Botanical Studies (2009) 50: 389-402.
MOLECULAR BIOLOGY
Marker-based analysis of genome structure and DNA methylation in a watermelon (Citrullus lanatus) ploidy series
Chun-Guo WANG1, Hui LI2, Zhen-Yi XUE1, Cheng-Bin CHEN1, Yu GU3, De-Ling SUN3, and Wen-Qin SONG1*
1 College of Life Sciences, Nankai University, Tianjin, 300071, P.R. China
2Department of Horticulture, Tianjin Agricultural University, Tianjin, 300384, P.R. China
3Tianjin Vegetable Research Institute, Tianjin, 300382, P.R. China
(Received August 26, 2008; Accepted April 9, 2009)
ABSTRACT. Polyploidization is a major driver of plant genome evolution and speciation. However, most investigations have focused on allopolyploidization, and little is known about the genetic and epigenetic impact imposed by autopolyploidization. In the present study, an autoploidy series (2x, 3x and 4x) in watermelon (Citrullus lanatus) was constructed. The genome structure changes and DNA methylation adjustments at CCGG sites in a genome-wide level were analyzed in the three different autoploidy watermelons by the ISSR, SRAP, and MSAP methods. Compared with diploids, no obvious genetic changes were discovered in autotriploids. Extensive changes occurred in autotetraploids in which sequence eliminations were predominant. However, analysis of DNA methylation level indicated that the total DNA methylation levels were not significantly different between diploid and autotetraploid. Autotriploid showed a trend of low DNA methylation level. It implied that the alterations of DNA methylation levels were not linear with the autopolyploidy level in watermelon. Further analysis suggested that about half of total sites experienced adjustments of DNA methylation patterns in different ploidy watermelons. Interestingly, autotriploid still showed an obvious trend of demethylation. These results indicated some differences from other investigated autopolyploid plants in autopolyploid watermelon. Autotriploid watermelon, in particular, showed unusual characters of genome structure and DNA methylation status, which may be closely associated with the odd-ploidy effect and the excellent traits present in triploid plants.
Keywords: Autopolyploidization; Allopolyploidization; DNA methylation; MSAP; Watermelon.
INTRODUCTION
Polyploidy is remarkably common in the plant kingdom, resulting either from duplication of the entire genome (autopolyploidy) or through combination of genomes from different parental species (allopolyploidy). The most popular estimate of the proportion of polyploid in flowering plants is about 70% (Masterson, 1994; Wendel, 2000). However, recent investigations indicate that almost all higher plants have undergone one or more polyploidization events during their evolutionary history, even many classic diploid plant species including model plants Arabidopsis and maize, are of polyploid origin (Helentjaris et al., 1988; The Arabidopsis Genome Initiative, 2000; Vision et al., 2000; Blanc et al., 2003; Ermolaeva et al., 2003). Thus, polyploidization has been
*Corresponding authors: E-mail: songwq@nankai.edu.cn (Wen-Qin SONG); wangcg@nankai.edu.cn (Chun-Guo
WANG).
regarded as one of the most important driving forces for plant genome evolution and speciation (Lewis, 1980; Soltis et al., 2004; Adams and Wendel, 2005). Moreover, because some polyploid plants possess excellent crop traits, such as increased abiotic and biotic stress tolerance, early flowering time, increased organ size and biomass, many important crops are also polyploids (Levin, 1983;
Hilu, 1993; Liu and Wendel, 2002). The prominent
evolutionary significance and practical value of polyploidy in agriculture underscore the importance of exploring the molecular nature of polyploidization (Chen and Ni, 2006).
