Efficient embryo induction in cucumber ovary culture and homozygous identification of the regenetants using SSR markers

Several factors, i.e. the duration of thermal shock pretreatment at 35 °C, the concentrations of TDZ and the silver nitrate were investigated for their effects on embryo formation in a variety of cucumber (Cucumis sativus L) ovary culture. The results showed that a thermal shock for 3 days at 35 °C at the start of the culture resulted in higher frequency of embryo formation than 2 or 4 days. TDZ had a positive effect on the embryo formation. Highest embryo formation frequency (72.7%) was recorded by adding 0.04 mg/L TDZ into the induction medium. The results found that addition of AgNO₃ to induction medium had no significant effect on frequency of embryo formation but shortened embryo sprouting period and improved number of embryos formed in each ovary slice. All the experiment materials responded well to ovary culture, and there were no difference among genotypes used in this study. Among the forty regenerated plants obtained, two were identified as haploid plants (2n = x = 7), five were tetraploid plants (2n = 4x = 28), and the rest were diploid plants. Microsatellite markers (SSR) were used to analyze the homozygosity of the diploid plants, the putative chromosome-doubled haploids. Of the 33 diploid plants, 17 (51.5%) were identified as double haploids. Based on the above results, we have established a useful protocol for production of cucumber doubled haploids with ovary culture.     

Development of novel EST-SSR markers for cucumber (Cucumis sativus) and their transferability to related species

We report on the development of novel simple sequence repeat (SSR) markers from publicly available Cucumis sativus expressed sequence tags (ESTs) and on their transferability among related species. In total, 533 di- to penta-type SSRs were identified from 6344 cucumber ESTs retrieved from GenBank. Identified SSRs mainly comprised of di- and tri-nucleotide repeats, of which AG and AAG motifs were much abundant. A total of 392 SSR-containing unigenes (non-redundant ESTs/consensus sequences) were suitable for primer design. From these, 35 primer pairs were designed as representative samples and 28 were usable markers. Twenty-six out of 28 usable markers revealed polymorphism among 21 cucumber accessions with 2–7 alleles detected (mean = 3.77) and their polymorphism information content (PIC) values ranged from 0.091 to 0.748 (mean = 0.388). The polymorphism observed herein partially arose from the null alleles which occurred at the multiple homoeoloci detected by the markers. Transferability of the 28 EST-SSR markers was investigated in four other cucurbits: melon, watermelon, pumpkin and gourd which showed frequency of 92.9%, 57.1%, 53.6% and 60.7%, respectively. The EST-SSR markers developed herein will complement the currently available genomic SSR markers and may be useful for genetic studies in cucumber and related 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.     

Resistance of Cucumis sativus cultiv ars to Cladosporium cucumerinum

A set of 249 Cucumis sativus cultivars and lines was tested for resistance to Cladosporium cucumerinum. The cultivars were grouped according to resistance or susceptibility. A clear-cut reaction was observed in 85% of the samples (resistance, 34%; susceptibility, 51%), the rest of the cultivars showing intermediate or heterogeneous reactions. Some results indicate the possibility that physiological races of C. cucumerinum exist.     

Comparative analysis of genetic diversity in Tunisian apricot germplasm using AFLP and SSR markers

Eighty-one accessions representing apricot germplasm in Tunisia were collected from different areas of cultivation and fingerprinted using amplified fragment length polymorphism (AFLP) and microsatellites (SSR) markers. A total of 339 polymorphic markers were revealed using 5 AFLP primers combinations and 24 SSR loci. AFLP and SSR markers expressed a high level of polymorphism allowing the distinction of the accessions with an efficiency coefficient of discrimination of 100% for AFLP and 97% for SSR markers. Genetic diversity structure was assessed with AFLPs and SSRs markers separately then with combined matrix data by the help of hierarchical clustering elaborated using Wards method based on Nei and Li (1979) distances. Comparison of the obtained dendrograms revealed a phylogeographic structure into two major groups with significant conservation between the observed subgroups in relation with the geographic origin of the accessions. The relative efficiency of the markers in determining the genetic relationships among apricot accessions has been assessed and a combination of AFLPs and SSRs markers was the most effective. In addition, Mantel test based on genetic distances indicated highly significant correlation between AFLP-SSR data and each of the AFLP and SSR ones, with Pearson correlation values of r = 0.873 and r = 0.692, respectively, revealing the higher efficiency of the combination of both molecular techniques (AFLP and SSR) to estimate the levels of genetic variability among apricot germplasm.     

