Journal of Heredity Advance Access originally published online on February 2, 2008
Journal of Heredity 2008 99(3):304-315; doi:10.1093/jhered/esm122
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ISSR and Isozyme Characterization of Androgenetic Dihaploids Reveals Tetrasomic Inheritance in Tetraploid Somatic Hybrids between Solanum melongena and Solanum aethiopicum Group Gilo
From the CRA-ORL, Research Unit for Vegetable Crops, Via Paullese 28, I-26836 Montanaso Lombardo (LO), Italy (Toppino and Rotino); the CRA-ORT, Research Center for Vegetable Crops, PO Box 48 I-84098 Pontecagnano (SA), Italy (Mennella and D'Alessandro); the CRA-GPG, Genomic Research Center Via S. Protaso 302, I-29017 Fiorenzuola d'Arda (PC), Italy (Rizza); and the Université Paris Sud, Ecologie, Systématique, Evolution, UMR8079, CNRS, AgroParisTech, Bât. 360, F-91405 Orsay, France (Sihachakr)
Address correspondence to Giuseppe L. Rotino at the address above, or e-mail: giuseppeleonardo.rotino{at}entecra.it.
Gene exchanges between Solanum melongena and its allied relative Solanum aethiopicum are a crucial prerequisite for introgression of useful traits from the allied species into the cultivated eggplant. In order to evaluate the extent of genetic recombination between the 2 species, biochemical and molecular markers were employed. A dihaploid population obtained through anther culture of the corresponding tetraploid somatic hybrids was genetically analyzed. The extent of disomic/tetrasomic inheritance and segregation ratios of 3 isozyme systems and intersimple sequence repeat (ISSR) markers were evaluated. The dihaploids, being derived from microspores, allowed for simple, complete, and accurate analyses. The segregation of 280 ISSR markers (110 aethiopicum-specific, 104 melongena-specific, and 66 monomorphic) were evaluated in 71 dihaploids. According to the genetic constitution (simplex/duplex/triplex), almost 64% of the fragments revealed the tetrasomic and/or disomic inheritance. With regard to the assigned species-specific fragments, 68% and 4% were unambiguously the result of tetrasomic and disomic inheritance, respectively. Twenty-four of the 66 monomorphic ISSRs were inherited according to random chromatid segregation. The phenotypes of glucose-6-phosphate dehydrogenase (G-6-PDH), 6-phosphogluconate dehydrogenase (6-PGDH), and shikimate dehydrogenase (SKDH) were studied in 70 dihaploids and inferences were made about the allelic state of their 5 loci. The isozyme markers segregated in the dihaploids in a distorted manner, their segregations did not fit in with any of the expected segregation ratios. However, tetrasomic inheritance might be suggested for G-6-PDH 2 and SKDH 1 loci. Our results demonstrated that gene exchanges occurred readily in the somatic hybrids between S. melongena and S. aethiopicum gr. Gilo.
Genetic improvement of many economically important crops has frequently been based on the introgression of useful agronomic traits from their wild and cultivated relatives (Tanksley and McCouch 1997). Genetic recombination between chromosomes of the involved species is crucial for the introgression of useful genes from the allied to the cultivated species (Bernacchi et al. 1998). The presence of multivalents at meiosis is a necessary but not sufficient condition for genetic recombination because crossing-over does not always occur after chromosome pairing (Sybenga 1999). Analyses of species-specific markers have been employed to demonstrate the hybridity of sexual or somatic interspecific hybrids and also to reveal the occurrence of recombination between chromosomes of the parental species in their progenies (Williams et al. 1993; McGrath et al. 1994; Fulton et al. 1997; Garriga-Calderé et al. 1997; Bletsos et al. 1998; Barone et al. 2002).
Eggplant (Solanum melongena L.) is susceptible to numerous diseases and pests, particularly bacterial and fungal wilts, nematodes and insects (Sihachakr et al. 1994). Resistance to most pathogens is also found in the cultivated eggplant, but its levels are partial and often insufficient for effective utilization in breeding programs (Messiaen 1989; Rotino et al. 1997). Therefore, interspecific hybridization of eggplant with its wild and cultivated relatives has been considered a valid option for transfer and introgression of useful agronomic traits, such as disease resistance (Sihachakr et al. 1994). Among the relatives of eggplant which have so far been used in breeding programs, Solanum aethiopicum has become attractive for resistance against bacterial and fungal wilts, caused by Ralstonia solanacearum (Hebert 1985) and Fusarium oxysporum f. sp. melongenae (Mochizuki and Yamakawa 1979; Cappelli et al. 1995), respectively. Therefore, both sexual and somatic hybrids of eggplant with S. aethiopicum have been developed. The resultant tetraploid somatic hybrids were, however, distinctly higher in fertility than the corresponding diploid sexual hybrids (Ano 1991; Daunay et al. 1993; Collonnier et al. 2001). Likewise, chromosome doubling of the sexual hybrids of eggplant with S. aethiopicum gr. Aculeatum and gr. Gilo has resulted in the development of synthetic amphidiploids with significantly improved fertility, due to better chromosome balance at meiosis. Those amphidiploids formed multivalents at meiosis, and genetic analysis of their selfed progenies revealed the segregation of isozyme and molecular markers (Isshiki et al. 2000; Isshiki and Taura 2003).