A s eries of recent inves tig ations focusing on allopolyploidy indicate that widespread genomic changes occur concomitantly with hybridization and genome doubling - the two most important events of
allopolyploidization (Wendel, 2000; Ozkan et al., 2001;
Liu and Wendel, 2002). It was first reported in Brassica that extensive genomic changes occurred in low-copy nuclear sequences in each of F2 to F5 generations of synthetic allotetraploids among different species (Song et al., 1995). Following that, rapid sequences elimination,
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gene silencing, transposon activation, and other genome or chromosome changes were widely detected (Feldman
et al., 1997; Ozkan et al., 2001; Shaked et al., 2001;
Kashkush et al., 2002; Madlung et al., 2002; Mishima
et al., 2002; Pontes et al., 2004; Salina et al., 2004; Han
et al., 2005; Petit et al., 2007), as well as the changes of epigenetic modification, especially DNA methylation
(Matzke et al., 1999; Lee and Chen, 2001; Madlung et
al., 2002), and gene expression in newly synthesized allopolyploid wheat (He et al. , 2003 ), Arabidopsis (Wang et al., 2004; Wang et al. , 2006) and Brassica napus (Osborn et al. , 2003; Albertin et al. , 2007). However, not all investigations have detected genomic changes in allopolyploid plants (Paun et al., 2007). A representative exception is allopolyploid cotton. A survey of approximately 22, 000 genomic loci from nine sets of synthesized allotetraploid and allohexaploid cotton by AFLP and MSAP fingerprinting suggested genomic stasis and lack of rapid DNA methylation changes at symmetric CCGG sites (Liu et al., 2001). The exceptions can also be found in Brassica (Axelsson et al., 2000) and sugarcane (Jannoo et al., 2007). These contrasting examples indicate that allopolyploidization in plants may involve a diverse array of molecular e volutionary strategies reflecting both genomic constituents and taxonomic considerations (Liu et al. , 2001), further highlighting the complex molecular mechanism of allopolyploidization. However, despite considerable research attention, the underlying mechanisms remain poorly understood.
C ompared to allopolyploidization, relatively few studies have been devoted to elucidating the consequences of autopolyploidization, in which only genome doubling occurs, partly because for many years autopolyploidy was considered extremely rare and maladaptive (Stebbins, 1950). Contradicting the view of autopolyploidy as an evolutionary dead-end, as asserted by Soltis et al. (2007), is the fact that the number of angiosperm species undergoing autopolyploidy has been grossly underestimated. In addition to Zea diploperennis and Z. perennis, at least five autopolyploids, i.e., Tolmiea menziesii, Galax urceolata, Chamerion angustifolium, Heuchera grossulariifolia and Vaccinium corymbosum, should be regarded as distinct species. Furthermore, several autopolyploid plants are successful crop species of major importance, including alfalfa, potato, cassava, watermelon, banana and apple cultivars, and sugar beet (Soltis and Soltis, 1993; Ming
et al. , 2001; NeSmith and Duval, 2001; Paterson,
2005). Substantial evidence indicates the potential of autopolyploids to create more new crop species or cultivars than their diploid progenitors and supports the need for further research to extend the utilization of autopolyploidy in agriculture.
In the present study, we constructed a set of autopolyploid (diploid, autotriploid and autotetraploid) watermelons (Citrullus lanatus) to detect possible genomic and epigenetic changes induced by autopolyploidization. Thereafter, we conducted a quantitative estimate of the frequency of genome-wide and epigenetic alterations
using inter-sample sequence repeat (ISSR), sequence-related amplified polymorphism (SRAP) and methylation-sensitive amplified polymorphism (MSAP) analyses. Finally, we established the characteristic alterations in genome structure and DNA methylation at CCGG sites. Possible causes for the observed changes as well as differences from other autopolyploid or allopolyploid plants were discussed.
MATERIALS AND METHODS
Plant materials
The plant materials used in this study include homozygous diploid watermelon (Citrullus lanatus), which is an inbred line, newly synthesized autotriploid and autotetraploid watermelon, named JKR-1, JKR-2 and JKR-3, respectively. JKR-3 was created by colchicines-doubling the diploid as described by Jaskani et al. (2004) while the autotriploid JKR-2 was generated by crossing diploid, JKR-1 with the autotetraploid, JKR-3 (as the female recipient of pollen). The ploidy level of JKR-2 and JKR-3 was identified by chromosome number in root tip mitosis. All these materials were kindly provided by Prof. Dingliang-Jiao, Tianjin Vegetable Research Institute, Tianjin, China, and synchronously grown in a greenhouse under normal light conditions (day/light temperature of 25/19°C, 16 h photoperiod). After 10 days the young leaves from each of the three different ploidy watermelons were used for the extraction of genomic DNA.
Karyotype analysis
Chromosome preparations were prepared using the squash technique as described previously (Urdampilleta et al., 2006). In brief, tender root tips from germinated seeds of diploid watermelon (JKR-1) and predicted autotriploid and autotetraploid watermelons, were cut and pretreated with 2 mM 8-hydroxyquinoline for 4 h at 20°C, fixed in ethanol: acetic acid (3:1, V:V) for 10 h. Following that, all root tips were hydrolyzed in 1 N HCl for 10 min at room temperature and thoroughly rinsed with distilled water. After that the root tips were placed on a frozen slide, squashed, and then stained with Carbor fuchsin. Photographs were taken with a photomicroscope (Nikon80i, Japan). At least eight cells with good metaphase structures from a single plant were examined by this method. Only the individuals with the correct number of chromosomes (e.g., diploid with 2n=2x=22, triploid with 2n=3x=33 and tetraploid with 2n=4x=44) were considered for further investigation.
