Is single bulb producing garlic Allium sativum or Allium ampeloprasum?

A set of control specimens of Allium sativum and Allium ampeloprasum from different geographic origins were analyzed using karyotype analysis and molecular markers (RAPD, M13 minisatellites and SNPs) to assess the gene-pool source of the single bulb garlic cultivar. Karyotype analysis showed that A. sativum and single bulb garlic were diploids, wild A. ampeloprasum was 2× (Sardinia and Basilicata) and 4× (Tremiti Island) while Great headed garlic landrace was 6×. Molecular diversity analyses confirmed the differentiation between A. sativum (14 specimens) and A. ampeloprasum species-complex (18 specimens). The “single bulb” market garlic cultivar, introduced into the European market from China, resulted to be nested in A. sativum genetic cluster. The cultivated forms of A. ampeloprasum (Leek and Great headed garlic) proved to be more differentiated than the wild one. Among the wild Mediterranean plant materials, the specimens of A. ampeloprasum from Sardinia demonstrated to be more differentiated than the wild genotypes from the west (Basilicata and Tremiti Island) based on either nucleotide variations of ITS1 or RAPD and M13 diversity. No SNP was observed, comparing a consensus sequence of 148 bp at ITS1 gene among three different specimens of A. sativum, while 18 mutated positions were detected in seven different specimens of A. ampeloprasum. The maximum number of variable positions were detected in the Sardinian wild A. ampeloprasum, followed by Leek and the wild A. ampeloprasum specimens from Basilicata and Tremiti (South Italy). Overall, it is clearly evident that the single bulb Chinese garlic cultivar is A. sativum. M13 marker demonstrated to be the most suitable in distinguishing the two different gene pools performing very well in terms of resolution, cost and ease of use.     

Application of genomic in situ hybridization for phylogenetic study between Mangifera indica L. and eight wild species of Mangifera

The phylogenetic relationship between mango (Mangifera indica L.) and eight wild species of Mangifera were analyzed by comparing signal intensity of genomic in situ hybridization (GISH) on somatic metaphase chromosomes of M. indica, using labeled DNA of eight wild Mangifera species. The eight wild species were divided into four groups based on intensity and number of hybridization signals on chromosomes of M. indica in GISH analyses. The probe of Mangifera sylvatica Roxb. gave the highest intensities on the chromosome of M. indica, indicating a close relationship between M. indica and M. sylvatica. For the other species, classification of GISH was comparable to that of the phylogenetic analysis using AFLP markers, as previously reported ( Eiadthong et al., 2000). This suggested a possibility that GISH analysis can be effectively used in the classification of Mangifera species.     

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.     

Conversion of chromosome-specific RAPDs into SCAR-based anchor markers for onion linkage maps and its application to genetic analyses in other Allium species

Integration of previously developed Allium cepa linkage maps requires the availability of anchor markers for each of the eight chromosomes of shallot (A. cepa L. common group Aggregatum). To this end, eight RAPD markers originating from our previous research were converted into SCAR markers via cloning and sequencing of RAPD amplicons and designing of 24-mer oligonucleotide primers. Of the eight pairs of SCAR primers, seven resulted in the amplification of single bands of the original RAPDs, and the remaining primer set amplified an additional band. The results of Southern hybridization using RAPD amplicons from genomic DNA of Japanese bunching onion (Allium fistulosum L.)—shallot monosomic addition lines indicated that five SCAR markers were single shallot chromosome-specific markers and were not detected in genomic DNA of A. fistulosum. The eight SCAR primer pairs were applied to other Allium species and exhibited three types of amplification profiles, namely RAPD amplicons observed only in shallot, in shallot and Allium vavilovii, and in several Allium species. A mapping study using 65 F₂ plants generated by the selfing of one interspecific cross A. cepa × Allium roylei individual integrated the SCAR marker SAOE17₅₀₀ into chromosome 5 as expected. The results of the present study show that the eight SCAR primer sets specific to shallot can facilitate the mapping in A. cepa and can also serve as anchor points between maps of different Allium species.     