In previous studies, tetraploid somatic hybrids between eggplant and S. aethiopicum gr. Gilo (Collonnier et al. 2001) have been brought back to the diploid status by anther culture, resulting in the development of a dihaploid population (Rizza et al. 2002). The androgenetic origin of the regenerated plants has been checked by means of ploidy determination and confirmed by using isozyme and intersimple sequence repeat (ISSR) markers (Rizza et al. 2002). The dihaploids obtained, together with hybrids derived from crossings between tetraploid eggplants and somatic hybrids with S. aethiopicum gr. Gilo, were employed in series of backcrosses to a diploid recurrent eggplant to introgress desirable traits from the close relative into the cultivated eggplant (Rotino et al. 2005). However, detailed information about genomic recombination is needed. Particularly, sufficient homeologous recombination is a prerequisite for the effective introgression of useful genes from S. aethiopicum gr. Gilo into cultivated eggplant. Therefore, the aim of this work was to monitor the interaction between the genomes of S. melongena and S. aethiopicum gr. Gilo by evaluating the extent of 1) disomic or tetrasomic inheritance for the various allelic compositions of the considered loci (i.e., simplex, duplex, and triplex), 2) random chromosome segregation, and 3) genetic recombination through random chromatid segregation at a number of tetrasomic loci. For this purpose, isozyme and ISSR markers were used to analyze the dihaploid population in order to detect interspecific recombination between the genomes of S. melongena and S. aethiopicum. Genetic analyses are easier and simpler for the dihaploids than for the sexual F2 population derived from allotetraploids as the dihaploids have the genetic constitution of the male gametes generated at meiosis.
 |
Materials and Methods |
|---|
 Top Materials and Methods ResultsDiscussionConclusionsFundingReferences |
|---|
Plant Materials
The androgenetic population employed was obtained by anther culture (Rizza et al. 2002) of 3 clones of the tetraploid somatic hybrids between S. melongena cv. Dourga and S. aethiopicum gr. Gilo (Collonnier et al. 2001): D-Sa 2/1, D-Sa 2/2, and D-Sa 2/3. Ploidy level of the androgenetic plants was checked by flow cytometry and chloroplasts counting in the guard cells (Rizza et al. 2002).
ISSR Characterization
Seventy-one dihaploids derived from D-Sa 2/1 (22), D-Sa 2/2 (38), and D-Sa 2/3 (11) were analyzed.
DNA was extracted from young leaves, using the Gene EluteTM Plant Genomic DNA Miniprep Kit, following the manufacturer's instruction (Sigma, St. Louis, MO). ISSR analysis was carried out using 33 primers of the UBC Primer set #9 (University of British Columbia) identified as polymorphic between the fusion parents (Rizza et al. 2002). Each reaction was performed in 20 µl containing 20 ng DNA, 1x buffer (Invitrogen, Carlsbad, CA), 2 mM MgCl2, 0.1 mM deoxynucleotide triphosphates (Applied Biosystems, Forster City, CA), and 0.8U Platinum Taq Polymerase (Invitrogen). Amplification reaction was run according to the following profile: 1 cycle of 3 min at 94 °C; 45 cycles of 30 s at 94 °C, 45 s at 55 °C, 2 min at 72 °C; and 1 cycle of 5 min at 72 °C.
Polymerase chain reaction products were separated by electrophoresis in a 1.2% (w/v) agarose gel containing 0.25 µg/m ethidium bromide at 55 V/cm for 4 h in TAE buffer. The amplified DNA fragments were observed under UV light and stored using the Gel Doc 1000 (Bio-Rad, Hercules, CA) apparatus.
Isozyme Analysis
Seventy dihaploids derived from D-Sa 2/1 (21), D-Sa 2/2 (38), and D-Sa 2/3 (11) were analyzed.
The extractions, separations, and staining activity of the 3 foliar isozymes glucose-6-phosphate dehydrogenase (G-6-PDH, E.C. 1.1.1.49 [EC] ), 6-phosphogluconate dehydrogenase (6-PGDH, E.C. 1.1.1.44 [EC] ), and shikimate dehydrogenase (SKDH, E.C. 1.1.1.2 [EC] 5) were performed according to Rizza et al. (2002).
Data Analyses
ISSR
Only distinct, unambiguous, well-resolved ISSR bands were scored as present (A) or absent (a). The amplified bands in the fusion parents were classified as "polymorphic markers" when found only in 1 of the 2 parents (either aet-specific or mel-specific) or as "monomorphic markers" when detected in both of them.
In a tetraploid hybrid, taking into account that ISSRs are dominant markers, quadruplex (AAAA) and triplex (AAAa) loci would behave always as monomorphic and the simplex (Aaaa) loci as polymorphic markers. In the case of duplex loci (AAaa), 2 alternatives are possible depending on the genotype of the parents: when the two parents are AA and aa genotypes (homozygous homologous chromosomes), the corresponding marker is polymorphic; if both parents are Aa (heterozygous homologous chromosomes), the corresponding marker is monomorphic.
The segregation ratios of both monomorphic and species-specific markers (expressed as the presence/absence of the corresponding fragments) were analyzed in the androgenic population, which has the genetic constitution of the microspores generated from meiosis in the somatic hybrids. According to the allelic composition of the tetraploid somatic hybrid, the expected phenotypic segregation ratios of gametes were hypothesized for each locus, considering both tetrasomic and disomic inheritance (Table 1). Based on all the segregation ratios that can theoretically be obtained in the androgenic population, the genotype of the loci and their type of heredity was determined. The informativeness of the markers was established, and the observed segregation ratio was fitted to at least one of the various hypotheses.
|
Segregation ratios of monomorphic markers corresponding to triplex loci are often not informative enough to distinguish between tetrasomic and disomic inheritance in a sexual F2 progeny because a very large selfed population is necessary to evidence the rare recessive genotypes originated from tetrasomic inheritance of the locus. However, the use of segregating androgenetic plants allows tetrasomic inheritance due to random chromatid segregation to be identified. The monomorphic bands corresponding to duplex AaAa loci of the somatic hybrid (both the parents having Aa genotype) show a segregation ratio of 3A:1a in the case of disomic inheritance and of 5A:1a or 11A:3a in the case of tetrasomic inheritance, when random chromosome or random chromatid segregation occurs, respectively.