DNA extraction
Genomic DNA was extracted from young fresh leaves of eight individuals from JKR-1, JKR-2, and JKR-3, respectively, by the CTAB procedure (Murray and Thompson, 1980) with some modifications. In brief, young leaves (200 mg) were collected and immediately ground in liquid nitrogen, before the frozen power was added to 2
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mL lysis buffer including 100 mM Tns-HCl (pH 8.0), 100 mM EDTA, 1.4 M NaCl, 0.2% P-mercaptoethanol, 2% PVP and 1xCTAB. After incubation at 56°C for 30 mm, 2 mL phenol: chloroform: isoamyl alcohol (25:24:1, V:V:V) was added. The following steps were as described by our previous study (Wang et al., 2007; Gu et al., 2008).
ISSR and SRAP analysis
Thirty IS S R primers (Wang et al. , 2008) were designed for PCR amplifications in JKR-1, JKR-2 and JKR-3. The optimized polymerase reaction conditions for 25 fil volume contained 10 mM Tns-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl?, 200 jiM dNTPs, 100 ng template DNA, 0.5 jiM primer and 1 unit Taq polymerase (TaKaRa, Japan). Amplification was performed in an Eppendorf Master cycler gradient (Eppendorf Netheler-Hinz, Hamburg, Germany) following a touchdown PCR program: initial denaturation at 94°C for 2 min; 35 cycles of denaturation at 94°C for 45 s, annealing at a specific annealing temperature for 1 min and extension at 72°C for 1 min 30 s, with a final extension of 8 min at 72°C. The annealing was performed with touchdown decrements of 1°C starting at 65°C for the first 10 cycles followed by 25 cycles at 55°C. The amplified products were roughly analyzed by electrophoresis in a 1.5% agarose gel before being separated on a neutral 4% polyacrylamide gel (29:1, acryamide:bisacrylamide) to improve the resolution. The polyacrylamide electrophoresis was carried out in 1xTBE buffer at room temperature and run at 240 V for 3 h, and then the gel was stained with Silver Stain Kit (ATTO, Japan). Only clear and reproducible bands that appeared in two independent PCR amplifications with different batches of DNA isolations were considered for the following analysis.
For SRAP analysis, eight forward primers and ten reverse primers were used (Table 1). Each combination of forward and reverse primers was screened for its polymorphism and reproducibility. The primer pairs were excluded if their banding patterns were difficult to score or if they failed to amplify consistently. The PCR
reaction mixtures (25 fil total volume) consisted of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl?, 200 jiM dNTPs, 100 ng template DNA, 0.8 (il each of 10 mM forward and reverse primers and 1 unit Taq polymerase (TaKaRa, Japan). Amplifications were carried out following the program as described by Li and Qurios (2001). The amplified products were also roughly analyzed by 1.5% agarose gel, and then separated on a neutral 4% polyacrylamide gel as mentioned in ISSR analysis.
MSAP analysis
The protocol used for MSAP analysis was essentially as previously reported (Xiong et al., 1999; Xu et al., 2000; Sha et al., 2005; Dong et al., 2006), with modifications. Briefly, for each sample of watermelon materials, i.e., JKR-1, JKR-2 and JKR-3, aliquots (400 ng) of DNA
were digested for 72 h at 37°C with 3 U EcoRI (TaKaRa,
Japan) and 3 U Hpall or Mspl (Promega, USA) in a final volume of 15 fl containing 1 x R-L buffer (0.05 mol/L
Tris-HCl, 0.05 mol/L MgAc2, 0.25 mol/L KAc). HpaII
and MspI are a pair of isoschizomers that recognize the same tetranucleotide sequence 5'-CCGG (a prominent site for methylation in both plants and animals) but have different sensitivity to the methylation states of cytosines: HpaII will not cut if either of the cytosines is fully (double-strand) methylated, whereas, MspI will not cut if the external cytosine is fully-or hemi-(single-strand) methylated (Xu et al., 2000). The digested fragments were then ligated to the adapter by adding a 5 fl ligation mixture, containing 30 pmol HpaII/MspI adapters, 30
pmol EcoR I adapters, 0.4 mM ATP, 0.8 U T4 DNA ligase
(TaKaRa, Japan), and 1 fil 5 x R-L buffer in a final volume of 20 fl, and this ligation mixture was incubated at 16°C for 10-12 h. It was then diluted 1:10 with sterile water and used as the template for the pre-amplification with E00 and H/M00 primers. The reaction was performed for 25 cycles of 30 s denaturation at 94°C, 1 min annealing at 56°C, and 1 min extension at 72°C. The volume of pre-amplification was 20 fl, containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgC^, 200 jiM
Table 1. Forward and reverse primers using in SRAP assay.
