Interspecific hybridization in vanilla and molecular characterization of hybrids and selfed progenies using RAPD and AFLP markers

Vanilla, Vanilla planifolia Andrews, is native to Mexico and Central America, but is now cultivated in other parts of the tropics. Continuous clonal propagation has resulted in very little variability for crop improvement programmes in vanilla. In this study, an attempt has been made to increase the spectrum of variation by interspecific hybridization with Vanilla aphylla, an Indian species which is tolerant to Fusarium. Interspecific hybrids were successfully produced and morphological characters and molecular profiles revealed the true hybridity of the progenies. Ten seedling progenies of V. planifolia, and four interspecific hybrids obtained from crosses between V. planifolia (female) and V. aphylla (male) using a number of different loci as markers were evaluated and 319 amplified fragment length polymorphisms (AFLPs) and 83 random amplified polymorphic DNAs (RAPDs) loci were marked. The profiles indicate similarity between the parents, selfed progenies and interspecific hybrids and that all the progenies tested were variable when compared to each other, which can be exploited for crop improvement in vanilla. This is the first report in vanilla, indicating that RAPD and AFLP profiles coupled with morphological characters can be utilized to assess the variability and hybrid nature of genotypes and of successful interspecific hybridization and production of hybrids between V. planifolia and V. aphylla.     

Cross-transferable polymorphic SSR loci in Prunus species

This work reports the transferability and polymorphism of previously reported SSRs in 10 Prunus species. The availability of a large number of SSRs in the genus Prunus makes marker choice random, while preventing comparison of results in fingerprinting studies. The availability of SSR markers, polymorphic in a wide sample of Prunus species, would facilitate marker choice, while allowing the comparison of results. In this work, microsatellite markers useful for analyzing 10 different Prunus species (P. persica, P. dulcis, P. armeniaca, P. domestica, P. insititia, P. salicina, P. cerasifera, P. avium, P. cersus and P. mahaleb) were searched through screening SSRs previously reported to be conserved and/or polymorphic in more than one Prunus species. A selected group of 13 SSRs, transferable to the 10 species, was analyzed in terms of their usefulness for analyzing these species. The amplification range, polymorphism and variability detected by these loci are reported. The information provided will be useful for Prunus genetic studies as well as conservation and management of Prunus germplasm resources.     

Rose (Rosa hybrida L.) EST-derived microsatellite markers and their transferability to strawberry (Fragaria spp.)

The ‘Genome database for Rosaceae (GDR)’ provides a large collection of expressed sequence tags (ESTs) harboring simple sequence repeats (SSRs) from several Rosaceae genera, including Rosa (rose). Primer pairs flanking SSR were designed for 312 unique Rosa ESTs based on GDR database. Eight rose (Rosa hybrida L.) genotypes were tested for PCR amplification, and 287 (92%) of the primer pairs generated allele-specific PCR bands that were readily scored. From 183 (63.7%) primer pairs that evidenced polymorphic alleles among the eight rose cultivars, 20 pairs evidencing EST sequence homology to known gene functions and high levels of polymorphism were selected and utilized for DNA fingerprinting and genetic diversity assessments of 47 rose hybrids. A total of 202 polymorphic bands were scored and generated unique fingerprints for each rose hybrid. The Nei–Li genetic similarity coefficients among 1081 pair-wise comparisons of 47 cultivars exhibited a broad range of genetic variations from 0.30 (‘Grand King’ and ‘Carnival’) to 0.99 (‘First Red’ and ‘Red Champ’). UPGMA cluster analysis divided 47 hybrids into five major groups and two sub-groups. The cross-species transferability of 273 EST-SSR primer pairs was evaluated using four genotypes of the strawberry, a genus member of the Rosaceae family. PCRs on the DNA samples of strawberry were successful for 165 primer pairs; among these, 123 pairs amplified 243 polymorphic bands. As surrogates of the marker transfer, the phenetic relationship among the four strawberry genotypes was evaluated. Genetic similarity coefficients varied from 0.78 (‘Maehyang’ and ‘Janghyee’) to 0.64 (‘Janghyee’ and ‘Pragana’). The results of cluster analysis showed that the three octaploid strawberry cultivars were quite similar, whereas the diploid ‘Pragana’ was related distantly at the genomic DNA level. The EST-SSR markers developed in the present study can be efficiently utilized for genetic diversity studies in Rosaceae.     

