Characterization of aphid resistance loci in black raspberry (<Emphasis Type="Italic">Rubus occidentalis</Emphasis> L.)

Viruses vectored by the aphid Amphorophora agathonica cause decline in black raspberry plant health resulting in a shortened life and poor fruit quality of the infected plantings. New aphid resistant cultivars could increase the longevity of plantings providing growers and processors with consistent fruit production. Recent exploration of the native range of black raspberry identified three sources of aphid resistance: Ag₄ from Ontario (ON), Canada, Ag₅ from Maine (ME), and a third source from Michigan (MI) with no formal designation. The objectives of this study were to assess segregation of these three sources of aphid resistance in populations with single and combined sources and develop markers that can identify each source of resistance. A genetic linkage map constructed for ORUS 4305 placed the ON aphid resistance locus on Rubus linkage group (RLG) 6. Segregation ratios in populations with single and combined sources, and linkage mapping in two populations (ORUS 4304 and ORUS 4812) segregating for the Ag₅ and MI sources, respectively, indicated that these three sources of resistance are each conferred by single dominant genes/alleles that are linked on RLG6. Confirmation of marker association in 16 validation populations identified four markers that could be used to predict resistance; however, none could distinguish between the ON and MI sources. These four markers may be useful for screening populations to enrich the field-planted progeny for aphid resistance. Fine mapping of the resistance loci is needed to develop functional markers at each of the resistance loci to enable pyramiding and durable aphid resistance.     

A chalcone synthase gene <Emphasis Type="Italic">AeCHS</Emphasis> from <Emphasis Type="Italic">Abelmoschus esculentus</Emphasis> regulates flavonoid accumulation and abiotic stress tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Chalcone synthase (CHS) is one of the key enzymes in flavonoid biosynthesis pathway in plants. However, the roles of AeCHS gene from Abelmoschus esculentus in flavonoid accumulation and tolerance to abiotic stresses have not been studied. In this study, the AeCHS gene was cloned from Abelmoschus esculentus. The open reading frame contained 1170 nucleotides encoding 389 amino acids. The coding region of AeCHS was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis to obtain transgenic plants. Overexpression of AeCHS increased the production of downstream flavonoids and the expression of related genes in the flavonoid biosynthesis pathway. It also improved resistance to salt and mannitol stresses during seed germination and root development. Further component and enzymatic analyses showed the decreased content of H₂O₂ and malondialdehyde and the increased activities of superoxide dismutase (SOD) and peroxidase (POD) in transgenic seedlings. Meanwhile, the expression level of AtSOD and AtPOD genes was up-regulated against salt and osmotic stresses. Together, our finding indicated that changing the expression level of AeCHS in plants alters the accumulation of flavonoids and regulates plantlet tolerance to abiotic stress by maintaining ROS homeostasis. The AeCHS gene has the potential to be used to increase the content of valuable flavonoids and improve the tolerance to abiotic stresses in plants.     

Ectopic expression and functional characterization of type III polyketide synthase mutants from <Emphasis Type="Italic">Emblica officinalis</Emphasis> Gaertn

Key message

Functional characterization and ectopic expression studies of chalcone synthase mutants implicate the role of phenylalanine in tailoring the substrate specificity of type III polyketide synthase.

Abstract

Chalcone synthase (CHS) is a plant-specific type III polyketide synthase that catalyzes the synthesis of flavonoids. Native CHS enzyme does not possess any functional activity on N-methylanthraniloyl-CoA, which is the substrate for acridione/quinolone alkaloid biosynthesis. Here, we report the functional transformation of chalcone synthase protein from Emblica officinalis (EoCHS) to quinolone and acridone synthase (ACS) with single amino acid substitutions. A cDNA of 1173 bp encoding chalcone synthase was isolated from E. officinalis and mutants (F215S and F265V) were generated by site-directed mutagenesis. Molecular modeling studies of EoCHS did not show any active binding with N-methylanthraniloyl-CoA, but the mutants of EoCHS showed strong affinity to the same. As revealed by the modeling studies, functional analysis of CHS mutants showed that they could utilize p-coumaroyl-CoA as well as N-methylanthraniloyl-CoA as substrates and yield active products such as naringenin, 4-hydroxy 1-methyl 2(H) quinolone and 1,3-dihydroxy-n-methyl acridone. Exchange of a single amino acid in EoCHS (F215S and F265V) resulted in functionally active mutants that preferred N-methylanthraniloyl-CoA over p-coumaroyl-CoA. This can be attributed to the increase in the relative volume of active sites in mutants by mutation. Moreover, metabolomic and MS analyses of tobacco leaves transiently expressing mutant genes showed high levels of naringenin, acridones and quinolone derivatives compared to wild-type CHS. This is the first report demonstrating the functional activity of EoCHS mutants with N-methylanthraniloyl-CoA and these results indicate the role of phenylalanine in altering the substrate specificity and in the evolution of type III PKSs.