Primer
Type
Sequence (5'-3')
Me5
Forward
TGAGTCCAAACCGGATA
Me6
Forward
TGAGTCCAAACCGGAGC
Me9
Forward
TGAGTCCAAACCGGAAT
Me10
Forward
TGAGTCCAAACCGGACC
Me11
Forward
TAGGTCCAAACCGGCTC
Me12
Forward
TAGGTCCAAACCGGCGT
Me14
Forward
TGAGTCCAAACCGGAGT
Me15
Forward
TAGGTCCAAACCGGGTC
Primer
Type
Sequence (5'-3')
Em1
Reverse
GACTGCGTACGAATTAAT
Em2
Reverse
GACTGCGTACGAATTTGC
Em3
Reverse
GACTGCGTACGAATTGAC
Em4
Reverse
GACTGCGTACGAATTAAC
Em5
Reverse
GACTGCGTACGAATTGCA
Em6
Reverse
GACTGCGTACGAATTCTT
Em7
Reverse
GACTGCGTACGAATTGAT
Em8
Reverse
GACTGCGTACGAATTTAA
Em9
Reverse
GACTGCGTACGAATTTAT
Em10
Reverse
GACTGCGTACGAATTTGC
"Me" indicated forward primers; "Em" indicated reverse primers.
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dNTPs, 1 fil diluted ligation product, 0.5 jiM of each E00 and H/M00 primers and 1 unit Taq polymerase (TaKaRa, Japan). These products were also diluted 10-fold with sterile water, and used as the template for selective amplification. Selective amplifications were conducted in a final volume of 20 fil, containing 1 fil of the diluted pre-amplification product and 0.5 jiM each of E and H/M primers; the remaining components were the same as those in the pre-amplification reaction. The reaction was carried out as described by Xiong et al. (1999). The denatured PCR products were separated on a 6% denaturing polyacrylamide gel at 35 W for 2 h. The gel was then stained with silver (Chalhoub et al., 1997). Only clear and reproducible bands that appeared in two independent PCR amplifications [starting from the DNA digestion step, (i.e., the first step of MSAP) were scored].
RESULTS
Validation of the ploidy level by Karyotype assay
The ploidy levels of all materials used in present study were determined by counting chromosome numbers in root
cells. A total of ten individuals randomly selected from each of three different ploidy watermelons were tested. At least eight cells from two independent root tips of each sample were used to count the number of chromosomes. The results indicated that in each cell, the corresponding chromosome numbers were completely consistent with previous reports (Beevy and Kuriachan, 1996), implying that all materials were stable with respect to ploidy level, and no significant changes in chromosome structure had occurred (Figure 1).
Alteration in genomic structure detected by ISSR and SRAP assays
The ISSR assay generated approximately 1000 scored bands, for an average of 333 bands per ploidy sample, representing 328 prospective genetic loci. Partial results are presented in Figure 2. Among these 328 loci, 182 sites were simultaneously detected in three different ploidy samples, meaning that the remaining 146 were polymorphic, and the polymorphism frequency was thus 44.5% (146/328). A comparison of sites detected in 3x with those in 2x, showed that 17.4% (57/328) of sites were changed, of which 7.6% (25/328) sites were specifically
Figure 1. Karyotype of different ploidy watermelon. (a) diploid watermelon, JKR-1 (2n=2x=22); (b) autotriploid watermelon, JKR-2 (2n=3x=33); (c) autotetraploid watermelon, JKR-3 (2n=4x=44); Scale bar=5 (im.
