pmol of the forward primer with the tailed sequence added, 1.6 pmol fluorescence-labeled additional primers (T7p-FAM, M13fVIC, or M13rNED), 1.6 pmol each unlabeled reverse primer, and 30–50 ng template DNA in a total volume of 10 L. A leader sequence (gtttctt) was appended to the 5 end of the reverse primers to minimize the appearance of stutter bands in the electropherograms (Brownstein et al., 1996). Thermocycling conditions were the following: 94 ◦ C for 4 min followed by 10 in cycles at 94 ◦ C for 45 s, 56.5 ◦ C for 1 min, 72 ◦ C for 45 s, 94 ◦ C for 45 s, 35 in cycles at 94 ◦ C for 45 s, 52 ◦ C for 1 min, 72 ◦ C for 45 s, and final extension step at 72 ◦ C for 7 min and separated and detected using an ABI prism 3100/xl genetic analyzer (Applied Biosystems, Foster City, CA, USA) with each capillary containing 1 L of PCR product diluted five times with distillate water, 0.1 L GeneScan-500 LIZ Size Standard (Applied Biosystems), and 8.9 L of HiDi formamide (Applied Biosystems) that was denatured at 95 ◦ C for 5 min. The fragment sizes were estimated using Gene Scan Software (Applied Biosystems).
2.6. Linkage analysis After scoring the segregation of the SSR markers and the pollen–pistil incongruity alleviation gene in F2 plants, each locus was tested for goodness of fit to the expected 3:1 ratios using a Chi-square test. The linkage map was constructed using JoinMap 4.0 (van Ooijen, 2006). A LOD of 4.0 was used to create linkage groups. Recombination values were converted to genetic distances using the Kosanbi mapping function (Kosanbi, 1943). The naming of linkage groups nomenclature corresponds to that in two melon reference maps (Diaz et al., 2011; Fukino et al., 2012) because no map of C. anguria exists, and C. anguria had the same chromosome number as melon.
3. Results 3.1. Pollen tube growth in interspecific cross under differential temperature condition Pollen tube length in the interspecific cross between melon line ‘MR-1 and two C. anguria lines, PI 320052 and PI 364475, was measured 8 h after pollination. When the pistils were incubated under normal-temperature conditions, the pollen–pistil incongruity was observed and the pollen tube length was 0.59 mm in both PI 320052 and PI 364475. However, the pollen tube elongation differed by the cross combinations when the pistils were pollinated under high-temperature conditions. When PI 320052 was used as pistillate parent, the pollen–pistil incongruity was alleviated and the pollen tube length was 1.95 mm. However, when PI 364475 or the F1 line was used as pistillate parents, pollen–pistil incongruity was
Y. Matsumoto, M. Miyagi / Scientia Horticulturae 146 (2012) 81–85 Table 1 Pollen tube length of Cucumis anguria lines and F1 under the normal and high temperature pollinations. Temperature conditions
Line name
Pollen tube length (mm)a
28 ◦ C
PI 320052 PI 364475
0.59 ± 0.03 0.59 ± 0.03
32 ◦ C
PI 320052 PI 364475 F1 (PI 320052 × PI 364475)
1.95 ± 0.10 0.60 ± 0.03 0.59 ± 0.04
a
Means ± S.E., twenty pistils were investigated per each line.
observed and the pollen tube length were, respectively 0.60 mm and 0.59 mm in PI 364475 and F1 line (Table 1). 3.2. Segregation of pollen–pistil incongruity alleviation in F2 population Pollen tube length in the interspecific crosses between ‘MR-1 and F2 populations was measured 8 h after pollination under the high-temperature condition. By the pollen tube length, these lines were classified into two groups, non-alleviated (0.5–1.0 mm) and alleviated (1.5–2.0 mm) lines. The numbers of non-alleviated and alleviated lines were, respectively 60 and 24 (Fig. 1). The observed segregation closely approximates the expected 3 (non-alleviated): 1 (alleviated) ratio (2 = 0.57; P = 0.45). In these two groups, nonsignificant responses were detected among all non-alleviated lines and PI 364475 and F1 line, and all alleviated lines and PI 320052, with P < 0.05 by t-test. Results suggest that this pollen–pistil incongruity alleviation was controlled by a single recessive gene, and we assigned pollen–pistil incongruity alleviation (pia) according to the rules of gene nomenclature for the Cucurbitaceae (Taja and Wehner, 2009). 3.3. Mapping of the pia gene The genotype of the alleviate gene in F2 plants were classified as two groups based on the tests described above: non-alleviated and alleviated. In F2 genetic analysis, 60 markers fitted well the
Fig. 1. Distribution of pollen tube length in 84 F2 plants derived from PI 320052 × PI 364475.