Because each polymorphic species-specific segregating marker can be present in the somatic hybrid as a simplex or a duplex locus, the expected gametic segregation ratios in the androgenetic progeny vary depending on the occurrence of disomic or tetrasomic inheritance. In the latter case, the ratios also vary depending on the presence or absence of crossing-over between the locus and the centromere.
Under disomic inheritance, a gametic segregation of 1A:1a would always be associated with the Aaaa simplex locus because of pairing only between homologous chromosomes, whereas in the case of a duplex locus AAaa, all Aa gametes are expected from the somatic hybrid. Under tetrasomic inheritance, which involves pairing between homeologus chromosomes, the expected segregation ratios are 5A:1a or 11A:3a for a duplex and 1A:1a or 13A:15a for a simplex locus, when random chromosome or random chromatid segregation takes place, respectively.
The goodness of fit of each hypothetical segregation ratio was tested by chi-square analyses for all the expected values of gamete segregation in a tetraploid somatic hybrid and the range of ratios which would support the hypothesized segregation ratios (depending on the genotype of the somatic hybrid and the type of heredity) are given (Tables 2 and 3). The observed segregation ratios after amplification with ISSR primers were then compared with the hypothesized segregation ratios, and type of inheritance was inferred from the result of the goodness of fit test.
|
|
Isozymes
In the anodal section of the gel, the fastest migrating zone of activity in each enzyme system was arbitrarily designated as the first locus (1) and the next zone as the second locus (2). The fastest migrating band at each locus was arbitrarily designated as the first allele (a) the next band was the second allele (b). The absence of bands in some activity zones was associated with the presence of "null" alleles (which are considered variants that lack enzymatic activity) at that locus.
Segregation ratios in electrophoretic phenotypes of the isozyme loci observed in the parents, somatic hybrids and, overall, in the dihaploid progeny were used to distinguish between disomic and tetrasomic inheritance. Chi-square analyses were used to test hypothesized genetic ratios.
|
Results |
|---|
|
TopMaterials and MethodsResultsDiscussionConclusionsFundingReferences |
|---|
ISSR Analysis
The ISSR analysis performed on the parental species detected mel-specific, aet-specific and common bands. From amplification with 33 reliable ISSR primers of the fusion parents and the 3 somatic hybrids, a total of 280 markers were evidenced, ranging from 3 to 12 bands per primer with an average of 8. Among these markers, 110 (39.3%) were aet-specific, 104 (37.1%) were mel-specific and 66 were monomorphic. The amplification patterns were very different between the fusion parents (Figure 1a). In most of cases, the somatic hybrids showed all the fragments owned by the parental species, demonstrating their hybrid nature. The hybrids exhibited identical amplification pattern for 84.3% of the fragments. Moreover, loss of parental fragments (Figure 1b) or appearance of bands absent in the fusion parents was observed in at least one somatic hybrid. Of the 44 (15.7%) cases of divergent fragment amplifications scored, 17 (6.1%) regarded simultaneously the 3 somatic hybrids.
|
The dihaploid population showed a notable degree of band segregation (Figure 1c). In the 71 androgenetic individuals, 10 primers amplified a total of 79 fragments which were all polymorphic, whereas the remaining 23 primers yielded about 70% polymorphic fragments (Table 4). The percentage of different markers detected in a single dihaploid ranged from 70% to 86%, with an average of 78%; these values were equally present in the dihaploids, irrespective of the somatic hybrid from which they derived.
|
The extent of disomic or tetrasomic inheritance was determined by following the segregation of each ISSR fragment in the androgenetic dihaploids. Chi-square goodness of fit tests (P > 0.05) performed to determine, among all the possible segregation ratios, which hypotheses significantly fitted to each observed frequency are shown in Tables 5 and 6 for polymorphic and in Table 7 for monomorphic fragments.
|
2 analysis, (P>0.05)
|
|
Among the 214 polymorphic fragments evaluated, 99 (48 aet-specific and 51 mel-specific) did not significantly fit any tested segregation ratio (Tables 5 and 6). For most of these fragments, the observed segregation did not clearly determine whether disomic or tetrasomic inheritance had occurred. This is the case, for example, of 32 (29.1%) aet-specific and 28 (26.9%) mel-specific markers, whose segregations were between the 2 ratios of 1A:0a and 5A:1a, corresponding to a duplex genotype in the somatic hybrid (Tables 2 and 5). For 13 fragments (7 aet-specific and 6 mel-specific), the observed segregation could neither be used to determine their type of heredity (disomic or tetrasomic) nor if they belonged to a duplex or simplex genotype. The remaining 26 (12.1%) fragments showed an aberrant distribution in the dihaploid population with segregation ratios lower than any expected ones. All these ISSR markers were not considered further.
A total of 115 species-specific fragments were clearly assigned to 1 or 2 segregation ratios and their allelic constitution and type of inheritance inferred. Solanum aethiopicum gr. Gilo and S. melongena contributed to these classes of loci with 62 aet-specific and 53 mel-specific ISSRs (Tables 5 and 6). Both aet-specific and mel-specific ISSR fragments were mainly duplex markers (72.6% and 69.8%, respectively) and tetrasomically inherited. Only 3 of 110 (2.7%) aet-specific and 2 of 104 (1.9%) mel-specific ISSRs were detected in all the dihaploids, showing no segregation at all. These ISSRs (2.3% of total markers) undoubtedly displayed disomic inheritance (Table 5).