WANG et al. — Genetic and epigenetic changes in watermelon
393
Figure 2. Amplification results of part ISSR primers in different ploidy watermelon (2x, 3x and 4x). indicated the number of ISSR primers; "M1" indicated the molecular weight marker; Arrows indicated the different amplification patterns detected by ISSR primers in three different ploidy watermelon.
present in 3x; 9.8% (32/328) of sites were not detected. Comparisons between 3x and 4x, and between 4x and 2x were also conducted. In the 3x versus 4x comparison, 119 of 328 sites were found to be polymorphic, for a polymorphism ratio of 36.3%. Of these sites, 27.5% sites (90/328) were confirmed in 3x but not in 4x, and only 8.8% sites (29/328) were absent. In other words, a comparison of 4x with 3x showed that 27.5% sites were eliminated in 4x, indicating that the absent sites were predominant. The results obtained from a comparison of 4x with 2x indicated that 30 of 328 site (9.2%) were only detected
in 4x. However, the number of absent sites was 83. Thus the percentage of absent ratio sites (25.3%) was higher than that of present sites (9.2%). These data and additional details are presented in Table 2.
The SRAP assay generated a total of 1, 256 bands by 35 primer pairs, ranging in size from 100 bp to 2,000 bp representing 491 scored genetic loci. Partial results were presented in Figure 3. Among theses sites, 237 (48.3%) were polymorphic, and 254 were monomorphic. Pair-wise comparisons were carried out as for the ISSR analysis. A comparison between 3x and 2x indicated that only 35 sites
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Table 2. Estimate of the genomic changes occurring in three difference ploidy (diploid, autotriploid and autotetraploid) watermelons
by ISSR assay.
Total sites Identified sites Polymorphic sites and corresponding ratio (%)
2x-3x-4x 328 182 146 (44.5%)
3x-2x 328 271 25+ (7.6%) 57 (17.4%)
32- (9.8%)
3x-4x 328 209 90+(27.5%) 119 (36.3%)
29- (8.8%)
4x-2x 328 215 30+(9.2%) 113 (34.5%)
83- (25.3%)
"+" indicated the sites were present; "-" indicated the sites were absent.
(7.1%) were present, and 50 sites (10.2%) were specifically absent in 3x. A comparison between 3x with 4x indicated that 193 of 491 sites (39.3%) were polymorphic, of which 29.1% sites were present and only 10.2% sites were absent in 3x. The assay yielded similar results for a comparison between 4x and 2x. Thus, compared to 2x, only 9.2% sites (45/491) were present in 4x, a much small percentage than
the 30.5% (150/491) of absent sites (Table 3).
Evaluation of DNA methylation levels and patterns at CCGG sites by MSAP assay
In MS AP assays, seven EcoRI and eight HpaII/ MspI primer pairs were employed. Among 56 primer combinations, 23 pairs were selected for further study
based on their ability to produce clear and completely reproducible bands in independent amplification reactions with different batches of DNA isolations. Approximately 1,200 bands were amplified in each of the three different ploidy watermelons, representing 647, 655 and 581 distinguishable genetic loci in 2x, 3x and 4x, respectively. On average, 60 bands (equal to 30 sites,) per primer pair were observed (Figure 4). Among all of these sites detected by MSAP method, the total methylation polymorphism
ratios in 2x, 3x and 4x were 28.0%, 22.9% and 27.4%,
respectively (Table 4). In 2x, 121 of 181 methylation sites were fully methylated, corresponding to a fully methylated ratio of 18.7% (121/647), while the hemi-methylated ratio was only 9.3% (60/647). In 3x, the fully methylated and
Figure 3. Amplification results of part SRAP primers in different ploidy watermelon (2x, 3x and 4x). Arrows indicated the different amplification patterns detected by SRAP primers in three different ploidy watermelon.
WANG et al. ― Genetic and epigenetic changes in watermelon 395
Table 3. Estimate of the genomic changes occurring in three difference ploidy (diploid, autotriploid and autotetraploid) watermelons
by SRAP assay.
Total sites
Identified sites
Polymorphic sites and corresponding ratio (%)
2x-3x-4x
491
254
237 (48.3%)
3x-2x
491
406
35+ (7.1%)
85 (17.3%)
50- (10.2%)
3x-4x
491
298
143+ (29.1%)
193 (39.3%)
50- (10.2%)
4x-2x
491
296
45+ (9.2%)
195 (39.7%)
150- (30.5%)
"+" indicated the sites were present; "-" indicated the sites were absent.
Table 4. DNA methylation levels in different ploidy (diploid, autotriploid and autotetraploid) watermelons, based on MSAP analysis using 23 primer pairs.