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expected 3:1 or 1:2:1 ratios (P < 0.05), although two markers did not fit the expected ratios (P < 0.01). All markers deviated from Mendelian rations were not used for the linkage analysis. Linkage analysis using a minimum LOD score of 4.0 caused nine major and one minor linkage groups. SSR markers mapped on melon reference maps (Diaz et al., 2011; Fukino et al., 2012) were used as an anchor points for map comparison. Consequently, our linkage groups (LG I–LG XII) were named according to the linkage groups by the melon reference maps. The remaining group (LG XIII) included those in which the common SSR marker was not mapped. The resulting map contained 14 LGs spanning 311.3 cM, with an average marker distance of 6.9 cM. The LGs ranged in size from 56.2 cM (LG XII) to 0.6 cM (LG XIB); 23 markers were in common with the reference maps (Diaz et al., 2011; Fukino et al., 2012). The locations of 23 of those 24 common markers were almost identical in the melon LGs to those in the reference maps. The pia gene was located respectively on LG I between CSN221 and SSR19844 at distances of 3.7 cM and 4.5 cM from CSN221 and SSR19844 (Fig. 2).
4. Discussion The pollen–pistil incongruity is a pre-zygotic barrier between self-fertilizing species (Hogenboom, 1984). Attempts to promote pollen tube growth in interspecific crosses through temperature conditions were also conducted in Lilium (Ascher and Peloquin, 1968), Tulipa (Kho and Baër, 1971), Brassica (Matsuzawa, 1983), Oryza (Sitch and Romero, 1990), and Cucumis (Franken et al., 1988; Matsumoto et al., 2012). However pollen tube growthpromoting effects at higher temperatures have rarely been reported in interspecific crosses. Ascher and Peloquin (1968) concluded that incubation of styles at 39 ◦ C suppressed self-incompatibility, and had no effect on interspecific incompatibility. In Tulipa, 14 ◦ C was reported as the optimum temperature for interspecific crosses (Kho and Baër, 1971). In Brassica, good results were obtained at 25 ◦ C (Matsuzawa, 1983). Sitch and Romero (1990) reported that temperature had no effect on pollen tube growth in crosses of Oryza spp., but that had been stimulated in one cross combination, and at 35 ◦ C only. In our previous study, pollen–pistil incongruity between wild Cucumis spp. and melon alleviated pollen tube growth and enhanced fruit setting under 32–34 ◦ C conditions (Matsumoto et al., 2012). However, in these studies, the genetic mechanism of the pollen–pistil incongruity alleviation effects has not been revealed. This study revealed the inheritance mode of the pollen–pistil incongruity alleviation gene, pia, and the chromosome location. It was single recessive and located between the two SSR markers, CSN221 and SSR19844, on LG I. Short-term high-temperature treatment of pollen or pistils before pollination has been reported as effective in suppressing self-incompatibility of several species such as Lilium longiflorum, Raphanus sativas, and Brassica campestris (Matsubara, 1984; Okazaki and Hinata, 1987). In Brassica, the pollen–pistil recognition of self-incompatibility depends on ligand–receptor interaction (Nasrallah, 2000; Takayama and Isogai, 2003). Therefore, it might be true that pia gene can affect the protein structure in the recognition or the downstream pathway of the incongruity and suppress the incongruity response. To clarify the fundamental mechanism of the pollen–pistil incongruity, high-resolution mapping of the gene must be studied at molecular level. Selection of plants available for the interspecific crosses with melon by phenotype alone is difficult because evaluation of this trait is influenced by temperature conditions, and its mode of inheritance is recessive. The CSN221 and SSR19844 markers are anticipated for use as powerful tools for breeding lines with alleviated pollen–pistil incongruity in the interspecific crosses with melon. Recently, an interspecific hybrid between C. anguria and
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Fig. 2. Linkage map derived from the F2 population of a PI 320052 × PI 364475 cross. The SSR markers are shown on the right of each linkage group. Distances between SSR markers are indicated in centimorgans on the left. Linkage group nomenclature corresponds to that in the melon reference linkage maps (Diaz et al., 2011; Fukino et al., 2012). Bold type indicates markers in common with the reference linkage map, and the asterisk indicates marker in different positions on the two maps.