Univocal tetrasomic inheritance was clearly identified in 78 loci, whereas in 32 ISSRs (17 aet-specific and 15 mel-specific, fitting 1A:1a and 1A:1a/13A:15a), the disomic type of heredity may be predicted as well (Table 5 and 6).
Taking into account the tetrasomically inherited ISSRs, random chromatid or chromosome segregation were clearly distinguished when the detected value statistically fitted one single expected segregation ratio (Table 5). This is the case of 23 ISSRs which fit a 5A:1a ratio in agreement with random chromosome inheritance and of 10 ISSRs displaying random chromatid inheritance with a ratio of 11A:3a or 13A:15a. The majority of cases (45 ISSRs), however, showed a segregation which fits both the 5A:1a and 11A:3a ratios, thus precluding the possibility to establish whether random chromatid or random chromosome was involved in their segregation.
Taking into consideration the 66 monomorphic ISSRs between the parental lines, 31 markers were present in the 71 dihaploids (1A:0a ratio), whereas the other 35 displayed a certain degree of segregation (data not shown). Among the segregating ISSRs, 24 (36.4%) cases of tetrasomic inheritance according to random chromatid segregations (27A:1a ratio) were unambiguously detected. This was clear evidence of crossing-over between nonsister chromatids at the heterozygous locus of homeologous chromosomes. Segregation in the remaining 11 ISSRs did not allow their allelic constitution and type of inheritance to be determined and were considered as unassigned or aberrant segregating loci (Tables 3 and 7).
Isozymes Analysis
The segregation ratios of the studied isozymes in the dihaploids were markedly distorted and, consequently, they did not significantly fit in with any of the expected segregation ratios.
G-6-PDH
Zymograms of eggplant and S. aethiopicum fusion parents both showed 2 activity zones. Somatic hybrids exhibited the parental activity bands with no intermediate mobility ones, whereas a variable number of activity bands was evidenced in the dihaploid plants (Figure 2). Moreover, the zymograms of the somatic hybrids suggested that this enzyme was active as a monomer and controlled by 2 independent loci as reported in many plants (Kephart 1990). In fact, for monomeric enzymes, codominant inheritance leads to the presence of all 4 parental bands in the somatic hybrids, which then segregate in the dihaploid progeny.
|
The zymogram analysis of dihaploids indicated that eggplant was heterozygous or homozygous at G-6-PDH 1 locus (–b or bb) and heterozygous at G-6-PDH 2 locus (a–), whereas S. aethiopicum was heterozygous at both the G-6-PDH loci (1ab and 2ab). Therefore, the somatic hybrids exhibited the allelic composition –bab or bbab for G-6-PDH 1 and a–ab for G-6-PDH 2 (Table 8).
|
The phenotypes at G-6-PDH 1 locus detected in the dihaploids were 49 a, 15 ab, and 6 b. Considering the hypothesis of –bab allelic composition of the somatic hybrids at this locus, a disomic or tetrasomic random chromosome inheritance might be suggested because of the absence of the null (––) alleles. Nevertheless, the random chromatid type of tetrasomic segregation would also not be ruled out in the hypothesis of somatic hybrids with bbab allelic composition (Table 8).
For G-6-PDH 2 locus, taking into account both the a–ab allelic configuration in the somatic hybrid and the presence of null (––) phenotypes observed in the segregating dihaploids, tetrasomic inheritance with random chromatid segregation is the only possible type of inheritance that may be suggested (Table 8).
6-PGDH
Solanum melongena showed only 2 fast migrating bands in the 6-PGDH zymogram, whereas S. aethiopicum exhibited an unique anodal band at the 6-PGDH 1 locus and 2 at 6-PGDH 2 locus (Figures 2 and 3). Somatic hybrids showed the parental activity bands confirming the monomeric pattern of the enzyme. The allelic configuration of 6-PGDH 1 locus, inferred from the electrophoretic phenotypes of dihaploids, fusion parents, and somatic hybrids did not lead to discriminate between disomic or tetrasomic inheritance (Table 8). At the 6-PGDH 2 locus, somatic hybrids showed the allelic configuration ––ab (Figure 2; Table 8). The observed ab phenotype in the dihaploid population ruled out the possibility of disomic inheritance, but the distorted segregation of this locus was poorly fitting in with random chromosome and random chromatid segregation (Table 8).
|
SKDH
As regards the SKDH 1 locus, the isozyme patterns of eggplant and S. aethiopicum showed 2 fast and 2 slow migrating bands, respectively. Somatic hybrids showed all parental bands (monomeric enzymes), and the phenotypes scored in the dihaploid progeny confirmed the involvement of a single encoding gene (Figure 2). Similarly to G-6PDH 2 and 6-PGDH 2, the segregation ratio observed in the dihaploids ruled out the possibility of disomic inheritance but, at the same time, did not statistically fit in with either random chromosome or random chromatid inheritance.