EST-PCR markers developed for highbush blueberry are also useful for genetic fingerprinting and relationship studies in rabbiteye blueberry

The pedigrees of most rabbiteye blueberry (Vaccinium virgatum) cultivars can be traced back to four wild selections, ‘Ethel’, ‘Clara’, ‘Myers’, and ‘Black Giant’; thus, they result from a very narrow germplasm base and are highly related. Until now randomly amplified polymorphic DNA (RAPD) has been the only type of molecular marker used in rabbiteye blueberry. Here we have tested whether a type of sequence-tagged site (STS) marker which utilizes specific ∼20-mer primers from expressed sequence tags (ESTs) of highbush blueberry (V. corymbosum), called EST-PCR markers, are useful for genetic fingerprinting and relationship studies in rabbiteye blueberry. Of 44 EST-PCR primer pairs, from an assortment of genes expressed in flower buds of cold acclimated and non-acclimated plants, and shown to amplify polymorphic fragments among a collection of highbush genotypes, 40 (91%) resulted in successful amplification, and 33 of those (83%) amplified polymorphic fragments among the rabbiteye genotypes. The average number of scorable bands per primer pair was two. A dendrogram constructed from genetic similarity values, based on the EST-PCR marker data, tended to group siblings and parent/progeny together, generally agreeing with pedigree information. A group of 20 markers from five EST-PCR primer pairs distinguished all the genotypes in this study. These markers are as easy to generate and as affordable as RAPDs, but are based on actual gene sequences, and should have general utility for DNA fingerprinting, genetic diversity, and mapping studies.     

Identification of parental species of the Alstroemeria cv. ‘Jubilee’ using AFLP marker technique

Twelve Alstroemeria species, two hybrids, one cv. ‘Jubilee’, an anther-cultured plant from cultivar ‘Jubilee,’ and Bomarea salsilla and Leontochir ovallei (the latter two were chosen as outgroup) were evaluated using the AFLP marker technique in order to identify putative parental genotypes of the Alstroemeria cv. ‘Jubilee’ and of known interspecific hybrids, and to estimate their genetic relationships within the genus Alstroemeria. A total of 297 AFLP markers were scored by using the primer combination (E + ACCA/M + CTAG). In order to discriminate all Alstroemeria genotypes, cluster analysis (UPGMA) and principal coordinates analysis were performed. The Alstroemeria cv. ‘Jubilee’, of which the parents are unknown, had genetic distance (GD) 0.54 from Alstroemeria exserens, GD 0.57 from Alstroemeria garaventae, GD 0.62 from Alstroemeria gayana, and GD 0.66 from Alstroemeria hookeri cumminghiana. Thus, these four species are considered as putative parental genotypes. An interspecific hybrid (Alstroemeria aurea × Alstroemeria inodora), showed the smallest genetic distance from A. aurea (GD 0.56) and A. inodora (GD 0.45). The Alstroemeria ligtu group was distantly allocated from other Chilean species. We conclude that the AFLP marker technique appears to be a satisfactory tool for identifying the parental genotypes of interspecific hybrids in Alstroemeria.     

Development of novel chloroplast microsatellite markers for Dendrobium officinale, and cross-amplification in other Dendrobium species (Orchidaceae)

Nine pairs of polymorphic chloroplast microsatellite primers were developed for Dendrobium officinale Kimura et Migo, an endangered herb. Levels of polymorphism were tested across a total of 55 individuals from four natural populations (12–15 individuals per population). Allele numbers varied from two to four per locus, while the number of haplotypes ranged from four to six per population. Transferability of the nine polymorphic chloroplast microsatellite primers was checked on an additional set of 51 Dendrobium individuals (belonging to 17 different species). Three markers could be transferable to all the species tested, while the remaining six markers successfully cross-amplified in most species tested. Moreover, polymorphism of the nine chloroplast microsatellite primers was tested across Dendrobium moniliforme (L.) Sw. and Dendrobium loddigesii Rolfe. All of them were polymorphic in D. moniliforme, while seven of which were polymorphic in D. loddigesii. These polymorphic chloroplast microsatellite primers developed for D. officinale will be a useful tool for the study of genetic diversity, population genetic structure, evolution of D. officinale and establishment of effective conservation strategies.     