Cucumis zeyheri was developed (Skálová et al., 2008). C. zeyheri was not fertilized by the cross with melon (Matsumoto et al., 2012). However, if the gene were introgressed into C. zeyheri, it could be fertilized by the cross with the melon. The distances between the markers and gene are 3.7 cM and 4.5 cM, respectively, so adding more markers in this region would enable us to conduct more accurate selection of this trait. We constructed a C. anguria linkage map using F2 plants from the cross between PI 320052 and PI 364475. SSR markers were usually codominant and were therefore mapped accurately using F2 plants compared with dominant markers. Some common SSR markers with melon reference maps were useful to identify the same linkage groups as that developed by Diaz et al. (2011) and Fukino et al. (2012). Among the common SSR markers, few markers exist in different positions on the two maps. Therefore, these markers could be anchor points for map comparison between C. anguria and melon. A melon linkage map would be useful to add more markers in this
region. Furthermore, the SSR marker SSR19844 was mapped on cucumber chromosome 7 (Zhang et al., 2012). This chromosomal information might be available if synteny between C. anguria and cucumber were also observed. C. anguria is also consumed as ‘West Indian Gherkin’ or ‘Maxixe’ mainly in Brazil and the United States (Mangan et al., 2008). However, regarding the breeding of ‘West Indian Gherkin’, it had been conducted by phenotype selection (Modolo and Costa, 2004) because no linkage map has been reported in C. anguria. This linkage map can also contribute the breeding research related to ‘West Indian Gherkin’. Acknowledgement We are grateful to Asc. Prof. Dr. Tsutomu Kuboyama, College of Agriculture, Ibaraki University, Japan, for his valuable technical advice.
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Mapping of a gene conferring alleviation of pollen–pistil incongruity found in an interspecific cross between Cucumis anguria L. and Cucumis melo L. (melon) Yuichi Matsumoto a,∗ , Makoto Miyagi b a b
United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183-8509, Japan Plant Biotechnology Institute, Ibaraki Agricultural Center, Kasama, Ibaraki 319-0292, Japan
a r t i c l e
i n f o
Article history: Received 20 June 2012 Received in revised form 15 August 2012 Accepted 16 August 2012 Keywords: Cucumis spp. Reproductive barrier Pollen tube Interspecific cross Mapping
a b s t r a c t In Cucumis, when melon (Cucumis melo L.) was crossed with Cucumis anguria L., PI 320052, at 24–30 ◦ C, pollen tube growth was arrested in the style. However, at 32 ◦ C and 34 ◦ C, the pollen–pistil incongruity was alleviated. Pollen tubes penetrated into ovules and fruit set was observed. However, these studies have not revealed the genetic and molecular mechanism of pollen–pistil incongruity alleviation. This study reveals the mode of inheritance and identifies molecular markers linked with the gene. We developed the F1 and F2 lines from the alleviate line and non-alleviate line, and evaluated the mode of inheritance of the gene. We constructed the linkage map by the phenotypes and conducted simple sequence repeat (SSR) analysis. We demonstrated that the pollen–pistil incongruity alleviation gene is single recessive and that it is located on LG I between CSN221 and SSR19844 at a distance of 3.7 cM and 4.5 cM from CSN221 and SSR19844, respectively. Therefore we assigned the gene as pollen–pistil incongruity alleviation (pia). This is the first report on the mapping of a gene conferring the pollen–pistil incongruity alleviation in interspecific crosses © 2012 Elsevier B.V. All rights reserved.