|
Discussion |
|---|
|
TopMaterials and MethodsResultsDiscussionConclusionsFundingReferences |
|---|
Molecular phylogenetic studies in Solanum based on chloroplast DNA and mitochondrial DNA (Sakata and Lester 1997; Isshiki et al. 2003) as well as genomic DNA analysis (Furini and Wunder 2004; Rotino GL, Cavallanti F, Toppino L, unpublished data) demonstrated a close relationship between S. melongena and S. aethiopicum gr. Gilo. These indications are also in agreement with 1) the sexual compatibility between eggplant and this species (Ano et al. 1991; Behera and Singh 2002), 2) the improved fertility of the tetraploid synthetic and somatic hybrids confirmed by the cytological evidences of multivalent formation at meiosis (Isshiki and Taura 2003), and 3) the wide variation in phenotypic, biological, and physiological features expressed by the androgenetic dihaploids derived from their somatic hybrids (Rizza et al. 2002; Rotino et al. 2005). However, those previous results did not clarify whether the interaction between melongena and aethiopicum genomes was limited to parental chromosomal segregation or expanded to intergenomic (i.e., between homeologous chromosomes) recombination during meiosis as well. To elucidate this aspect, investigations were extended to study an androgenetic dihaploid population by using structural dominant genetic molecular markers (ISSR) and functional codominant biochemical markers (isozymes). The availability of microspore-derived population has enabled the genetic constitution of the male gametes originated from the meiosis to be directly monitored.
ISSR
ISSR primers (Zietkiewicz et al. 1994) were an efficient tool for the genetic analysis of the somatic hybrids S. melongena (+) S. aethiopicum gr. Gilo and their androgenetic dihaploid progeny. ISSR markers have been employed in many species for fingerprinting and phylogenetic studies, gene tagging, and mapping (Trojanowska and Bolibok 2004). ISSRs appear to be quite evenly dispersed in the plant genome, although they were supposed to display a higher frequency in specific regions (simple sequence repeat hot spot). Inheritance of ISSRs follows Mendelian rules as demonstrated in chickpea (Ratnaparkhe et al. 1998).
The loss of parental molecular markers and the appearance of novel ones are common phenomena detected in newly synthetic or natural allotetraploids. In fact, rapid genomic and chromosomal rearrangements and changes in gene expression associated with allopolyploidy have been observed in several species belonging to different botanical families (e.g., Brassica, Triticum, and Solanum) in both sexual and somatic hybrids (Barone et al. 2002; Chen and Ni 2006; Adams 2007).
The majority of mel-specific and aet-specific ISSR markers, whose segregations significantly (P > 0.05) fitted in with the expected ratios, were duplex loci and followed mainly a tetrasomic (68%) type of inheritance. Only a small number (5 ISSRs corresponding to 4% of the assigned loci) segregated univocally according to a disomic type of inheritance. The remaining 28% (32 ISSRs) equally fitted in with both tetrasomic (random chromosome) and disomic inheritance (Table 6). Among the 78 tetrasomically inherited polymorphic markers, both random chromatid (9 duplex and 1 simplex ISSR) and random chromosome (23 duplex ISSRs) segregation were clearly evidenced. A part of the remaining 45 ISSR (58% of the total polymorphic tetrasomic loci) fitting both 5A:1a and 11A:3a was likely subjected to recombination events. Indeed, all these cases fall between the 2 limits of tetrasomic inheritance in a tetraploid genotype represented by random chromosome and random chromatid segregation. In the first case, random assortment of the 4 homologous chromosomes occurs, whereas in the second case random assortment of the 8 sister chromatids takes place. In the latter case, with a certain frequency, there is recombination between the centromere and a given locus (double reduction) with an increased production of homozygous gametes. Most likely, a larger dihaploid population could enable a higher number of markers to be statistically assigned to 1 of the 2 classes. Similar considerations were reported for the somatic hybrid between potato and Solanum commersonii (Barone et al. 2002).
Because the genetic composition of dihaploid plants is identical to the gametes of the tetraploid parent, the triplex loci (classified as monomorphic) were highly informative for determining tetrasomic inheritance. In fact, the gametic segregation of 27A:1a and 5A:1a was clearly shown in 45% of the monomorphic ISSRs. Moreover, 24 (80%) of the 30 tetrasomically inherited ISSRs significantly displayed a type of segregation (27A:1a, random chromatid), involving crossing-over of the homeologous chromosomes. This kind of information is hardly obtainable in the sexual F2 progeny because a very large population and many efforts are needed to show the corresponding segregation ratio of 738A:1a (Barone et al. 2002).
Isozymes
Segregation of the genes coding for G-6-PDH, 6-PGDH, and SKDH isozymes occurred in the dihaploid progeny. However, goodness of fit tests gave chi-square values poorly fitting with all the tested segregation models of disomic and tetrasomic inheritance. Nevertheless, tetrasomic inheritance might be predicted for G-6-PDH 2, 6-PGDH 2, and SKDH 1 loci because the allelic composition of parents and somatic hybrids and, specially, the detection of certain phenotypes (e.g., the null phenotype for G-6-PDH 2 locus) in the dihaploids ruled out the hypothesis of disomic inheritance.
Tetrasomic inheritance has been documented using isozyme in a number of plant species including Medicago sativa L. (Quiros 1982), Solanum tuberosum L. (Martinez-Zapater and Oliver 1984; Quiros and McHale 1985), Tolmiea menziesii L. (Soltis DE and Soltis PS 1988), Heuchera micrantha Douglas (Soltis DE and Soltis PS 1989), Vaccinium corymbosum L. (Krebs and Hancock 1989), and Solanum spp. (Isshiki 1996). Likewise, disomic inheritance in allopolyploids has been reported in tetraploid species, such as Tragopogon mirus Ownbey and Tragopogon miscellus Ownbey (Roose and Gottlieb 1976), and Prunus cerasus L. (Beaver and Iezzoni 1993), in hexaploid Triticum aestivum L. for triplicated loci (Hart 1983) and octaploid Fragaria x ananassa for quadruplicated loci (Arulsekar et al. 1981).