Methylated CCGG sites
Watermelon Total sites
Fully methylated sites Hemi-methylated sites Total
Diploid
647
121 (18.7%)
60 (9.3%)
181 (28.0%)
Autotriploid
655
80 (12.2%)
70 (10.7%)
150 (22.9%)
Autotetraploid
581
82 (14.1%)
77 (13.3%)
159 (27.4%)
ej2-(h/m)kt ejs-(h/m)a2 ejt-(h/m)w v(h/m)b:
Figure 4. Different DNA methylation patterns detected in MSAP assay. "H2, H3 and H4", respectively, indicated that total DNA of diploid (2x), autotriploid (3x) and autotetraploid (4x) was degested by EcoRI- HpaII; "M2, M3 and M4", respectively, indicated that total DNA of diploid (2x), autotriploid (3x) and autotetraploid (4x) was degested by EcoRI- Mspl; "Ma" indicated the molecular weight marker (50-bp ladder); Arrows indicated the different DNA methylation patterns in Table 5 and Table 6.
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hemi-methylated ratios were 12.2% (80/655) and 10.7%
(70/655), respectively. Similar results were observed in 4x, where the frequency of fully methylated sites was 14.1%, and the hemi-methylated ratio was 13.3% (Table 3 ). However, it should be noticed that several other states of DNA methylation―including unmethylated CCGG, full methylation of the external cytosine, and hemi-methylation of the internal cytosine―could not be unequivocally distinguished by the MSAP method. In some cases then, the methylation percentages will likely be underestimated (Cervera et al., 2002; Dong et al., 2006). This fact notwithstanding, MSAP's evaluation of two major methylation states at CCGG sites, i.e., full-methylation of the internal cytosine and hemi-methylation of internal cytosine, is still reliable and efficient (Knox and
Ellis, 2001; Baurens et al., 2003; Guo et al., 2007).
All bands detected in three samples were scored as a binary character for absence (0) or presence (1). If a comparison between 3x or 4x with 2x was conducted, a total of four types of band patterns, including fifteen subtypes, was observed (Table 5). Type A showed methylation patterns that were unchanged in 3x or 4x
versus 2x. About half of the total patterns detected belonged to this type (3x vs. 2x: 56.5%; 4x vs 2x: 43.9%). Type B showed changes in patterns associated with the hypermethylation of certain sites in 3x or 4x versus 2x. A comparison of 3x with 2x, showed that 19.5% sites in 3x hypermethylated. However, a comparison between 4x and 2x indicated that the frequency of hypermethylation had increased to 33.9% in 4x. Type C represented samples in which demethylation events occurred in 3x or 4x versus 2x. A comparison of 3x with 2x, showed that 22.1% sites exhibited a decrease in methylation levels. A similar low methylation level (20.5%) was obtained in a comparison of 4x with 2x. Another type detected in the present study was type D, which corresponded to patterns unresolved by the MSAP method. Type D samples were not considered in subsequent assays. A similar analysis was conducted for 2x or 4x with 3x, and the detailed results are presented in Table 6. Based on the polymorphic band patterns, 35 fragments which may represent DNA methylation sites were successfully cloned and sequenced. Of these, six sequences indicated homology to known transposable elements by Blast analysis (data not shown).
Table 5. Patterns of DNA methylation and comparison between autotriploid or autotetraploid watermelon with diploid watermelon.
"="indicated the patterns of DNA methylation at CCGG sites were unchanged; " t " indicated the patterns of DNA methylation at CCGG sites were changed due to hypermethlyation events in 3x or 4x versus 2x; " i " indicated the patterns of DNA methylation at CCGG sites were changed due to demethylation events in 3x or 4x versus 2x; " " indicated the alternations of DNA methylation patterns were unidentified by MSAP method.
WANG et al. — Genetic and epigenetic changes in watermelon 397
Table 6. Patterns of DNA methylation and comparison between diploid or autotetraploid watermelon with autotriploid watermelon.
"="indicated the patterns of DNA methylation at CCGG sites were unchanged; " t " indicated the patterns of DNA methylation at CCGG sites were changed due to hypermethlyation events in 2x or 4x versus 3x; " i " indicated the patterns of DNA methylation at CCGG sites were changed due to demethylation events in 2x or 4x versus 3x; " " indicated the alternations of DNA methylation patterns were unidentified by MSAP method.