1. Introduction Pollen–pistil incongruity, a pre-zygotic barrier between selffertilizing species (Hogenboom, 1984), has been reported in many taxa such as Tulipa (Kho and Baër, 1971), Nicotiana (Kuboyama et al., 1994), Oryza (Suputtitada et al., 2000), and Cucumis (Kishi and Fujishita, 1970; Kho et al., 1980). In Cucumis, pollen–pistil incongruity is often observed in interspecific crosses between melon (Cucumis melo L.) and wild Cucumis spp. In these crosses, although pollens germinate and pollen tubes enter into the stigma, most pollen tubes are arrested in the style and fertilization is rarely observed. Wild Cucumis spp. reportedly has resistance for some melon pests. Some examples are downy mildew (Pan and More, 1996), powdery mildew (Lebeda, 1984; Pan and More, 1996), fusarium wilt (Pan and More, 1996; Matsumoto et al., 2011), and root-knot nematode (Sigüenza et al., 2005). Therefore, methods to bypass these reproductive barriers are available for expansion of usable genetic resources in melon breeding. Hedhly et al. (2005) reported temperature as an important factor affecting pollen performance during the progamic phase: from
∗ Corresponding author. Present address: Plant Biotechnology Institute, Ibaraki Agricultural Center, Kasama, Ibaraki 319-0292, Japan. Tel.: +81 299 458330; fax: +81 299 458351. E-mail address: [email protected] (Y. Matsumoto). 0304-4238/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scienta.2012.08.021
pollination to fertilization. Regarding pollen–pistil incongruity, the alleviated effect by the temperature has been reported in interspecific crosses of Lilium (Ascher and Peloquin, 1968), Tulipa (Kho and Baër, 1971), Brassica (Matsuzawa, 1983), Oryza (Sitch and Romero, 1990), and Cucumis (Franken et al., 1988; Matsumoto et al., 2012). In Cucumis, we observed that when melon was crossed with Cucumis anguria L. at 24–30 ◦ C, pollen tube growth was arrested in the style. However, at 32 ◦ C and 34 ◦ C, the pollen–pistil incongruity was alleviated and pollen tubes penetrated into ovules and fruit set was observed (Matsumoto et al., 2012). However, the genetics of this pollen–pistil incongruity alleviation in interspecific cross was not studied. Such inheritance study is important to overcome reproductive barrier, and expansion of usable genetic resources. Hence we examined mode of inheritance of pollen–pistil incongruity alleviation and using molecular market made effort to locate gene for the pollen–pistil incongruity alleviation in Cucumis. For this study, we examined the mode of inheritance and the chromosome location of the gene conferring the pollen–pistil incongruity alleviation in C. anguria.
2. Materials and methods 2.1. Plant materials Melon line ‘MR-1 was obtained from the Institut National de la Recherche Agronomique (INRA), France, and used as the pollen
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parent. The C. anguria L. lines PI 320052 and PI 364475 were obtained from Germplasm Resources Laboratory, USDA, ARS, Beltsville, USA, and used. Eighty-four F2 plants from the cross between PI 320052 and PI 364475 were developed for mapping the population. 2.2. Pollen tube observation Pollination was conducted using mature flowers emasculated one day before anthesis. The flowering pistils of PI 320052 and PI 364475 were collected, put on a wetted paper towel, pollinated with ‘MR-1 pollen, and then incubated at controlled temperatures of 28 ◦ C (normal temperature) or 32 ◦ C (high temperature). The incubated pistils were collected 8 h after pollination and macerated by autoclaving in 50 g L−1 aqueous sodium sulfite and stained with aniline blue before squashing under a cover glass (Jefferies and Belcher, 1974). Pollen tubes were observed under a fluorescence microscope (blue–violet excitation filter cube, UMWBV, BX50; Olympus Corp., Tokyo, Japan). Photographs of pollen tubes were taken using a digital camera (DS-Fi1; Nikon Corp., Tokyo, Japan) and acquired with imaging software (NIS-Elements D; Nikon Corp., Tokyo, Japan). The images were registered on image tiling software (e-Tiling; Mitani Corp., Fukui, Japan). The distance from the stigma to the longest pollen tube was regarded as the approximate pollen tube length. 