Aneuploid strawberry (2n = 8x + 2 = 58) was developed from homozygous unreduced gamete (8x) produced by second division restitution in pollen

Unreduced gamete formation is significant in the evolutionary development of complex polyploidy series found in wild strawberry, genus Fragaria (Rosaceae). Also, it is important for genetics and breeding in strawberry plants to elucidate the mechanism of unreduced gamete formation. The objective of this study was to search for ploidy anomalies resulting from artificial diploid × octoploid crosses, and examine the mechanism through which these unreduced gametes were produced. Five everbearing cultivars of Fragaria vesca L. diploid (2n = 2x = 14) were crossed with pollen from six June-bearing cultivars of Fragaria × ananassa Duch., octoploid (2n = 8x = 56). A total of 3000 mature seeds, 100 from each of the 30 parental combinations were sown at 23 °C/20 °C (day/night) under artificial lighting with a 16 h day. The seedlings were transplanted to pots and grown in a greenhouse. Reproductive and morphological observations, flow cytometry analyses, chromosome counts and DNA analyses using CAPS markers were performed to identify the genetic background of the offspring. Most of the seed (79%) did not germinate or died soon after germination. Of the seedlings produced, 7% seemed to be pure F. vesca based on morphological characteristics, flow cytometry analyses and chromosome counts; 14% were pentaploids (2n = 5x = 35), 0.1% were hexaploids (2n = 6x = 42), and 0.03% (one individual) was aneuploid (2n = 8x + 2 = 58). Electrophoresis banding patterns obtained by CAPS marker analysis were heterozygotic in the 8x pollen parent but homozygotic in the aneuploid progeny. Judging from the chromosome counts and the CAPS marker analysis, the aneuploid was the result of a homozygous unreduced pollen grain (8x) crossed with an incomplete chromosome compliment from the egg. Because of the homozygosity, the unreduced male gamete must have been derived from second division restitution (SDR) in the octoploid pollen parent.     

Pollination with laser-irradiated pollens breaks cross-incompatibility between zicaitai (Brassica campestris var. purpurea) and ornamental kale (Brassica oleracea var. acephala) to produce hybrids with the aid of ovule culture

The objective of this study was to produce interspecific hybrids between an Ogura-cytoplasmic male sterile (CMS) line of zicaitai (Brassica campestris var. purupurea, 2n = 20) and cultivars of ornamental kale (Brassica oleracea var. acephala, 2n = 18) to develop a CMS system for hybrid seed production. Pollination with pollen grains of ornamental kales irradiated at a power output of 9.0 mW with a He–Ne laser for 3 min could overcome the cross-incompatibility between the species concerned. Intact hybrids could be efficiently produced from ovules cultured on Murashige and Skoog media supplemented by 0.2 mg l⁻¹ 6-benzyladenine. Chromosome number of hybrids was confirmed to be 2n = 19. Hybrids resembled ornamental kales in leaf morphology and in vernalization response. Pollens of hybrids had a sterile appearance. Moreover the hybridity of the putative hybrids was confirmed by RAPD data on a DNA fragment of 820 bp.     

Estimation of individual leaf area,fresh weight,and dry weight of hydroponically grown cucumbers (Cucumis sativus L.) using leaf length,width, and SPAD value

Non-destructive and mathematical approaches of modeling can be very convenient and useful for plant growth estimation. To predict individual leaf area, fresh weight, and dry weight of a cucumber (Cucumis sativus L.), models were developed using leaf length, leaf width, SPAD value, and different combinations of these variables. Eight regression equations, commonly used for developing growth models, were compared for accuracy and adaptability. The three nonlinear models developed were as follows: individual leaf area (LA) = −210.61 + 13.358W + 0.5356LW (R² = 0.980^***), fresh weight (FW) = −2.72 + 0.0135LW + 0.00022LWS (R² = 0.956^***), and dry weight (DW) = 0.25 − 0.00102LS + 0.000077LWS (R² = 0.956^***), where L is the leaf length, W the leaf width, S the SPAD value, and LWS = L × W × S. For validation of the model, estimated values for individual leaf area, fresh weight, and dry weight showed strong agreement with the measured values, respectively. Leaf dry weight, especially, was estimated with a higher degree of accuracy through the use of a SPAD value, as well as leaf length and width. Therefore, it is concluded that models presented herein may be useful for the estimation of the individual leaf area, fresh weight, and dry weight of a cucumber with a high degree of accuracy.     