For G-6-PDH 1 and 6-PGDH 1 loci, none of the segregation models hypothesized could clearly be ruled out because of the strongly distorted segregation observed.
At G-6PDH 2 locus, a random chromatid-type tetrasomic inheritance appeared the most likely segregation that could explain the presence of 6 null (––) phenotypes in the dihaploids. The null phenotype may be originated because of the lack of a correct interaction between the subunities coded by the 2 species. This may be caused by either subcellular compartmentalization of isozymes or the inability of polypeptides coded by different genetic alleles to form an active enzyme. This phenomenon was most frequently observed in isozymes revealed by nonspecific stains, such as reduced form of nicotinamide adenine dinucleotide–dehydrogenase and reduced nicotinamide adenine dinucleotide phosphate–dehydrogenase, acid phosphatase, and esterase (Wendel and Weeden 1989).
At 6-PGDH 2 locus, no dihaploids exhibited the expected b and "null" electrophoretic phenotypes, probably because the codominant inheritance for allozymes was not always detected (Cullis 1979; Weeden and Robinson 1986).
In order to explain the behavior of the alleles at SKDH 1 locus in the dihaploids (Figure 2), it is useful to consider that many enzymes routinely display multiple-banded phenotypes that segregate as units; these are frequently reported for various plant isozymes, particularly SKDH (Tanksley 1984; Weeden 1984; Harry 1986; Jarret and Litz 1986; Wendel and Weeden 1989). It is not known whether these multiple-banded allozymes arise in vivo through posttranslational modification or in vitro by chemical or conformational changes. Due to their frequency, the theory of "gene duplications" appears unable to explain their origin (Wendel and Weeden 1989). The tetrasomic gametic ratios 1:4:1 or 3:8:3 (aa:ab:bb) should be expected for SKDH 1 locus in the dihaploid progeny of an allotetraploid plant, but the overproduction of gametes showing ab genotype determined the distortion from the expected random chromosome or random chromatid segregation ratio.
The distorted segregation observed at these 5 loci could be explained by a higher relative abundance of some gametes carrying specific alleles (genotypes a– or aa and ab). This occurrence was also reported by Isshiki et al. (2000) for the segregation of isozymes in selfed progenies of a synthetic amphidiploid between Solanum integrifolium (=S. aethiopicum gr. Aculeatum) and S. melongena. The occurrence of gametic selection could explain the abundance of those alleles and their distorted segregation. Similar evidences were showed by monitoring the segregation and recombination of isozymes in rice, among androgenetic and F2 plants produced from 6 F1 hybrids and in doubled haploid lines obtained from the japonica x indica cross (Guiderdoni et al. 1989; Guiderdoni 1991).
|
Conclusions |
|---|
|
TopMaterials and MethodsResultsDiscussionConclusionsFundingReferences |
|---|
Segregation of triplex, duplex, and simplex ISSR loci clearly demonstrated the occurrence of crossing-over between homeologous chromosomes. Distorted segregation of allozymes was also clearly detected in the dihaploids and in some cases a tetrasomic-type inheritance might have occurred. These findings clearly show that interspecific recombination took place in the somatic hybrid between S. melongena and S. aethiopicum gr. Gilo. The demonstration of the interspecific gene exchanges offers the possibility of widely exploiting the genetic variability of S. aethiopicum for the improvement of the cultivated eggplant by introgression of alien genes. Backcrossed lines resistant to F. oxysporum f. sp. melongenae obtained from dihaploids are currently employed to develop marker assisted selection for Fusarium wilt resistance and also for a detailed molecular, biochemical, and nutritional characterization to uncover other possible useful genes and features introgressed from S. aethiopicum into eggplant.
|
Funding |
|---|
|
TopMaterials and MethodsResultsDiscussionConclusionsFundingReferences |
|---|
European Commission International Cooperation-Developing Countries (INCO-DC) INCO-DC project "Development of eggplant lines resistant to fungal and bacterial wilts," European Union (contract no. ICA4-CT-2001-10064); Italian Ministry of Food, Agricultural and Forestal Policy with funds released by Comitato Interministeriale per la Programmazione Economica (CIPE, resolution 17/2003) in the framework of the project "Progetto di Ricerca per Potenziare la Competitività di Orticole in Aree Meridionali (PROM)".
|
Acknowledgments |
|---|
We thank Mrs M. G. Tacconi for technical assistance. In addition, we are grateful to A. Falavigna, C. Scotti, and anonymous reviewers for comments and/or suggestions to the earlier version of the manuscript.
|
Footnotes |
|---|
Corresponding Editor: John Stommel
Received June 28, 2007
Accepted December 6, 2007
|
References |
|---|
|
TopMaterials and MethodsResultsDiscussionConclusionsFundingReferences |
|---|
-
Adams KL. Evolution of duplicate genes in polyploid and hybrid plants. J Hered (2007) 98(2):136–141.
Ano G, Hebert Y, Prior P, Messiaen CM. A new source of resistance to bacterial wilt of eggplants obtained from a cross: Solanum aethiopicum L. x Solanum melongena L. Agronomie (1991) 11:555–560.[CrossRef][Web of Science]
Arulsekar S, Bringhurst RS, Voth V. Inheritance of PGI and LAP isozymes in octoploid cultivated strawberries. J Am Soc Hortic Sci. (1981) 106:679–683.