Analysis of the taxonomic subdivision within the genus Helleborus by nuclear DNA content and genome-wide DNA markers

Helleborus is a genus of herbaceous perennials belonging to the family Ranunculaceae. Within this genus six sections with a total of 22 species are found. The largest section Helleborastrum contains 16 species for which genetic relationships are still unclear. This study represents the first genetic analysis in the genus Helleborus, including the two newly described species H. liguricus and H. abruzzicus based on multilocus amplified fragment length polymorphism (AFLP) markers with a genome-wide distribution in combination with nuclear DNA content data. Chromosome analyses of roots tips revealed a number of 2n = 32 for the selected species, which was congruent with previous observations. The nuclear DNA content of Helleborus was estimated by flow cytometry applying propidium iodide staining and varied between 18 and 33 pg/2C, depending on the species. For AFLP analyses, 19 out of the 22 Helleborus species were studied using 10 AFLP primer combinations, resulting in a total of 1109 polymorphic bands among all species including the outgroup. The genetic distances between species varied between 0.034 and 0.330. Based on genetic distances a phenogram using the Neighbor-joining cluster method with bootstrap analysis was calculated. The results support the previously suggested division of the genus into six sections and thereby approve AFLP data to be applicable for phenetic analyses. Moreover, this genetic information is significant for the development of future Helleborus breeding strategies.     

Molecular analysis of introgression lines from Cucumis hystrix Chakr. to C. sativus L

Two stable introgression lines (ILs), IL56 and IL73 with fourteen chromosomes, derived from a cross between the wild relative Cucumis hystrix Chakr. (2n = 24) and the cultivated cucumber “beijingjietou” (Cucumis sativus L., 2n = 14) were characterized using SSR and AFLP markers. Twenty-three SSR primers were used to detect the introgression from C. hystrix to C. sativus, and one locus at 210 bp was revealed and assigned to introgressive fragment of C. hystrix genome in IL56. Substantial genomic changes in these two lines were detected by AFLP analysis with thirty different primer combinations. C. hystrix-specific bands, novel bands for the two parents and deleted bands in C. sativus were observed. One hundred and forty-seven polymorphic bands were observed, but only four and five introgressed bands were found in IL56 and IL73, respectively. Although representing only a small proportion of the C. hystrix genome, these two introgression lines with downy mildew and fusarium wilt resistance will be valuable to resistant genetics and breeding.     

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.     

Using bulked AFLP analysis to assess genetic diversity in Echinacea species

Echinacea is an allogamous genus, thus its cultivars or populations are genetically heterogeneous. Using amplified fragment length polymorphism (AFLP) to estimate the genetic diversity of Echinacea is generally limited by the large number of individual plants and the higher cost that need to be processed. In the present study, effectiveness of several sizes of DNA bulking (10, 15, 20, 25 and 30 individuals) with 20, 36 and 55 primer pairs was compared using AFLP in determining the genetic diversity of Echinacea species. The results indicated that the use of bulked DNA-based AFLP analysis by using the selected eight primer pairs was capable of detecting genetic diversity between the tested Echinacea species, provided that the potential presence of low frequency variants was ignored and a possible bias in the estimates of genetic similarity was accepted. The assessments showed that a bulk of 15 individuals could detect sufficient AFLP variations at most genomic sites. Additionally, 20 primer pairs could generate sufficient polymorphic fragments to achieve high resolving power of AFLP for the tested Echinacea species.     

Interspecific relationships of Lycoris (Amaryllidaceae) inferred from inter-simple sequence repeat data

Inter-simple sequence repeat (ISSR) markers were used to evaluate interspecific relationships of Lycoris. Twenty-four samples were included in the study representing a total of 20 among species and varieties. Thirteen primers produced 228 discernible DNA fragments, 205, or 89.91%, of which were polymorphic, indicating a high level of interspecific genetic variation in Lycoris. Our UPGMA cluster analysis recognized four major groups of species, which were consistent with morphological and karyotype observations. The first group included species with telocentric and metacentric chromosomes, while the second consisted of species with a haploid genome of 11 subtelocentric chromosomes. The third group contained species with a mixture of subtelocentric, telocentric and metacentric chromosomes. The last group included the Japanese and Korean species. Lycoris anhuiensis was clustered within accessions of Lycoris longituba, suggesting that it could be recognized as a variety of L. longituba. Our ISSR data also suggested that L. straminea may be a hybrid between Lycoris chinensis and Lycoris radiata var. pumila, and that L. caldwellii, an allotriploid, may be of a hybrid origin of L. chinensis and L. sprengeri.     