DISCUSSION
As a major driver of plant genome evolution, polyploidization, has been studied by numerous research programs, with allopolyploidization, in particular, the focus of a number of studies. Considerable evidence indicates that allopolyploidization events can result in rapid genetic changes (Song et al., 1995; Han et al., 2003;
Madlung et al., 2005; Petit et al., 2007). One conceivable
explanation for these changes is the "genomic shock" hypothesis proposed by McClintock (1984). In this hypothesis, when allopolyploidization events occur, two or more divergent, but related, genomes are brought together in a single cell nucleus. The cytoplasmic-nuclear equilibrium established in the progenitor is destroyed and different genomic interactions take place. Reestablishing the cytoplasmic-nuclear balance and stabilizing multiple genomes in a single nucleus may necessitate certain adjustments for allopolyploid plants. However, previous investigations indicated that genetic alterations were not prevalent in autopolyploid plants. In addition, the function and the expression patterns of duplicated genes were also
less affected (Albertin et al., 2005; Wang et al., 2006;
Stupar et al., 2007). Interestingly, the results of our present ISSR and SRAP analyses of a watermelon autoploidy series demonstrated some inconsistencies with respect to the literature. As in previous reports, the present data indicated that no obvious genomic structure changes had occurred in the autotriploids (Table 2, Table 3). However, comparisons of the autotetraploid with diploid or autotriploid yielded the opposite results, indicating that the genomic structure in the autotetraploid had dramatically changed (Table 2, Table 3). Approximate 40% of total detected sites showed these changes; although some sites were specifically present, more sites were specifically eliminated in the autotetraploid. As a result, the genomic changes that occurred in 4x watermelon resembled those in most allopolyploid plants, where site elimination events are prominent. These data essentially indicated that, although autotriploid and autotetraploid watermelons were both polyploids, the characteristic genomic changes were different. Taken together with the exceptions presented by allopolyploid cotton (Liu et al., 2001), sugarcane (Jannoo
et al., 2007), and the review of Paun et al. (2007), the
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results presented here allow us to boldly predict that the ability to respond to "polyploidization"―that is, "genomic shock"―differs from plant to plant and may be closely associated with the polyploidy level. A similar opinion is expressed by Soltis and Soltis (1999). Accordingly, different plants may possibly possess their own critical ploidy value. If this is the case, at a polyploidy level below the critical plant-specific ploidy value, polyploid plants need not adjust their genomic structure on a large scale. Once the polyploidy level crosses the critical ploidy value, rapid genetic changes are required to stabilize the genomes and reconcile nuclear-cytoplasmic compatibility. One of the major genetic alterations is rapid DNA sequence elimination, which allows polyploid plants to reduce some redundant sequences and possibly reestablish stable cytoplasmic-nuclear interactions. Based on this hypothesis, allopolyploidization might give rise to more extensive "genomic shock" for most plants than would autopolyploidization. Consequently, the critical ploidy value is low. Once the allopolyploidization events occur, the genetic changes are necessary. The apparent exceptions of allopolyploid cotton, sugarcane and others, may be explained by arguing that the tolerance of the divergent genomes to nuclear or cytoplasmic-nuclear interactions in these plant species is sufficient to allow them to cope with "genomic shock." Under these conditions, rapid genomic structural alterations or other extensive genetic changes are apparently unnecessary, implying that the critical ploidy value, if it exists, may be higher in these plants. However, the question of whether the concept of a critical ploidy value can truly be applied to the whole plant kingdom awaits further investigations into the character of the genomic alterations that accompany polyploidization. Based on our hypothesis, during autopolyploidization in watermelon, triploidy may present the critical ploidy value. Accordingly, the ploidy levels above this value, such as autotetraploidy, provoke rapid genomic changes while ploidy levels less than or equal to this value, including autotriploidy, are not accompanied by dramatic genomic changes.
Unlike the results of genomic structure analysis, the estimates of DNA methylation levels and patterns s ugges ted that the frequency of DNA methylation sites was similar between diploid and autotetraploid, while autotriploid exhibited a clear trend toward lower methylation levels, implying that the level of DNA methylation at CCGG sites was non-linearly related to ploidy level in watermelon. Further analysis of DNA methylation patterns indicated that about half of the observed sites underwent alterations in DNA methylation patterns during autopolyploidization, suggesting that the changes related to DNA methylation modification were more extensive and visible than the alterations of genomic structure. Of particular interest, no extensive alterations in genomic level occurred in autotriploid versus diploid watermelon, but the changes in DNA methylation patterns were obvious. DNA methylation, commonly occurring at the C5 position of cytosine residues
predominantly in symmetric CG and CNG sequences (Finnegan et al., 1998) or in many cases observed in non-symmetric CNN sites (Steward et al., 2002) (where N is A, T or C), is one of the key epigenetic regulation mechanisms in plants and is closely associated with gene silencing, transposon activation, and genomic imprinting (Wassenegger, 2000). This may imply that priority of genetic changes, epigenetic regulation (i.e., alterations of DNA methylation levels and patterns) occurs in response to the "genomic shock" resulting from allopolyploidization or autopolyploidization. In other words, below a certain ploidy level, polyploid plants need not extensively change their genomic structure, and can rely instead on changes in epigenetic regulation to maintain the genomic stability. However, if the ploidy level exceeds the tolerance limits of the plant, both rapid genomic changes and epigenetic regulation may be necessary. It suggests that epigenetic modification is an early event in plant evolution and is significant for the stability of genomic structure and control of gene expression.
