Evaluation of genetic variability in choosen apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis> Borkh.) cultivars by ISSR-PCR analysis

The aim of the study was to determine the genetic variability in eight apple cultivars: Delikates, Cortland, James Grieve, Lired, Jonathan, Golden Delicious, Jonagold, and Idared from the collection of Fruit Growing Research Station in Rajkowo of the West Pomeranian University of Technology, Szczecin. The cultivar Delikates was obtained from the crossing of two cultivars: Cortland, and James Grieve, whereas cultivar Lired is a James Grieve’s sport. The second one cultivar—Jonagold was obtained from the crossing of Jonathan and Golden Delicious. The cultivar Idared is a hybrid obtained from the crossing of Jonathan and Wagener. Out of 40 primers, 17 were chosen for the final study. Those amplified a total of 183 loci (872 amplicons) out of which 34 (18.5%) were monomorphic, 128 (69.5%) were polymorphic and 22 (12%) cultivar-specific. Specific ISSR products were detected for each apple cultivar. A dendrogram was constructed using the UPGMA method which revealed two distinct clusters: I—Delikates, Cortland, James Grieve and Lired, II—Jonathan, Golden Delicious, Jonagold and Idared. Genetic similarity between Delikates, Cortland and James Grieve was 68.6, 70.8%, respectively and between cultivar Jonagold, Jonathan and Golden Delicous was 79.8, 85.2%, respectively.     

Genetic linkage maps of two apple cultivars (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis> Borkh.) based on AFLP and microsatellite markers

Genetic linkage maps for two apple cultivars were constructed using AFLP and SSR markers and the pseudo-testcross mapping strategy. The F₁-mapping population was produced by crossing the cultivar Braeburn to the cultivar Telamon and consisted of 257 individuals. Out of the 182 AFLP primer combinations screened, a total of 48 were selected. Using these, 463 AFLP markers segregating 1:1 in the progeny were identified, of which 231 were heterozygous in Telamon and 232 in Braeburn. Eighty-five AFLP markers present in both cultivars (3:1 segregation) were scored in the whole mapping population. Twenty-one SSR primer pairs were tested, which clearly screened 23 loci (some multi-locus markers). This resulted in the identification of 3 loci heterozygous only in Telamon (1:2:1), 5 loci heterozygous only in Braeburn (1:2:1) and 15 loci which were heterozygous in both cultivars (1:1:1:1). Two linkage maps were produced. The Telamon map comprised 259 markers (242 AFLPs and 17 SSRs) divided into 17 linkage groups. The total map length was 1039 cM with a marker density of 4.0 cM. At = 0.05, 8.9% of the mapped loci showed distorted segregation. The Braeburn map consisted of 264 markers (245 AFLPs and 19 SSRs) mapped on 17 linkage groups and spanning 1245 cM. The average distance between two markers was 4.7 cM and segregation distortion was observed for 18.6% of the mapped markers ( = 0.05). Fourty-six markers common to both maps (32 AFLPs and 14 SSRs) allowed the identification of 16 homologous linkage groups. The seventeenth pair of homologous linkage groups from Telamon and Braeburn was identified by 2 SSR markers which were in common to the genetic linkage maps of Fiesta and Discovery, two other apple cultivars.     

Genome-wide analysis and identification of the <Emphasis Type="Italic">SMXL</Emphasis> gene family in apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis>)

Strigolactones (SLs) are a recently discovered type of plant hormone that controls various developmental processes. The DWARF53 (D53) protein in rice and the SMAX1-LIKE (SMXL) family in Arabidopsis repress SL signaling. In this study, bioinformatics analyses were performed, and 236 SMXL proteins were identified in 28 sequenced plants. A phylogenetic analysis indicated that all potential SMXL proteins could be divided into three groups and that the SMXL proteins may have originated in Bryophytes. An analysis of the SMXL chromosomal locations suggested that gene duplication events at different times led to expansion of the SMXL family members in Angiospermae. Subsequently, the gene structure and protein modeling of MdSMXLs showed that they are highly conserved. The expression patterns of MdSMXLs indicated that they were expressed in different organs of apple (stems, roots, leaves, flowers, and fruits) at varying levels and that MdSMXLs may participate in the SL signaling pathway and the response to abiotic stress. This study provides a valuable foundation for additional investigations into the function of the SMXL gene family in plants.     