Cross-amplification of SSR markers developed from Allium sativum to other Allium species

For genetic analysis of the genus Allium, which is composed of diverse species, we acquired 50 transferable and polymorphic microsatellite markers from A. sativum and tested them for transferability in five Allium species. Among the 50 simple sequence repeat (SSR) loci, the dinucleotide motif was the most prevalent, with a ratio of 50% (25/50), and (GT)n was more frequent than (GA)n within the dinucleotide motif. The average number of amplified alleles ranged from 1.452 to 1.910 and the accessions of A. tuberosum had a maximum of 4.8 alleles per accession with the GB-AS-104 SSR marker. Whereas A. porrum belonging to the Allium section revealed 73.0% transferability, A. altaicum and A. fistulosum appertaining to different sections showed low transferability, with a ratio of 47.6% and 48.0%, respectively. The phylogenetic results for these SSR markers did not deviate from previous classifications of the genus Allium. As the rate of successful amplification of SSR markers generally correlates with genetic distance, these SSR markers are potentially useful in the analysis of genetic relationships between or within Allium species.     

A preliminary genetic linkage map of chrysanthemum (Chrysanthemum morifolium) cultivars using RAPD,ISSR and AFLP markers

A genetic linkage map of chrysanthemum (Chrysanthemum morifolium) was constructed by genotyping 142 F₁ progeny of the bi-parental cross ‘Yuhualuoying’ × ‘Aoyunhanxiao’ with a combination of RAPD, ISSR and AFLP markers in a double pseudo-testcross mapping strategy. A total of 567 polymorphic markers, including 153 RAPDs, 61 ISSRs and 353 AFLPs, were used in linkage mapping. 336 of 567 (60%) markers were grouped on the two parental maps, leaving 231 (40%) markers unlinked. In the ‘Yuhualuoying’ linkage map, 210 markers including 116 testcross and 94 intercross markers were placed in 12 major and 32 minor (8 triplets and 24 doublets) linkage groups, covering 1034 cM with an average map distance of 6.2 cM between adjacent markers. In ‘Aoyunhanxiao’ linkage map, 190 markers consisting of 113 testcross and 77 intercross markers were resolved into 9 major and 24 minor linkage groups, with genome coverage of 1095 cM and a mean inter-marker separation of 6.9 cM between adjacent markers. Six pairs of homologous linkage groups were established on the basis of 64 intercross markers shared by the two parental maps. The maps lay a foundation for further quantitative traits loci (QTL) mapping and marker-assisted breeding of chrysanthemum.     

A SCAR marker linked to the N gene for resistance to root knot nematodes (Meloidogyne spp.) in pepper (Capsicum annuum L.)

The root-knot nematodes (Meloidogyne spp.) are important nematode pests and cause serious diseases in pepper in the world. No molecular markers linked to the nematodes resistance N gene have been reported. In this paper, ‘Carolina Wonder’ (Capsicum annuum L.), a sweet pepper line resistant to root-knot nematode with N gene, ‘20080-5-29’ (C. annuum L.), an inbred line susceptible to root-knot nematode with good horticultural characteristics, and their F₂ progeny with 320 individuals were used as materials. Evaluation of resistance and susceptibility of parental lines, F₁ and F₂ progeny inoculated with root-knot nematodes (Meloidogyne incognita) were carried out. ‘Bulked segregant analysis’ method was used to search for polymorphic markers from 512 pairs of AFLP primers. Based on the assessment of resistance and susceptibility and polymorphism of the AFLP marker in F₂ population, the genetic linkage distance between the AFLP marker and the N gene was estimated. One AFLP marker E39/M41-339 was obtained and transferred to a SCAR marker amplifying a 315 bp DNA fragment linked to the N resistant allele and a 331 bp fragment linked to the N⁺ susceptible allele. The distance between the molecular marker and the nematodes resistance N gene is 6.3 cM. This research delivered a valuable tool for the marker assisted selection of nematodes resistance in pepper.     