Reaction of cucurbits species to infection with Zucchini lethal chlorosis virus

Zucchini lethal chlorosis virus (ZLCV) is a tospovirus that infects species of cucurbits in Brazil and is transmitted by the thrips Frankliniella zucchini. The purpose of the present work was to evaluate the reaction of seven species of cucurbits to infection with ZLCV under field and greenhouse conditions in Piracicaba County, SP. In the field experiment, ZLCV infection occurred naturally. In the greenhouse, plants were mechanically inoculated with ZLCV at the cotyledonal stage. Evaluations were based on symptoms expression and detection of the virus by PTA-ELISA. The percentages of infected plants for field and greenhouse assays, respectively, are indicated in parenthesis for each species/cultivar: Cucurbita pepo var. Caserta (72.9 and 70.7); C. maxima var. Alice (16.0 and 10.4); C. maxima var. Exposição (0.0 and 0.0); C. moschata var. Menina Brasileira (29.2 and 18.2); C. maxima × C. moschata hybrid Takaiama (63.4 and 42.7); Citrullus lanatus var. Crimson Sweet (25.0 and 25.0); Cucumis sativus var. Safira (14.3 and 41.2); and C. anguria (21.4 and 22.7).     

Parental chromosome differentiation in citrangequat [Fortunella sp. × (Citrus sinensis × Poncirus trifoliata)] using CMA banding patterns

The efficiency of chromomycin A₃ (CMA) staining was examined for parental chromosome differentiation in citrange [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] and citrangequat (Fortunella sp. × citrange). All of the accessions analyzed had the same chromosome number of 2n = 18. CMA staining revealed six characteristic banding patterns on the basis of the number and position of CMA positive bands (CMA⁺) as follows; A: two terminal and one proximal band, B: one terminal and one proximal band, C: two terminal bands, D: one terminal band, E: no band, and F: one proximal band. Chromosome CMA banding patterns of the accessions were 1A + 1B + 2C + 13D + 1F in Fortunella margarita, 2B + 2C + 7D + 7E in ‘Fukuhara’ orange, 2B + 10D + 6E in Poncirus trifoliata, 1B + 1C + 10D + 6E in citrange and 1A + 1C + 11D + 4E + 1F in citrangequat. The results of this study confirmed the intergeneric and tri-generic hybridity of citrange and citrangequat, respectively.     

Production of inter-section hybrids between Primula filchnerae and P. sinensis through ovule culture

Inter-section hybrids were obtained in the reciprocal crosses between Primula filchnerae (2n = 2x = 24) of Sect. Pinnatae and P. sinensis ‘Fanfare’ (2n = 2x = 24) of Sect. Auganthus by rescuing ovules on half-strength (1/2) Murashige and Skoog's (MS) medium supplemented with 50 g l⁻¹ sucrose, 2.5 g l⁻¹ gellan gum, 0.1 mg l⁻¹ α-naphthaleneacetic acid (NAA), 0.1 mg l⁻¹ 6-benzyladenine (BA) and 50 mg l⁻¹ gibberellic acid (GA₃). In ovule culture, germination occurred with radicle elongation but no plumule was observed. The radicle kept on the initial medium showed root proliferation with callus formation. When the calluses were transferred to (1/2)MS media containing 30 g l⁻¹ sucrose and 3 g l⁻¹ gellan gum, without plant growth regulators (PGRs) or with 1 mg l⁻¹ zeatin and 0.1 mg l⁻¹ NAA, plantlets were regenerated. The plants thus obtained were confirmed to be hybrids through flow cytometry (FCM) and random amplified polymorphic DNA (RAPD) analyses. The hybrid obtained when P. filchnerae was used as the maternal parent was diploid, whereas hexaploid hybrid was obtained when using P. sinensis as the maternal parent. The hexaploid hybrid might be produced through chromosome doubling of a triploid originated from the fertilization of P. sinensis with unreduced pollen of P. filchnerae.     