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.     

RAPD and ITS-based variability revealed in Atriplex species introduced to semi-arid zones of Morocco

Variability related to RAPD markers and sequences of nrDNA ITS region has been studied in seven species of the genus Atriplex (A. amnicola, A. canescens, A. halimus from Morocco (MAR) and from USA, A. lentiformis, A. nummularia, A. semibaccata and A. undulate). As a whole, the results show a high variability among the species. The biggest diversity was obtained by RAPD data, followed by ITSs. According to RAPD markers, two major groups can be distinguished, one formed by A. semibaccata and A. undulata, the least similar to the rest of species. A. lentiformis was closer to A. halimus. Phylogenetic analysis confirmed the divergence of A. semibaccata from all species and the closeness of A. lentiformis to A. halimus. The intraspecies variability was also high, as 39 individual RAPD haplotypes were obtained by analyzing 40 plants. Genetic diversity was bigger among species (60.23%) than among individuals (39.77%). The amplification of ITS region leads to three well-defined clades. The heterogeneous and larger clade includes all A. amnicola individuals and some individuals from A. nummularia, A. lentiformis, A. halimus USA, A. halilmus MAR and A. undulata. Both RAPDs and ITS analyses revealed with some exceptions, that all individuals from one species grouped together. RAPDS turn out to be more appropriate than ITS to differentiate Atriplex species. The two markers gave rise to the same species relationships, but to a different structure of the Atriplex genetic diversity. AMOVA analysis estimated interspecies differences for 11.20% of the total ITS variation.     

Development of the female gametophyte in the sterile ecotype of the bolting Allium sativum L.

Manifestations of female sterility were investigated in a sterile bolting ecotype of Allium sativum (garlic). Morphological studies revealed interference during the formation of integuments around the nucellus and the lack of a micropylar channel and anatomical studies revealed disorders in female gametophyte formation. As the consequence of developmental abnormalities ovule abortion was observed. Finally, significant differences were revealed between 2D protein profiles of sterile ovules of A. sativum and fertile ovules of Allium tuberosum. Further possibilities of application of modern proteomic tools to identify proteins responsible for developmental changes in sterile ovules of A. sativum are discussed.     

Comparison of the use of morphological,protein and DNA markers in the genetic characterization of Iranian wild Prunus species

In this study, the genetic diversity of four Iranian wild Prunus species including Prunus eleagnifolia, Prunus hauskenchtii, Prunus scoparia and Prunus lycioides were investigated using morphological, protein and DNA markers. DNA markers included nuclear and chloroplast SSRs and self-incompatibility (S) allele amplification. At the morphological level, leaf width showed significant differences between the four wild Prunus species. Concerning fruit and kernel characters, their values are relatively similar indicating the high degree of homoplasy described in Prunus. Nuclear SSR markers have been the most abundant markers with a higher polymorphism in comparison with morphological, protein and chloroplast SSR markers. Results also indicated the high variability present in the S locus. On the other hand, the correlation between the clustering based on DNA markers and protein were in general low. Dendogram performed using nuclear and chloroplast SSR indicated a more diffuse clustering between the wild almond species probably due to the natural introgression of genes observed in these wild almond species. Data from the analysis of the total protein seems to be more accurate to establish taxonomy relationships in these very close wild species.     

Correlation of saponin content and Fusarium resistance in hybrids from different ploidy levels of Lilium Oriental

Lilium Oriental is of great commercial value, but large losses in production can result from its susceptibility to diseases caused by Fusarium ssp. Here we report a mutant of Lilium Oriental resistant to Fusarium, Cai-74, which was generated from crossing tetraploid (from ‘Star fighter’) and diploid (‘Cor. Amore’ × ‘Acapulco’) plants. Hybrid progeny were screened via inoculating Fusarium oxysporum into tissue cultured plantlets in a greenhouse. Although Cai-74 had a saponin content of 3.81 mg g⁻¹, which is much higher than its parents, the highly Fusarium-resistant wild species Lilium dauricum had the highest content of 4.11 mg g⁻¹ among the tested genotypes. Cai-74 had a 3A karyotype rather than 3B found in normal plants of Lilium Oriental. Taken together, our results suggest that Cai-74 bears a chromosomal variation in its structure, and that the saponin content in the lily scales correlate with the Fusarium resistance of Lilium Oriental. Cai-74 could be used as a gene resource for breeding Fusarium-resistant cultivars of Lilium Oriental.     