Syrian pear (Pyrus syriaca) as a pollinator for European pear (Pyrus communis) cultivars

In Israel four European pear cultivars are grown: ‘Spadona’ is the main cultivar and ‘Coscia’, ‘Gentile’ and ‘Spadochina’ are its pollinators. However, molecular S-genotyping revealed that ‘Spadona’ is semi-compatible with its three pollinators. This explains, at least in part, the relatively low pear yield in Israel. The Syrian pear (Pyrus syriaca) grows wild in Israel and blooms intensively, overlapping the blooming of the cultivated European pears. Cross-fertilization between Syrian pear and ‘Spadona’ was shown to be efficient suggesting that Syrian pear might be a potent pollinator for ‘Spadona’. Twenty-six Syrian pear seedlings, from different sites in north-east Israel were S-genotyped identifying 11 that are fully compatible with the four European pear varieties cultivated in Israel. By this screening, 24 different S-RNases were cloned; ten of them are new, whereas the other fourteen had been identified previously. In addition, seedlings of two wild pear species were also S-genotyped. Two seedlings from Pyrus betulifolia and one from Pyrus korshinskii were found to be genetically compatible with the four European pear cultivars. From these seedlings four S-RNases were cloned, two are new, one had been cloned previously and one was identical to an S-RNase allele cloned from Syrian pear in this work.     

Polymorphisms of amplified mitochondrial DNA non-coding regions in Diospyros spp.

Universal primers were used to amplify mtDNA non-coding regions in Diospyros spp. including 6 related species and 20 genotypes of Diospyros kaki Thunb. The results showed: (1) 32 universal primers successfully amplified either introns or intergenic regions in Diospyros spp. A total of 119 bands were obtained, in which 110 were polymorphic. (2) Twenty three universal primers were used to analysis genetic diversity at the level of intra-specific, which revealed that the mitochondrial genomes had abundant variation during recombination. Chinese, Japanese PCNA genotypes were separated distinctly from each other by clustering analysis. (3) Two Chinese PCNA genotypes of Japanese persimmon, ‘Baogaitianshi’ and ‘Eshi No.1’, have unique bands to other materials, which showed they would have derived from the same female parent according to the maternal inheritance of mitochondrial genome.     

Conservation of Vanilla species, in vitro

Vanilla (Vanilla planifolia) is a crop of great commercial importance as the source of natural vanillin, a major component of flavor industry. The primary gene pool of V. planifolia is narrow and is evidently threatened due to destruction of its natural habitats making the secondary gene pool important as a source of desirable traits especially for resistance to diseases. Many species of vanilla are considered rare and endangered hence an urgent need to conserve them, arises. Effective procedures for micropropagation and in vitro conservation by slow growth in selected species of vanilla, are described. Synthetic seed technology was standardized by encapsulating 3–5 mm in vitro regenerated shoot buds and protocorms in 4% sodium alginate, which could be stored up to 10 months with 80% germination in sterile water at 22 ± 2 °C. In vitro conservation technology of Vanilla was standardized and shoot cultures could be maintained for more than 1 year without subculture, on slow growth medium, i.e. Murashige and Skoog medium supplemented with 15 g l⁻¹ each of sucrose and mannitol in sealed culture vessels at 22 ± 2 °C. These cultures were maintained in vitro for more than 7 years with yearly subculture. The conserved material could be retrieved and multiplied normally in MS medium with 1.0 mg l⁻¹ BA and 0.5 mgl ⁻¹ IBA. The in vitro conserved plants showed good growth and developed into normal plants. This synseed and in vitro conservation system can be utilized for conservation and exchange of vanilla genetic resources.     