     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Genetics and fine mapping of a yellow-green leaf gene (<Emphasis Type="Italic">ygl</Emphasis>-<Emphasis Type="Italic">1</Emphasis>) in cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">capitata</Emphasis> L.)

Cabbage (Brassica oleracea var. capitata L.) is one of the most popular cultivated vegetables worldwide. Cabbage has rich phenotypic diversity, including plant height, head shape, head color, leaf shape and leaf color. Leaf color plays an important role in cabbage growth and development. At present, there are few reports on fine mapping of leaf color mutants in B. oleracea. In this study, a naturally occurring yellow-green leaf cabbage mutant (YL-1), derived from the self-pollinated progenies of the hybrid ‘Hosom’, was used for inheritance analysis and gene mapping. Segregation populations including F₂ and BC₁ were generated from the cross of two inbred lines, YL-1 and 01–20. Genetic analysis with the F₂ and BC₁ populations demonstrated that the yellow-green leaf color was controlled by a single recessive nuclear gene, ygl-1. Insertion–deletion (InDel) markers, designed based on the parental re-sequencing data, were used for the preliminary mapping with BSA (bulked segregant analysis) method. A genetic map constructed with 15 InDels indicated that ygl-1 was located on chromosome C01. The ygl-1 gene is flanked by InDel markers ID2 and M8, with genetic distances of 0.4 cM and 0.35 cM, respectively. The interval distance between two markers is 167 kb. Thus, it enables us to locate the ygl-1 gene for the first time in B. oleracea. This study lays the foundation for candidate gene prediction and ygl-1gene cloning.     

The genetic structure of red raspberry (<Emphasis Type="Italic">Rubus idaeus</Emphasis> L.) populations in Lithuania

The red raspberry (Rubus idaeus L.) is widely distributed in Lithuania and occupies a range of habitats. The presence of coadapted gene pools in local populations of R. idaeus is a question of interest not only to plant scientists, but also to plant breeders. In this study, we investigated the genetic structure of R. idaeus and the influence of local habitats on the genetic diversity within and among populations. Nineteen populations of R. idaeus were sampled from different habitats in various agroclimatic subregions of Lithuania, and analyzed using RAPD markers. 113 RAPD bands were identified among 315 individuals; 84.31% of these were polymorphic. The mean values of Shannon’s information index for different populations ranged from 0.341 to 0.455. Nei’s gene diversity established within populations averaged 0.266. An AMOVA revealed 74% of genetic variation among individuals within populations of R. idaeus, and 23% among populations. The remaining genetic variation was distributed among populations from different agroclimatic subregions (3%). The results of this study suggest that the genetic structure of R. idaeus populations in Lithuania may be influenced partially by isolation by distance as well as by local environmental conditions.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of grapevine (<Emphasis Type="Italic">Vitis vinifera</Emphasis> L.) with a novel stilbene synthase gene from Chinese wild <Emphasis Type="Italic">Vitis pseudoreticulata</Emphasis>

A novel stilbene synthase gene (STS), cloned from Chinese wild Vitis pseudoreticulata (W. T. Wang) and responsible for synthesis of the phytoalexin resveratrol in grapevine, was successfully transferred into V. vinifera L. cv. Thompson Seedless via Agrobacterium tumefaciens-mediated transformation. Using transformation procedures developed in the present study, 72% GFP-positive germinated embryos were produced with about 38% of transformed embryos regenerated into normal plantlets. Integration of the STS gene into the transgenic plants was verified by PCR and Southern blot analysis. Expression of the STS gene was detected by high performance liquid chromatography (HPLC), which showed that the resveratrol concentration in the transgenic plants was 5.5 times higher than that in non-transformed control plants. Chaohong Fan and Ni Pu contributed equally to this work.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Cytogenetic characterization of <Emphasis Type="Italic">Amaranthus caudatus</Emphasis> L. and <Emphasis Type="Italic">Amaranthus hybridus</Emphasis> subsp<Emphasis Type="Italic">. cruentus</Emphasis> (L.) Thell.

The present study is aimed to identify genetic variability between two species of Amaranthus viz., A. caudatus and A. hybridus subsp. cruentus, two economically important species, cultivated mainly for grain production. Karyomorphological studies in Amaranthus are scarce, probably due to higher number of small sized chromosomes. Karyomorphological studies were conducted using mitotic squash preparation of young healthy root tips. Karyological parameters and karyotypic formula were established using various software programs and tabulated the karyomorphometric and asymmetry indices viz., Disparity index, Variation coefficient, Total forma percentage, Karyotype asymmetry index, Syi index, Rec index, Interchromosomal and Intrachromosomal asymmetry index and Degree of asymmetry of karyotypes. The mitotic chromosome number observed for A. caudatus was 2n = 32 with a gametic number n = 16 and A. hybridus subsp. cruentus was 2n = 34 with a gametic number n = 17. In A. caudatus the chromosome length during somatic metaphase ranged from 0.8698 to 1.7722 μm with a total length of 39.1412 μm. In A. hybridus subsp. cruentus the length of chromosome ranged from 0.7756 to 1.9421 μm with a total length of 44.9922 μm. Various karyomorphometry and asymmetry indices analyzed revealed the extend of interspecific variation and their evolutionary status.     