Genetic characterization of asparagus doubled haploids collection and wild relatives

The genus Asparagus is very large consisting of around 150 species found as herbaceous perennials, tender woody shrubs and vines. The cultivated species (Asparagus officinalis L., diploid) is a highly prized vegetable, grown in different environments ranging from cool temperate zones to deserts, Mediterranean climates and tropical areas. As a consequence, Asparagus breeders have developed different cultivars that differ for their morpho-agronomic traits, habit and ploidic status (few triploid and tetraploid cultivars are used). Several breeding methods are used for developing cultivars, among which a well developed in vitro anther culture technique produces homozygous clones useful for F₁ hybrids constitution. A fluorescent based AFLP (amplified fragment length polymorphism) technique were applied with the aim to assess genetic diversity among a collection of 173 doubled haploid (DH) androgenetic clones, five Asparagus wild species and interspecific hybrids obtained among the cultivated species and two wild relatives. The average number of AFLP fragments generated per primer set was 105, varying in size from 50 to 550 bp. A total of 1054 AFLP fragments were detected, 20% of which were polymorphic. Genetic similarity based on DNA polymorphisms, showed that a few number of AFLP primer combinations are able to distinguish the cultivated DH clones from the wild species. Indeed, from one DH clone for each anther donors and the wild species were used to construct a dendrogram using Dice's coefficient and the unweighted pair group method with the arithmetic mean (UPGMA). Genetic distances among all DH clones were calculated using the C.S. Chord distance; and a neighbour-joining (NJ) consensus tree was constructed in order to support the breeder for parental genotype choice for asparagus hybrid constitution.     

Evaluation of genetic relationships among Iranian pistachios using microsatellite markers developed from Pistacia khinjuk Stocks

To evaluate the genetic relationships among wild and cultivated Pistacia species grown in Iran and the analysis of genetic variation among Iranian pistachio genotypes, two DNA libraries enriched for dinucleotide (AG)_n and trinucleotide (ATG)_n microsatellite motifs were developed from Pistacia khinjuk genome. Following screening of clones by colony PCR technique, 44 clones were sequenced and 27 pairs of primers designed from flanking regions of the repeats. The examination of primer pairs, designed from P. khinjuk sequences, showed successful cross-species amplification within the genus Pistacia. A dendrogram constructed on the basis of the Minimum Evolution clustering algorithm revealed that Pistacia vera has closer relationships with P. khinjuk, than with Pistacia integerrima, Pistacia palaestina, Pistacia atlantica and Pistacia mutica. The dendrogram further distinguished the wild Sarakhs pistachio from the rest of P. vera genotypes suggesting that the domesticated genotypes of P. vera are evolved from P. vera var. Sarakhs and then this wild genotype likely develops to other local pistachios. Hence, it seems that the wild Sarakhs pistachio plays an important role in evolutionary trend of the edible pistachios in Iran. The results indicated that microsatellites developed in P. khinjuk are distributed in the genome of indigenous pistachio species including P. vera genotypes and therefore they will be useful in characterization of Iranian pistachio genotypes.     