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.     

Fingerprinting of three typical macrosperma Italian lentil (Lens culinaris Medik.) landraces using fluorescence-based AFLP markers

Italian lentil landraces are principally cultivated for self or local consumption. Most of them are disappearing, particularly macrosperma types by being less required by the market. A pre-requisite for the conservation and the efficient use of genetic resources is the better understanding of the extent and the distribution of the existing genetic variation, useful for future breeding programmes. Our study was undertaken to analyse and quantify the genetic diversity within and among three macrosperma Italian lentil landraces (Onano, Altamura and Villalba), using fluorescent AFLP markers. AFLP markers generated information to differentiate among closely related genotypes and group within the same cluster individuals belonging to the same landrace. The total genetic diversity (H_T), the genetic diversity within population (H_S) and the extent of differentiation between populations (D_ST) were 0.198, 0.155 and 0.043, respectively. The fixation index (G_ST = 0.219) showed that about 78% of the observed total genetic variation can be attributed to within population differences and around 22% is due to differences among populations. The gene flow estimate (N_m = 1.774) and the mean genetic distance value (0.077) suggested narrow genetic base among the analysed populations, confirming the tendency of Italian lentil landraces to group together. The present study showed that fluorescence-based AFLP technique is a biotechnological tool that can provide significant insights for research in genetic diversity of lentil landraces and their subsequent conservation and utilization in breeding programs.     

Genetic diversity and population genetic structure of pomegranate (Punica granatum L.) in Iran using AFLP markers

Pomegranate (Punica granatum L.) is one of the oldest known edible fruits. It is native to Iran and spread from Iran to other areas. In this study amplified fragment length polymorphism (AFLP) was used to detect intra- and inter-population genetic diversity of pomegranate. A group of 67 accessions belonged to 4 populations from Iran was studied using eight primer combinations. A total of 221 scorable bands were amplified, of which, 118 (54.13%) were polymorphic. Resolving power (Rp) ranged from 5.70 to 9.21, and the average of polymorphism information content (PIC) per primer pair was 0.40. According to Nei's gene diversity and allelic statistics, Isfahan population had a highest genetic diversity (H = 0.3646, I = 0.5327, N_e = 1.6467). Coefficient of gene differentiation between populations (GST) was 0.124, indicated that mainly proportion of genetic variation (87.6%), was within populations and the remaining (12.4%) of the variation was among populations that, also supported by analysis of molecular variance (AMOVA). The gene flow (Nm) varied from 0.969 to 10.404 between pair-wise populations and was 3.504 among all of the populations. The Jaccard similarity coefficient between individuals ranged from 0.26 to 0.88. The UPGMA dendrogram clustered all 67 accessions into 6 groups. In some cases accessions from same region were grouped together but in most cases, there was gene exchange. To study the genetic relationships among populations, a principal coordinate analysis (PCoA) based on Nei's genetic distances was performed. Results of this study showed that AFLP marker can be a useful tool for investigating the genetic diversity of pomegranate genotypes.     

Different mechanisms to obtain higher fruit growth rate in two cold-tolerant cucumber (Cucumis sativus L.) lines under low night temperature