Analysis of DNA methylation during spontaneous rooting from the stem apex in <Emphasis Type="Italic">Rubus idaeus</Emphasis>

Rubus idaeus L. is of great economic value. Some varieties of Rubus idaeus have a unique feature of spontaneous rooting from the stem apex. To determine whether DNA methylation is associated with the spontaneous rooting process, variations in the methylation at stem apex during four root developmental stages were investigated, using the methylation-sensitive amplification polymorphism (MSAP) technique and the bisulfite sequencing analysis (BSA). The results showed that the DNA methylation levels and patterns were significantly different between the four developmental stages. A total of 824 CCGG amplified sites were detected by MSAP. MSAP screening revealed that the level of DNA methylation at stages I to IV was 34.95, 36.04, 36.29, and 37.50%, respectively. The number of methylated sites and their methylation levels tended to decrease at stages III and IV (root differentiation and elongation) compared with those at stage I (stem elongation). After cloning and sequencing of the 16 polymorphic differentially methylated DNA fragments, BLAST search results indicated that they might be involved in differentiation of the lateral root primordium, plant defense, signal transduction, and energy metabolism. Results of the qRT-PCR and BSA analyses confirmed that methylation of some key genes was closely associated with their expression at the different developmental stages. These findings could be useful for future studies on the potential role of DNA methylation in spontaneous rooting from the stem apex, implying its importance in rooting regulation and rapid expansion of raspberry populations.     

Isolation and linkage mapping of NBS-LRR resistance gene analogs in red raspberry (<Emphasis Type="Italic">Rubus idaeus</Emphasis> L.) and classification among 270 Rosaceae NBS-LRR genes

Plant R genes confer resistance to pathogens in a gene-for-gene mode. Seventy-five putative resistance gene analogs (RGAs) containing conserved domains were cloned from Rubus idaeus L. cv. ‘Latham’ using degenerate primers based on RGAs identified in Rosaceae species. The sequences were compared to 195 RGA sequences identified from five Rosaceae family genera. Multiple sequence alignments showed high similarity at multiple nucleotide-binding site (NBS) motifs with homology to Drosophila Toll and mammalian interleukin-1 receptor (TIR) and non-TIR RNBSA-A motifs. The TIR sequences clustered separately from the non-TIR sequences with a bootstrap value of 76%. There were 11 clusters each of TIR and non-TIR type sequences of multiple genera with bootstrap values of more than 50%, including nine with values of more than 75% and seven of more than 90%. Polymorphic sequence characterized amplified region and cleaved amplified polymorphic sequence markers were developed for nine Rubus RGA sequences with eight placed on a red raspberry genetic linkage map. Phylogenetic analysis indicated four of the mapped sequences share sequence similarity to groupTIR I, while three others were spread in non-TIR groups. Of the 75 Rubus RGA sequences analyzed, members were placed in five TIR groups and six non-TIR groups. These group classifications closely matched those in 12 of 13 studies from which these sequences were derived. The analysis of related DNA sequences within plant families elucidates the evolutionary relationship and process involved in pest resistance development in plants. This information will aid in the understanding of R genes and their proliferation within plant genomes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biolistic transformation of cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.) with the <Emphasis Type="Italic">phyA</Emphasis> gene from <Emphasis Type="Italic">Aspergillus ficuum</Emphasis>

Transgenic cotton with an increased level of phytase activity was generated from cotton (Gossypium hirsutum L.) cv. ND94-7 by subjecting shoot-apex explants to particle bombardment. These tissues were transformed with plasmid pC-KSA2300 carrying a selectable marker (for kanamycin) and a target gene (phytase, or phyA, from Aspergillus ficuum). Primary plants were regenerated in a medium containing 75 mg l⁻¹ kanamycin. Of 1,534 shoot apices, 52 (3.4%) survived on this selection medium. Southern and Northern blot analyses confirmed that phyA was stably integrated and expressed in those primary transgenics. The progenies of the primary transgenic plants were found to have a 3.1- to 3.2-fold increase in root extracellular phytase activity, resulting in improved phosphorus (P) nutrition. Growth also was enhanced when they were supplied with phytate, and their P content was equivalent to that of wildtype plants supplied with inorganic phosphate. These results demonstrate that the expression of phyA in cotton plants improves their ability to utilize organic P in response to a deficiency.     

Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.