Barone A, Li J, Sebastiano A, Cardi T, Frusciante L. Evidence for tetrasomic inheritance in a tetraploid Solanum commersonii (+) S. tuberosum somatic hybrid through the use of molecular markers. Theor Appl Genet. (2002) 104:539–546.[CrossRef][Web of Science][Medline]
Beaver JA, Iezzoni AF. Allozyme inheritance in tetraploid sour cherry (Prunus cerasus L.). J Am Soc Hortic Sci. (1993) 118:873–877.
Behera TK, Singh N. Inter-specific crosses between eggplant (Solanum melongena L.) with related Solanum species. Sci Hortic (2002) 95:165–172.[CrossRef]
Bernacchi D, Beck-Bunn T, Emmatty D, Inai S. Advanced backcross QTL analysis of tomato. II. Evaluation of near-isogenic lines carrying single-donor introgressions for desireable wild QTL alleles derived from Lycopersicon hirsutum and L. pimpinellifolium. Theor Appl Genet. (1998) 97:170–180.[CrossRef][Web of Science]
Bletsos FA, Roupakias DG, Tsaktsina ML, Scaltsojannes AB, Thanassoulopoulos CC. Interspecific hybrids between three eggplant (S. melongena L.) cultivars and two wild species (S. torvum Sn & S. sisymbriifolium). Plant Breed (1998) 117:159–164.[CrossRef]
Cappelli C, Stravato VM, Rotino GL, Buonaurio R. Sources of resistance among Solanum spp. to an Italian isolate of Fusarium oxysporum f. sp. melongenae. EUCARPIA, IXth Meeting on Genetics and Breeding on Capsicum and Eggplant; 1995 Aug 21–25; Budapest (Hungary) (1995) 221–224.
Chen ZJ, Ni Z. Mechanism of genomic rearrangements and gene expression change in plant polyploids. Bioessay (2006) 28:240–252.[CrossRef][Web of Science][Medline]
Collonnier C, Mulya K, Fock I, Mariska I, Servaes A, Vedel F, Siljak-Yakovlev S, Souvannavong V, Ducreux G, Sihachakr D. Source of resistance against Ralstonia solanacearum in fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L. Plant Sci. (2001) 160:301–313.[Medline]
Cullis CA. Segregation of the isozymes of flax genotrophs. Biochem Genet. (1979) 17:391–401.[CrossRef][Web of Science][Medline]
Daunay MC, Chaput MH, Sihachakr D, Allot M, Vedel F, Ducreux G. Production and characterization of fertile somatic hybrids of eggplant (Solanum melongena L.) with Solanum aethiopicum L. Theor Appl Genet. (1993) 85:841–850.[Web of Science]
Fulton TM, Nelson JC, Tanksley SD. Introgression and DNA analysis of Lycopersicon peruvianum, a wild relative of the cultivated tomato, into Lycopersicon esculentum, followed through three successive backcross generations. Theor Appl Genet. (1997) 95:895–902.[CrossRef][Web of Science]
Furini A, Wunder J. Analysis of eggplant (Solanum melongena)-related germplasm: morphological and AFLP data contribute to phylogenetic interpretations and germplasm utilization. Theor Appl Genet. (2004) 108:107–208.
Garriga-Calderé F, Huigen DJ, Filotico F, Jacobsen E, Ramanna MS. Identification of alien chromosomes through GISH and RFLP analysis and the potential for the establishment of potato lines with monosomic additions of tomato chromosomes. Genome (1997) 40:666–673.[Medline]
Guiderdoni E. Gametic selection in anther culture of rice (Oryza sativa L.). Theor Appl Genet. (1991) 81(3):406–412.[Web of Science]
Guiderdoni E, Glaszmann JC, Courtois B. Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.). Euphytica (1989) 42(1–2):45–53.[CrossRef][Web of Science]
Harry DE. Inheritance and linkage of isozyme variants in incense-cedar. J Hered (1986) 77:261–266.
Hart GE. Genetics and evolution of multilocus isozymes in hexaploid wheat. Isozymes Curr Top Biol Med Res. (1983) 10:365–380.[Web of Science][Medline]
Hebert Y. Résistance comparée de 9 espèces du genre Solanum au flétrissement bactérien (Pseudomonas solanacearum) et au nématode Meloidogyne incognita. Intérêt pour l'amélioration de l'aubergine (Solanum melongena L.) en zone tropicale humide. Agronomie (1985) 5(1):27–32.[CrossRef][Web of Science]
Isshiki S. Genetic variation of isozymes and its application to breeding in eggplant and its wild relatives. Bull Fac Agric Saga Univ. (1996) 80:1–46.
Isshiki S, Okubo H, Fujieda K. Segregation of isozymes in selfed progenies of a synthetic amphidiploid between Solanum integrifolium and S. melongena. Euphytica (2000) 112:9–14.[CrossRef][Web of Science]
Isshiki S, Suzuki S, Yamashita K. RFLP analysis of mitochondrial DNA in eggplant and related Solanum species. Genet Resour Crop Evol. (2003) 50:133–137.[CrossRef]
Isshiki S, Taura T. Fertility restoration of hybrids between Solanum melongena L. and S. aethiopicum L. Gilo Group by chromosome doubling and cytoplasmic effect on pollen fertility. Euphytica (2003) 134:195–201.[CrossRef][Web of Science]
Jarret RL, Litz R-E. Enzyme polymorphism in Musa acuminate Colla. J Hered (1986) 77:183–188.