One cold-sensitive cultivar (Jinyan 4) and two cold-tolerant inbred lines (NY-1 and XC-1) of cucumber (Cucumis sativus L.) were subjected to temperatures of 28 °C/22 °C (day/night, control) or 28 °C/12 °C (day/night, cold treatment) in a 10 h photoperiod (7:00–17:00). Under control conditions, cucumber fruits grew fast during afternoon and early night, and slow during late night and morning. Under 28 °C/12 °C conditions, the two cold-tolerant inbred lines maintained relatively higher fruit growth rates than the cold-sensitive cultivar by different mechanisms. Compared to Jinyan 4, NY-1 fruits had higher growth rates during cold nights while XC-1 fruits grew faster during the next day. Under the 28 °C/12 °C temperature regime, the assimilate accumulation in the fruits of all tested genotypes followed a similar trend with the corresponding fruit growth rates. After a cold night treatment, the net CO₂ assimilation rates of one- and two-fruit plants, which had increased sink demand, were higher than that of plants without fruits in all tested genotypes. This response indicates that feedback inhibition might be an important reason for the reduction of photosynthesis on the next day. In addition, after a cold night treatment, the levels of exportable sugars (sucrose and stachyose) in mature leaves of XC-1 were higher than those measured in Jinyan 4 and NY-1, which might explain why XC-1 fruits had faster assimilate accumulation rates in the next morning. Higher activity of sucrose-phosphate synthase, a key enzyme of sucrose and stachyose biosynthesis, constituted an additional evidence that faster sucrose and stachyose biosynthesis in mature leaves may occur in XC-1 than in Jinyan 4 and NY-1 at that time. In conclusion, our results showed that cucumber genotypes may use different mechanisms to enhance their cold tolerance.     

AFLP-based genetic relationships in the Mediterranean myrtle (Myrtus communis L.)

Myrtle is an important plant species of the Mediterranean maquis, and is widely exploited for its aromatic properties. It is used in Italy for the production of a typical liqueur, for cut foliage and as an ornamental pot plant. We report the use of amplified fragment length polymorphism (AFLP) profiling to estimate genetic similarities within myrtle germplasm collected from six Italian regions, and from the Botanical Gardens of six other Mediterranean countries (including the outgroup Myrtus communis subsp. tarentina). Five AFLP primer combinations identified 122 polymorphic fragments analysing 92 individual samples, most of them (56%) were informative in discriminating among the populations. The AFLP patterns indicated that the majority of the variation occurs among rather than within populations (G_ST = 0.61). A neighbour-joining (NJ) tree separated the populations into two main branches: the first one grouped some of the Italian populations with those from Spain and Portugal; the second one included a Southern Italian subcluster together with samples from Greece, Israel, France and Croatia. A principal coordinate analysis supported the two major branches identified in the NJ analysis and showed the separation of Western and Eastern Mediterranean populations along the first axis. The Italian populations did not cluster in a single clade, but rather form distinct regional groups. The present analysis suggests that Italy represents a botanical transition zone between the Western and Eastern Mediterranean region in Myrtus communis.     

Regeneration and characterization of Swertia chirata Buch.-Ham. ex Wall. plants from immature seed cultures

First generation immature seeds (R₁) were collected from a field transferred micropropagated plant and seeds were induced to develop organogenic calli in Swertia chirata, a traditional revenue earning medicinal plant. Half strength MS medium with different growth regulators namely, BA, Kn (2.22–4.44 μM), NAA (2.69–5.37 μM), and 2.26 μM 2,4-D were used to induce callus and organogenesis. Isolated shoots produced roots either in the same medium or in presence of NAA (2.69–10.74 μM) or IBA (2.46–9.8 μM). Fully developed plantlets were successfully transplanted to soil and the fertile seed bearing plants developed. Occasionally plants derived from more than 56 weeks old calli showed some morphological variations. Such variations in regenerated plants is not reflected in their chromosomal constitution, with normal 2n = 26 chromosomes. Likewise, no variation was observed in DNA fingerprinting patterns among the short-term raised culture regenerants, which were morphologically similar to that of the donor plant illustrating their genetical uniformity and clonal fidelity. On the contrary, variation in DNA fingerprinting patterns was observed in long-term culture raised plants.     

The inheritance of two novel subgynoecious genes in cucumber (Cucumis sativus L.)

Cucumber (Cucumis sativus L.), which is a vegetable crop, has served as the model system for sex expression in flowering plants, and the inheritance of sex expression in cucumber is well documented. However, the genetics of subgynoecism expression in cucumber had rarely been described. In this study, we investigated the inheritance of subgynoecious traits in cucumber plants with the inbred cucumber lines of subgynoecious (C. sativus L. var sativus cv 97-17 and S-2-98) as the materials. Genetic analysis had showed the two subgynoecious inbred lines were controlled by one pair of recessive gene and one pair of incompletely dominant gene, which were designated presently as mod-F2 and Mod-F1, respectively. Furthermore, the mod-F2 and Mod-F1 loci, which enhance the intensity of femaleness, also inherited independently with F and M genes.