The autotriploid is still an interesting subject of study, despite the fact that no extensive genomic structural changes occurred relative to the diploid (Table 2, Table 3). For example, the autotriploid showed lower DNA methylation levels compared to the diploid and the autotetraploid (Table 4). Moreover, demethylation events in the autotriploid were predominant (Table 5, Table 6). As we know, low DNA methylation levels and demethylation in plants are generally associated with the expression of genes; in contrast, the repression of gene expressions is always associated with high DNA methylation status (Dieguez et al., 1997; Cao and Jacobsen, 2002; Wada et al., 2004). Given this, the low DNA methylation levels and the trend of demethylation of numerous sites in autotriploid watermelon may suggest up-regulation of many genes. This phenomenon may be explained by the "odd-ploidy effect" first observed by Guo et al. (1996) (Auger et al., 2005; Stupar et al., 2007). It may also be associated with the formation of several excellent traits, such as rapid growth, a large vegetative organ and seedlessness in the autotriploid watermelon, as well as in other triploid plants. However, numerous investigations have been focused on allopolyploidization and even-ploidy plants. Little is known about the odd-ploidy plants, specifically, triploids, which often hold excellent traits in some crops (Hamada, 1963; Kagan-Zur et al., 1990; Bhojwani and Razdan, 1996; Sreekumari et al., 1999; Thomas et al., 2000). Therefore, more investigations focused on odd-ploidy plants are required to further elucidate the molecular mechanism of odd-ploidy effects, especially the triploidy effect.
Acknowledgments. We are grateful to Dr. Dingliang Jiao, Tianjin Vegetable Research Institute, Tianjin, China, for kindly providing three different ploid watermelons and a greenhouse to seed all materials. We also thank Dr. Feng Zhang, University of Minnesota and Dr. Ai Li, Nankai University for discussion and critical reading of the manuscript. This work was supported by a grant
WANG et al. — Genetic and epigenetic changes in watermelon
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from the Doctoral Program of Higher Education of China for Young Teachers (No.20070055091) and two grants from the National Key Basic Research Program "973"
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不同倍性西瓜(Citrullus lanatus)基因組結構和DNA甲基化基
於標記技術的分析
王春國1 李慧2 薛振毅1 陳成彬1 古瑜3 孫德嶺3 宋文芹1

1中國天津南開大學生命科學學院
2中國天津農學院
3中國天津蔬菜研究所

多倍化是植物基因組進化和物種形成的主要驅動力，在植物界中廣泛存在。然而，多數研究都聚
焦在植物的異源多倍化過程。有關植物同源多倍化過程中的遺傳及表觀遺傳變化報導很少。為此，本研
究以西瓜的同源多倍體系列（2x 、3x 、4x)為材料，利用ISSR 、SRAP和MSAP方法探究西瓜同源多
倍化過程中的基因組結構變化及全基因組內CCGG位點處的DNA甲基化水準與模式變化特徵。結果表
明，儘管與二倍體相比，在三倍體西瓜內未檢測到明顯的基因組結構變化，而四倍體西瓜的基因組結構
變化十分顯著，並且變化以DNA序列消減為主。有趣地是，二倍體與四倍體西瓜相比DNA甲基化水
準卻未發生大規模的調整，而三倍體西瓜顯示出較為明顯的低DNA甲基化水準趨勢，表明西瓜同源多
倍化過程中其基因組DNA甲基化水準變化與其倍性不成線性關係。進一步;分析證明，在檢測到的遺傳
位點中，約有一半的位點DNA甲基化模式在不同倍性的西瓜中發生了調整。三倍體西瓜依然顯示出明
顯地去甲基化趨勢。綜上結果證明，西瓜同源多倍化過程中發生的遺傳和表觀遺傳改變與已報導的一些
植物的同源多倍化有所不同，特別是三倍體西瓜，其表現出獨特的基因組結構及DNA甲基化狀態變化
特徵，而這可能與植物的奇倍性效應及三倍體西瓜優良性狀的形成密切相關。
關鍵詞：同源多倍化；異源多倍化；DNA甲基化；MSAP;西瓜。