2.3. Segregation of expression types Pistils that pollinated under the high temperature condition were observed for pollen tube length. For these observations, 20 flowers of two parental line (PI 320052 and PI 364475) and F1 line, and 10 flowers of 84 F2 lines were examined. Pollen tube length was log-transformed and calculated using analysis of variance (ANOVA) via a least-squares fitting model. A Tukey–Kramer test was performed as a post hoc test for ANOVA to compare lines. For these analyses, statistical software (JMP ver. 9.0.0; SAS Institute Inc., Cary, NC, USA) was used. In these analyses, a line which showed a non-significant difference from PI 320052 (alleviated parent) was regarded as an alleviated line. A line which showed a non-significant difference from PI 364475 (non-alleviated parent) was regarded as a non-alleviated line. The observed ratios in the segregated F2 populations were tested for deviations from the expected ratios using the Chi-square goodness of fit test, with significance inferred for P < 0.05. 2.4. DNA extraction Total DNA was extracted according to Mukai and Yamamoto (1997) with some modifications. Briefly, each 2-cm-long young leaf tip from a single plant was put in a 1.5 ml tube. Then 290 L of extraction buffer (100 mM Tris–HCl (pH 8.0), 500 mM NaCl, 50 mM EDTA (pH 8.0), and 10 mM 2-mercapto ethanol) was added. The leaf was chopped with sharp plastic chips and a tube was incubated at 65 ◦ C for 10 min. After incubation, 100 L of 5 M potassium acetate were added and incubated at 4 ◦ C 15 min. After centrifuging, the supernatant was recovered and 300 L of 2-propanol was added. After centrifuging, the supernatant was discarded and the DNA pellet was rinsed with 300 L of 70% ethanol. The DNA pellet was dried and dissolved in 100 L of 1/10 TE buffer. 2.5. SSR analysis Sixty-two simple sequence repeat (SSR) markers developed by cucumber (Cucumis sativus L.) (Fukino et al., 2008; Watcharawongpaiboon and Chunwongse, 2008; Ren et al., 2009) and melon (Danin-Poleg et al., 2001; Chiba et al., 2003; Ritschel
et al., 2004; Gonzalo et al., 2005; Fukino et al., 2007; Cucurbit Genomics Database, http://www.icugi.org/), and which showed polymorphism between PI 320052 and PI 364475 were used to construct a genetic linkage map. PCR reaction was performed using the modified post labeled method (Schuelke, 2000) as follows: SSR forward primers were modified using 5 -concatenation with the T7 promoter sequence (aatacgactcactatagg), with the M13 forward sequence (tgtaaaacgacggccagt), or with the M13 reverse promoter (caggaaacagctatgacc) based on Moriya et al. (2010). For these three sequences, we also created tailed sequences labeled with fluorescent chemicals: FAM with the T7 promoter sequence (T7p-FAM), VIC with the M13 forward sequence (M13f-VIC), and NED with the M13 reverse sequence (M13r-NED). The PCR reaction mix contained 5.0 L of GoTaq Colorless Master Mix (Promega Corp., Madison, WI, USA), 0.4 pmol of the forward primer with the tailed sequence added, 1.6 pmol fluorescence-labeled additional primers (T7p-FAM, M13fVIC, or M13rNED), 1.6 pmol each unlabeled reverse primer, and 30–50 ng template DNA in a total volume of 10 L. A leader sequence (gtttctt) was appended to the 5 end of the reverse primers to minimize the appearance of stutter bands in the electropherograms (Brownstein et al., 1996). Thermocycling conditions were the following: 94 ◦ C for 4 min followed by 10 in cycles at 94 ◦ C for 45 s, 56.5 ◦ C for 1 min, 72 ◦ C for 45 s, 94 ◦ C for 45 s, 35 in cycles at 94 ◦ C for 45 s, 52 ◦ C for 1 min, 72 ◦ C for 45 s, and final extension step at 72 ◦ C for 7 min and separated and detected using an ABI prism 3100/xl genetic analyzer (Applied Biosystems, Foster City, CA, USA) with each capillary containing 1 L of PCR product diluted five times with distillate water, 0.1 L GeneScan-500 LIZ Size Standard (Applied Biosystems), and 8.9 L of HiDi formamide (Applied Biosystems) that was denatured at 95 ◦ C for 5 min. The fragment sizes were estimated using Gene Scan Software (Applied Biosystems).
2.6. Linkage analysis After scoring the segregation of the SSR markers and the pollen–pistil incongruity alleviation gene in F2 plants, each locus was tested for goodness of fit to the expected 3:1 ratios using a Chi-square test. The linkage map was constructed using JoinMap 4.0 (van Ooijen, 2006). A LOD of 4.0 was used to create linkage groups. Recombination values were converted to genetic distances using the Kosanbi mapping function (Kosanbi, 1943). The naming of linkage groups nomenclature corresponds to that in two melon reference maps (Diaz et al., 2011; Fukino et al., 2012) because no map of C. anguria exists, and C. anguria had the same chromosome number as melon.