Selection of arbuscular mycorrhizal fungi for citrus growth promotion and Phytophthora suppression

In order to reduce unnecessary amount of P-fertilizer and severity of Phytophthora root rot in citrus orchards, the experiment was set up. Thirteen indigenous arbuscular mycorrhiza (AM) fungi species were isolated from rhizosphere soil of citrus orchards in Thailand and were then propagated into three host plants [sorghum (Sorghum bicolor), maize (Zea mays), and leek (Allium cepa)] by trap culture. We also tested whether indigenous AMF species (13 different species) could colonize into three cultivars of citrus scions and rootstocks (Shogun: Citrus reticulata Blanco cv. Shogun; Tangerine: C. reticulata; and C-35 citrange: Citrus sinensis × Poncirus trifoliata). With root colonization rates, the results indicated that Acaulospora tuberculata and Glomus etunicatum provided the best colonization in all citrus cultivars. We selected, therefore, those AMF species to verify their influences on citrus growth and Phytophthora root rot resistance. Three cultivars of citrus scions and rootstocks, Shogun, Tangerine and C-35 citrange, were inoculated with two effective indigenous AMF species, G. etunicatum or A. tuberculata in order to determine the influences on citrus growth. The plants were investigated to determine the mycorrhizal efficiency index (MEI), AM colonization, P content, and other parameters. Co-inoculation of AMF species (G. etunicatum or A. tuberculata) with Phytophthora nicotianae was also carried out in Shogun scion/C-35 citrange rootstocks. The results of citrus growth revealed that Shogun and Tangerine inoculated with G. etunicatum produced the highest MEI. Tangerine and C-35 citrange amended with fertilizers and G. etunicatum showed the highest P content in leaves. This indicated that G. etunicatum has an influence on citrus growth and P uptake, suggesting it to be the highly effective strain. Shogun scion/C-35 citrange rootstock combinations that were inoculated by both P. nicotianae and different AM fungi (G. etunicatum or A. tuberculata) showed root injury at low level of root rot symptom. However, the part of Shogun scions grafted on rootstocks showed severe symptom of shoot die back in treatment inoculated with P. nicotianae alone, while treatment inoculated with different AM species (G. etunicatum or A. tuberculata) and P. nicotianae rendered lower shoot die back symptoms than that of Phytophthora treatment. The low level of shoot die back symptom was shown at first, then healthy young shoot was restored. Our results indicated the facts that different host plants and different AMF species produced different outcomes of growth and pathogen resistance. The application of both AM isolates, therefore, has an enormous potential to be produced the inoculum for citrus orchards.     

Development of ITS sequence based SCAR markers for discrimination of Paphiopedilum armeniacum, Paphiopedilum micranthum, Paphiopedilum delenatii and their hybrids

Paphiopedilum armeniacum, Paphiopedilum micranthum and Paphiopedilum delenatii are endangered orchid species. These three Paphiopedilum species and their hybrids are difficult to distinguish morphologically. In this study, rDNA-ITS (internal transcribed spacer) sequences were used to design species-specific SCAR (sequence characterized amplified regions) markers to distinguish P. armeniacum, P. micranthum, P. delenatii and their respective hybrids. The developed markers efficiently amplified 600 bp DNA product for P. armeniacum and its hybrids (SCAR-600armF/Pap-ITS2R), 300 bp product for P. delenatii and its hybrids (SCAR-300delF/Pap-ITS2R) and 700 bp product for P. micranthum and its hybrids (SCAR-700micF/Pap-ITS2R). The effectiveness of designed species-specific markers was also confirmed by using multiplex polymerase chain reaction amplification with a combination of developed three SCAR markers.     

Confirmation of hybrid origin in Arisaema (Araceae) using molecular markers

A population of hybrids between Arisaema triphyllum subsp. stewardsonii and A. dracontium was investigated using molecular markers to document the hybrid origin. Total genomic DNA was extracted from A. triphyllum, A. dracontium, and the hybrids, and subjected to sequence analysis of various regions of intergenic spacer and genes of chloroplast or intergenic spacer and genes of nuclear ribosome for single nucleotide polymorphisms (SNPs). Clustering was performed using the unweighted pair group method with averages (UPGMA) tested by bootstrap (BS). Hybrid origin could not be easily confirmed in some regions. However, dendrograms constructed with a combined sequence analysis of A. triphyllum 26S ribosomal RNA gene [sequence identification (SI) 467] plus A. tortuosum phytochrome C-like (phyC) gene (SI 468) were very similar to dendrograms constructed from sequences of all regions. This suggests that selecting SI 467 and SI 468 would be practical to identify hybrid origins involving two parental species. Clustering of hybrids together with the female parent in most target regions suggests that, in Arisaema, cpDNA is considered maternally inherited.