Elimination of a new ampelovirus (GLRaV-Pr) and Grapevine rupestris stem pitting associated virus (GRSPaV) from two Vitis vinifera cultivars combining in vitro thermotherapy with shoot tip culture

A new virus species designated as Grapevine leafroll associated virus-Pr (GLRaV-Pr), which is classified in a distinct phylogenetic group of the genus Ampelovirus (Closteroviridae), was recently characterized from Greek grapevine cultivars. Elimination studies of GLRaV-Pr were carried out in two grapevine cultivars, ‘Mantilaria’ and ‘Prevezaniko’, co-infected with Grapevine rupestris stem pitting associated virus (GRSPaV, Flexiviridae). Both viruses were detected by nested RT-PCR assays. Virus elimination was achieved by combining in vitro thermotherapy with meristem (≤0.2 mm) or shoot tip culture (≤0.5 cm). The survival and regeneration rate of meristems was very low. On the other hand, high survival rates were observed in the cultured shoot tips accompanied with high elimination rates for both viruses. Data obtained in this study indicate that virus elimination depends on the genotype of grapevine. The results confirmed that sanitation is easier for species of the Closteroviridae family than for GRSPaV, whereas it seems that eradication of GLRaV-Pr and GRSPaV is feasible even with larger plant tissue parts if combined with an appropriate thermotherapy profile in vitro.     

‘Swingle’ citrumelo propagation by cuttings for citrus nursery tree production or inarching

‘Swingle’ citrumelo [Citrus paradisi MacFaden × Poncirus trifoliata (L.) Raf.] has been extensively used as a rootstock in several citrus growing regions of the World, including Southern Brazil where ‘Rangpur’ lime (Citrus limonia Osbeck) is still the predominant variety despite being affected by several important pathogens. In this case, ‘Swingle’ citrumelo is used to produce nursery trees to establish new orchards or to be inarched in adult and healthy groves in order to change the rootstock. We report herein a system to produce trees on ‘Swingle’ citrumelo more rapidly by budding onto non-rooted cuttings, as well as assessing potential to rapidly multiply ‘Swingle’ through rooting of non-budded cuttings. Therefore, two potential products are described: budded trees and rooted rootstock cuttings. ‘Valencia’ sweet orange [Citrus sinensis (L.) Osbeck] was budded at different heights on cuttings derived from eight-month old rootstocks. Grafted and additional non-budded cuttings were then treated with indole-3-butyric acid (500 mg L⁻¹) or left untreated before rooting. Three types of cuttings were evaluated: softwood, semi-hardwood and hardwood. The use of nursery trees derived from pre-budded hardwood cuttings of ‘Swingle’ citrumelo is an alternative grafting method on this cultivar. Softwood cuttings with one leaf pair were considered the most adequate material for rapid multiplication of ‘Swingle’ citrumelo by cutting. This could be particularly useful for inarching production or conventional budding after transplant of cutting-derived rootstocks.     

Identification of S-alleles in pear (Pyrus communis L.) cv. ‘Rocha’ and other European cultivars

In this work we report the cloning and identification of S-RNase alleles responsible for gametophytic self-incompatibility (GSI) of ‘Rocha’ pear and of 13 other European pear cultivars that might be used as its pollinators. Partial sequences of S-RNase alleles were amplified by PCR with specific primers hybridising in conserved regions of previously identified S-RNase alleles of Pyrus communis, cloned and sequenced and the S-genotype of eight pear cultivars was fully determined. Three cultivars (‘General Léclerc’ (SqSl), ‘Tosca’ (SbSl) and ‘Alexandrine Douillard’ (SbSk)) shared no S-alleles with ‘Rocha’ (SaSe) and shall be totally compatible with this cultivar. None of the cultivars analysed showed an identical amplification pattern to the one observed in ‘Rocha’, so the other cultivars shall be at least semi-compatible. One new allele was identified in P. communis cv. ‘Beurré d’Avril’ (designated as St). The determination of both S-RNase alleles of cvs ‘Rocha’, ‘Beurré Precoce Morettini’ (SeSk) and ‘Tosca’ and the identification of one S-RNase allele in cvs ‘Carapinheira’ (Sb), ‘Amêndoa’ (Se), ‘Pérola’ (Sk) and ‘Beurré d’Avril’ (St) are important contributions for the effort recently developed worldwide to establish groups of sexual compatibility among European pears.     