3. Results 3.1. Pollen tube growth in interspecific cross under differential temperature condition Pollen tube length in the interspecific cross between melon line ‘MR-1 and two C. anguria lines, PI 320052 and PI 364475, was measured 8 h after pollination. When the pistils were incubated under normal-temperature conditions, the pollen–pistil incongruity was observed and the pollen tube length was 0.59 mm in both PI 320052 and PI 364475. However, the pollen tube elongation differed by the cross combinations when the pistils were pollinated under high-temperature conditions. When PI 320052 was used as pistillate parent, the pollen–pistil incongruity was alleviated and the pollen tube length was 1.95 mm. However, when PI 364475 or the F1 line was used as pistillate parents, pollen–pistil incongruity was
Y. Matsumoto, M. Miyagi / Scientia Horticulturae 146 (2012) 81–85 Table 1 Pollen tube length of Cucumis anguria lines and F1 under the normal and high temperature pollinations. Temperature conditions
Line name
Pollen tube length (mm)a
28 ◦ C
PI 320052 PI 364475
0.59 ± 0.03 0.59 ± 0.03
32 ◦ C
PI 320052 PI 364475 F1 (PI 320052 × PI 364475)
1.95 ± 0.10 0.60 ± 0.03 0.59 ± 0.04
a
Means ± S.E., twenty pistils were investigated per each line.
observed and the pollen tube length were, respectively 0.60 mm and 0.59 mm in PI 364475 and F1 line (Table 1). 3.2. Segregation of pollen–pistil incongruity alleviation in F2 population Pollen tube length in the interspecific crosses between ‘MR-1 and F2 populations was measured 8 h after pollination under the high-temperature condition. By the pollen tube length, these lines were classified into two groups, non-alleviated (0.5–1.0 mm) and alleviated (1.5–2.0 mm) lines. The numbers of non-alleviated and alleviated lines were, respectively 60 and 24 (Fig. 1). The observed segregation closely approximates the expected 3 (non-alleviated): 1 (alleviated) ratio (2 = 0.57; P = 0.45). In these two groups, nonsignificant responses were detected among all non-alleviated lines and PI 364475 and F1 line, and all alleviated lines and PI 320052, with P < 0.05 by t-test. Results suggest that this pollen–pistil incongruity alleviation was controlled by a single recessive gene, and we assigned pollen–pistil incongruity alleviation (pia) according to the rules of gene nomenclature for the Cucurbitaceae (Taja and Wehner, 2009). 3.3. Mapping of the pia gene The genotype of the alleviate gene in F2 plants were classified as two groups based on the tests described above: non-alleviated and alleviated. In F2 genetic analysis, 60 markers fitted well the
Fig. 1. Distribution of pollen tube length in 84 F2 plants derived from PI 320052 × PI 364475.
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expected 3:1 or 1:2:1 ratios (P < 0.05), although two markers did not fit the expected ratios (P < 0.01). All markers deviated from Mendelian rations were not used for the linkage analysis. Linkage analysis using a minimum LOD score of 4.0 caused nine major and one minor linkage groups. SSR markers mapped on melon reference maps (Diaz et al., 2011; Fukino et al., 2012) were used as an anchor points for map comparison. Consequently, our linkage groups (LG I–LG XII) were named according to the linkage groups by the melon reference maps. The remaining group (LG XIII) included those in which the common SSR marker was not mapped. The resulting map contained 14 LGs spanning 311.3 cM, with an average marker distance of 6.9 cM. The LGs ranged in size from 56.2 cM (LG XII) to 0.6 cM (LG XIB); 23 markers were in common with the reference maps (Diaz et al., 2011; Fukino et al., 2012). The locations of 23 of those 24 common markers were almost identical in the melon LGs to those in the reference maps. The pia gene was located respectively on LG I between CSN221 and SSR19844 at distances of 3.7 cM and 4.5 cM from CSN221 and SSR19844 (Fig. 2).