Kephart SR. Starch gel electrophoresis of plant isozymes: a comparative analysis of techniques. Am J Bot (1990) 77:693–712.[CrossRef][Web of Science]
Krebs S, Hancock J. Tetrasomic inheritance of isozyme markers in the highbush blueberry, Vaccinium corymbosum. L. Heredity (1989) 63:11–18.[CrossRef]
Martinez-Zapater JM, Olivier J-L. Genetic analysis of isozyme loci in tetraploid potatoes (Solanum tuberosum L.). Genetics (1984) 108:669–679.
McGrath JM, Wielgus S-M, Uchytill TF, Kim-Lee H, Haberlach GT, Williams CE, Helgeson JP. Recombination of Solanum brevidens chromosomes in the second backcross generation from a somatic hybrid with S. tuberosum. Theor Appl Genet. (1994) 88:917–924.[Web of Science]
Messiaen CM. L'aubergine. Le potager tropical, cultures speciales. (1989) Vol. 2. Paris: Collection Techniques vivantes, Agence de Coopération Culturelle et Technique, Presses Univer. 399p.
Mochizuki H, Yamakawa K. Resistance of selected eggplant cultivars and related wild species to bacterial wilt (Pseudomonas solanacearum). Bull Veg Ornamental Crops Res Stn Ano Mie (Japan) (1979) 6:1–10.
Quiros CF. Tetrasomic inheritance for multiple alleles in alfalfa. Genetics (1982) 101:117–127.
Quiros CF, McHale N. Genetic analysis of isozyme variation in diploid and tetraploid potatoes. Genetics (1985) 111:131–145.
Ratnaparkhe MB, Santra DK, Tullu A, Muehlbeur FJ. Inheritance of inter-simple sequence repeat polymorphisms and linkage with a Fusarium wilt resistance gene in chickpea. Theor Appl Genet. (1998) 96:348–353.[CrossRef][Web of Science]
Rizza F, Mennella G, Collonnier C, Sihachakr D, Kashyap V, Rajam MV, Presterà M, Rotino GL. Androgenetic dihaploids from somatic hybrids between Solanum melongena and S. aethiopicum group gilo as a source of resistance to Fusarium oxysporum f. sp. melongenae. Plant Cell Rep (2002) 20:1022–1032.[CrossRef][Web of Science]
Roose ML, Gottlieb LD. Genetic and biochemical consequences of polyploidy in Tragopogon. Evolution (1976) 30:818–830.[CrossRef][Web of Science]
Rotino GL, Perri E, Acciarri N, Sunseri F, Arpaia S. Development of eggplant varietal resistance to insects and diseases via plant breeding. Adv Hort Sci. (1997) 11:193–201.
Rotino GL, Sihachakr D, Rizza F, Vale’ G, Tacconi M-G, Alberti P, Mennella G, Sabatini E, Toppino L, D'Alessandro A, et al. Current status in production and utilization of dihaploids from somatic hybrids between eggplant (Solanum melongena L.) and its wild relatives. Acta Physiol Plant (2005) 27(4B):723–733.[CrossRef][Web of Science]
Sakata Y, Lester RN. Chloroplast DNA diversity in brinjal eggplant (Solanum melongena L.) and related species. Euphytica (1997) 97:295–301.[CrossRef][Web of Science]
Sihachakr D, Daunay MC, Serraf I, Chaput MH, Mussio I, Haicour R, Rossignol L, Ducreux G. Somatic hybridization of eggplant (Solanum melongena L.) with its close and wild relatives. In: Biotechnology in agriculture and forestry, somatic hybridization in crop improvement—Bajaj YPS, ed. (1994) Vol. I. Berlin: Springer-Verlag. 255–278.
Soltis DE, Soltis PS. Electrophoretic evidence for tetrasomic segregation in Tolmiea menziesii (Saxifragaceae). Heredity (1988) 60:375–382.[CrossRef][Web of Science]
Soltis DE, Soltis PS. Tetrasomic inheritance in Heuchera micrantha (Saxifragaceae). J Hered (1989) 80:123–126.
Sybenga J. What makes homologous chromosomes find each other in meiosis? A review and an hypothesis. Chromosoma (1999) 108:209–219.[CrossRef][Web of Science][Medline]
Tanksley SD. Linkage relationships and chromosomal locations of enzyme-coding genes in pepper, Capsicum annuum. Chromosoma (1984) 89:352–360.[CrossRef][Web of Science]
Tanksley SD, McCouch SR. Seed banks and molecular maps: unlocking genetic potential from the wild. Science (1997) 277:1063–1066.
Trojanowska MR, Bolibok H. Characteristic and comparison of three classes of microsatellite-based markers and their application in plants. Cell Mol Biol Lett. (2004) 9:221–238.[Web of Science][Medline]
Weeden NF. Distinguishing among white seeded bean cultivars by means of allozyme genotypes. Euphytica (1984) 33:199–208.[CrossRef][Web of Science]
Weeden NF, Robinson RW. Allozyme segregation ratios in the interspecific cross Cucurbita maxima x C. ecuadorensis suggest that hybrid breakdown is not caused by minor alterations in chromosome structure. Genetics (1986) 114:593–609.
Wendel JF, Weeden NF. Visualization and interpretation of plant isozymes. In: Isozymes in plant biology: advances in plant science series—Soltis DE, Soltis PS, eds. (1989) Vol. 4. Portland (OR): Dioscorides Press. 34–38.
Williams CE, Wielgus SM, Haberlach GT, Guenther C, Kim-Lee H, Helgeson JP. RFLP analysis of chromosomal segregation in progeny from an interspecific hexaploid somatic hybrid between Solanum brevidens and Solanum tuberosum. Genetics (1993) 135:1167–1173.[Abstract]
Zietkiewicz E, Rafalski A, Labuda D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification. Genomics (1994) 20:176–183.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||