Genetic diversity and relationships within Citrus and related genera based on sequence related amplified polymorphism markers (SRAPs)

Sequence related amplified polymorphism (SRAP) markers were used to detect molecular marker polmorphisms among 86 citrus and their relatives in Aurantioidea. Twenty-one SRAP primer combinations produced a total of 376 polymorphic fragments with an average of 17.9 per primer combination and an average polymorphism information content (PIC) of 0.86. The unweighted pair group method arithmetic average (UPGMA) analysis demonstrated that the accessions had a similarity range from 0.28 to 1.00 with a mean of 0.64. The subtribe Clauseninae (tribe Clauseneae) separated from the subtribes of the tribe Citreae. The subtribe Balsamocitrinae (tribe Citreae) was the most distant from the others. In the Citrinae, ‘primitive citrus fruit trees’ and ‘near citrus fruit trees’ groups did not clearly separate from each other but all genera in these groups were distinct. On the other hand, subgenus Papeda and subgenus Citrus were not separated clearly in the dendrogram. C. maxima, C. medica and C. reticulata separated into three distinct clusters in agreement with three ‘true basic species’ thesis. Similarity-based analyses supported the theory of few ancestral species in Aurantioidea.     

Pollen biology of ornamental ginger (Hedychium spp. J. Koenig)

An improved in vitro pollen germination assay was developed to assess the viability of stored Hedychium pollen. The effect of polyethylene glycol (PEG) (10, 15, and 20%, w/v) on pollen germination and tube growth was evaluated for Hedychium longicornutum and two commercial Hedychium cultivars, ‘Orange Brush’ and ‘Filigree’. Overall, the inclusion of PEG 4000 in the medium improved both pollen germination and tube growth for the three different genotypes tested and the results varied depending on genotype. In vitro germination was used to assess the viability of Hedychium pollen stored up to two months. Pollen nucleus status was determined for four Hedychium cultivars, ‘Orange Brush’, ‘Anne Bishop’, ‘Filigree’, and ‘Daniel Weeks’. Pollens of ‘Orange Brush’, ‘Anne Bishop’, and ‘Daniel Weeks’ were found to be binucleate but ‘Filigree’ was shown to possess both binucleate and trinucleate pollens. High pollen:ovule ratio values were obtained in several Hedychium taxa. The results obtained on the nuclear pollen status and pollen:ovule ratios will further our understanding of the pollination biology and help clarify the taxonomy and phylogeny of Hedychium species.     

Phylogenetic analysis in some Diospyros spp. (Ebenaceae) and Japanese persimmon using chloroplast DNA PCR-RFLP markers

Ten universal primer pairs of chloroplast genome were used to amplify non-coding regions of chloroplast DNA (cpDNA) in 7 Diospyros L. species including 29 genotypes and approximately 20.4 kb, 12.6% of the chloroplast genome were analyzed. The amplified products were digested by seven restriction enzymes. The results showed that there were abundant polymorphisms in inter-specific cpDNA within the genus Diospyros. However, it was not observed intra-species variations in 22 tested genotypes of Diospyros kaki (Japanese persimmon), except for ‘Male strain No. 9’. Persimmon had close relationship with Diospyros lotus and Diospyros glaucifolia, but distantly with Diospyros virginiana and Diospyros rhombifolia in diagram based on principal coordinates analysis and Wagner parsimony method. The discrepancy of digesting pattern in cpDNA suggested that the genotype Jinzaoshi was distinct with the remaining Diospyros spp., which revealed that Jinzaoshi may be a new species of the genus Diospyros.     

Adventitious root formation in Grevillea (Proteaceae), an Australian native species

Grevillea (Proteaceae) is a native Australian plant genus with high commercial value as landscape ornamentals. There has been limited research on the culture and propagation of Australian native species. The effect of indole-3-butyric acid (IBA) on the rooting of G.‘Royal Mantle’ and G.‘Coastal Dawn’ in winter, spring and summer was evaluated at University of Queensland Gatton, Southern Queensland in order to determine the rooting ability of this species in different seasons. Both Grevillea cultivars showed seasonal rooting. The more difficult-to-root G.‘Coastal Dawn’ had a reduced response to IBA application than G. ‘Royal Mantle’. Stem and leaf indole-3-acetic acid (IAA) levels were not different between cultivars, therefore rooting ability between the two cultivars does not appear to be due to the differences in endogenous IAA levels.