4. Discussion The pollen–pistil incongruity is a pre-zygotic barrier between self-fertilizing species (Hogenboom, 1984). Attempts to promote pollen tube growth in interspecific crosses through temperature conditions were also conducted in Lilium (Ascher and Peloquin, 1968), Tulipa (Kho and Baër, 1971), Brassica (Matsuzawa, 1983), Oryza (Sitch and Romero, 1990), and Cucumis (Franken et al., 1988; Matsumoto et al., 2012). However pollen tube growthpromoting effects at higher temperatures have rarely been reported in interspecific crosses. Ascher and Peloquin (1968) concluded that incubation of styles at 39 ◦ C suppressed self-incompatibility, and had no effect on interspecific incompatibility. In Tulipa, 14 ◦ C was reported as the optimum temperature for interspecific crosses (Kho and Baër, 1971). In Brassica, good results were obtained at 25 ◦ C (Matsuzawa, 1983). Sitch and Romero (1990) reported that temperature had no effect on pollen tube growth in crosses of Oryza spp., but that had been stimulated in one cross combination, and at 35 ◦ C only. In our previous study, pollen–pistil incongruity between wild Cucumis spp. and melon alleviated pollen tube growth and enhanced fruit setting under 32–34 ◦ C conditions (Matsumoto et al., 2012). However, in these studies, the genetic mechanism of the pollen–pistil incongruity alleviation effects has not been revealed. This study revealed the inheritance mode of the pollen–pistil incongruity alleviation gene, pia, and the chromosome location. It was single recessive and located between the two SSR markers, CSN221 and SSR19844, on LG I. Short-term high-temperature treatment of pollen or pistils before pollination has been reported as effective in suppressing self-incompatibility of several species such as Lilium longiflorum, Raphanus sativas, and Brassica campestris (Matsubara, 1984; Okazaki and Hinata, 1987). In Brassica, the pollen–pistil recognition of self-incompatibility depends on ligand–receptor interaction (Nasrallah, 2000; Takayama and Isogai, 2003). Therefore, it might be true that pia gene can affect the protein structure in the recognition or the downstream pathway of the incongruity and suppress the incongruity response. To clarify the fundamental mechanism of the pollen–pistil incongruity, high-resolution mapping of the gene must be studied at molecular level. Selection of plants available for the interspecific crosses with melon by phenotype alone is difficult because evaluation of this trait is influenced by temperature conditions, and its mode of inheritance is recessive. The CSN221 and SSR19844 markers are anticipated for use as powerful tools for breeding lines with alleviated pollen–pistil incongruity in the interspecific crosses with melon. Recently, an interspecific hybrid between C. anguria and
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Fig. 2. Linkage map derived from the F2 population of a PI 320052 × PI 364475 cross. The SSR markers are shown on the right of each linkage group. Distances between SSR markers are indicated in centimorgans on the left. Linkage group nomenclature corresponds to that in the melon reference linkage maps (Diaz et al., 2011; Fukino et al., 2012). Bold type indicates markers in common with the reference linkage map, and the asterisk indicates marker in different positions on the two maps.
Cucumis zeyheri was developed (Skálová et al., 2008). C. zeyheri was not fertilized by the cross with melon (Matsumoto et al., 2012). However, if the gene were introgressed into C. zeyheri, it could be fertilized by the cross with the melon. The distances between the markers and gene are 3.7 cM and 4.5 cM, respectively, so adding more markers in this region would enable us to conduct more accurate selection of this trait. We constructed a C. anguria linkage map using F2 plants from the cross between PI 320052 and PI 364475. SSR markers were usually codominant and were therefore mapped accurately using F2 plants compared with dominant markers. Some common SSR markers with melon reference maps were useful to identify the same linkage groups as that developed by Diaz et al. (2011) and Fukino et al. (2012). Among the common SSR markers, few markers exist in different positions on the two maps. Therefore, these markers could be anchor points for map comparison between C. anguria and melon. A melon linkage map would be useful to add more markers in this
region. Furthermore, the SSR marker SSR19844 was mapped on cucumber chromosome 7 (Zhang et al., 2012). This chromosomal information might be available if synteny between C. anguria and cucumber were also observed. C. anguria is also consumed as ‘West Indian Gherkin’ or ‘Maxixe’ mainly in Brazil and the United States (Mangan et al., 2008). However, regarding the breeding of ‘West Indian Gherkin’, it had been conducted by phenotype selection (Modolo and Costa, 2004) because no linkage map has been reported in C. anguria. This linkage map can also contribute the breeding research related to ‘West Indian Gherkin’. Acknowledgement We are grateful to Asc. Prof. Dr. Tsutomu Kuboyama, College of Agriculture, Ibaraki University, Japan, for his valuable technical advice.
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