Study of tree architecture of apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis> Borkh.) by QTL analysis of growth traits

Apple tree architecture is naturally very diverse, but for fruit production, certain tree habits are more desirable than others. Here we describe the results of a QTL analysis performed to study the genetic control of growth traits in apple. This was carried out on the progeny of a cross between two apple cultivars of contrasting tree architectures. “Telamon” has a columnar tree form and “Braeburn” has a more standard, “normal” growth habit. The growth traits were measured on the F ₁ seedlings of the Telamon × Braeburn population for two consecutive years of growth on own roots and for the first year of growth on M9 rootstock. QTL analysis was carried out using either the Kruskal–Wallis method or the Multiple QTL Method. For all but one growth characteristic, significant QTLs were detected. A major cluster of QTLs was located in the Co gene region of “Telamon”, confirming the major influence of the Co gene on tree architecture, although this influence changed as the plant material aged and was generally more pronounced for rootstock-grown plants. Additional QTL results suggest the occurrence of genes with pleiotropic effects on tree architecture. The observed QTL instability over different years and for different root systems indicates that the genetic control of tree architecture is largely influenced by environmental factors and probably changes as the tree matures. Finally, a major influence of the root system on all the traits determining tree architecture was clearly demonstrated.     

Peculiarities of polyphenolic profile of fruits of Siberian crab apple and its hybrids with <Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">Domestica</Emphasis> Borkh

For the first time, we have studied the polyphenolic profile of fruits of Siberian crabapple and its hybrids (F1, F2, and F3) with domestic apple cultivated in East Siberia. It is shown that the chemical composition of polyphenolics of Siberian crabapple fruits is overwhelmingly characteristic of the genus Malus; on the other hand, it has clearly identifiable features: low content of flavan-3-ols and derivatives of cinnamic acid; high content of procyanidin B1, phloridzin, anthocyanes, and quercetin glycosides. Procyanidin B2 was not found in both peel and flesh of Siberian crabapple. In the case of hybridization with domestic apple, the fruits of resulting cultivars show a substantial change in the flavan-3-ol content ratio because of the synthesizing of procyanidin B2 in tissues and an increase in (?)-epicatechin content.     

The isolation of the <Emphasis Type="Italic">IGT</Emphasis> family genes in <Emphasis Type="Italic">Malus × domestica</Emphasis> and their expressions in four idiotype apple cultivars

IGT family genes share the highly conserved motif GφL-(A/T) IGT in domain II and play an essential role in plant form. The tree architecture of apple (Malus ×?domestica Borkh.) affects fruit quality and yield. However, little information is available regarding IGT family genes in apple. Apple cultivars of four ideotypes (columnar, tip bearer, spur, and standard) were selected to characterize IGT family genes. Four IGT family members named MdoTAC1a, MdoTAC1b, MdoLAZY1, and MdoLAZY2 were found in the apple genome, sharing four conserved domains. In addition, MdoLAZY1 and MdoLAZY2 contain a fifth domain (EAR motif) at the C-terminus. There was no difference in the coding sequences of each gene in the four cultivars, but several mutated sites were found in their promoters. The four genes displayed lower expression levels in all tested tissues and organs of the columnar cultivar than in the other three cultivars, while expression levels of MdoTAC1a and MdoTAC1b in shoot tips and vegetative buds were highest in the standard cultivar, followed by spur, tip bearing, and columnar cultivars in decreasing order. These results indicate that IGT gene promoters are of great importance in the development of apple tree architecture and lay a theoretical basis for developing gene-specific markers for marker-assisted selection in breeding programs.     

Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis>)

Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant ‘McIntosh Wijcik’, which was discovered as a bud mutation from ‘McIntosh’, exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in ‘McIntosh Wijcik’ but not in ‘McIntosh’. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in ‘McIntosh Wijcik’ is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

The <Emphasis Type="Italic">rolB</Emphasis> gene promotes rooting <Emphasis Type="Italic">in vitro </Emphasis>and increases fresh root weight <Emphasis Type="Italic">in vivo</Emphasis> of transformed apple scion cultivar ‘Florina’

Transgenic apple (Malus × domestica Borkh.) Florina plants were obtained by Agrobacterium-mediated transformation. The efficiency of gene transfer was 7.9%, calculated as a number of explants producing at least one transgenic shoot, after co-cultivation of leaf explants from in vitro-grown shoots in a thin layer of the A. tumefaciens C58C1 strain with the binary vector pCMB-B:GUS. Polymerase chain reaction revealed that all the clones contained the nptII and rolB genes, while four of them did not contain the gus gene. Southern blot analysis confirmed the integration of the nptII and rolB genes, with one to three copies per genome being present. All independent rolB-transgenic lines were able to produce roots in vitro on the hormone free medium, while the plants, transformed with the vector pIB16.1, or untransformed control plants did not root, and only half of shoots of MM106 rootstock rooted on this medium. The average root number in the rolB-transgenic clones ranged from 4 to 7.7. Pretreatment with indole-3-butyric acid caused root formation in all transgenic and control plants and significantly increased root number in the rolB-transgenic lines, compared to untransformed plants. RolB-transgenic plants, grown in vivo in greenhouse for 2 years, did not differ phenotypically from the wild type line with the exception of root parts. All rolB-transformed plants produced altered root systems containing more fine roots leading to significantly increased fresh root weight in five plant lines.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

<Emphasis Type="Italic">TNF</Emphasis><Emphasis Type="Italic">α</Emphasis> and <Emphasis Type="Italic">LT</Emphasis><Emphasis Type="Italic">α</Emphasis> gene polymorphisms as additional markers of celiac disease susceptibility in a DQ2-positive population

TNFalpha and TNFbeta, or linfotoxin (LTalpha), are two molecules playing an important role in inflammation. Their genes map on Chromosome 6, between the HLA class II and class I loci. Polymorphisms in, or near, TNF genes have been associated with susceptibility to several autoimmune diseases. Studies of TNF genes in celiac disease (CD) have presented contradictory results. We have assessed the role of TNFalpha and linfotoxin alpha (TNFbeta) in CD and their relative value as CD markers in addition to the presence of DQ2. The TNFA -308 polymorphism and the polymorphism at the first intron of the LTA gene were typed in CD patients and healthy controls and the results were correlated with the presence of DQ2. Significant differences were found in genotype and allele frequencies for the TNFA and LTA genes between CD patients and controls, with an increase in the presence of the TNFA*2 and LTA*1 alleles in CD patients. These differences increase when DQ2-positive CD patients and DQ2-positive controls are compared. In DQ2-positive individuals, allele 2 (A) in position -308 of the promoter of TNFA and allele 1 (G) of the NcoI RFLP in the first intron of LTA are additional risk markers for CD.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

Two QTL characterized for soft scald and soggy breakdown in apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis>) through pedigree-based analysis of a large population of interconnected families

Soft scald and soggy breakdown are important postharvest physiological disorders of apple (Malus × domestica). ‘Honeycrisp’ and some of its offspring are particularly susceptible to developing these disorders. The purpose of this study was to identify molecular markers associated with high incidences of soft scald and soggy breakdown for use in marker-assisted breeding. Towards this aim, we employed a pedigree-based approach using mostly germplasm related to ‘Honeycrisp.’ Two quantitative trait loci (QTL) were consistently identified on linkage groups (LGs) 2 and 16 across the 2014 and 2015 harvest years. The same QTL were identified for both storage disorders, indicating that they may be physiologically related. ‘Honeycrisp’ is homozygous for an identical by state haplotype at the LG2 QTL that was consistently associated with a deleterious effect on soft scald and soggy breakdown incidence. This haplotype was traced through SNP-confirmed pedigrees to the following cultivars: ‘Grimes Golden,’ ‘Northern Spy,’ ‘Rome Beauty,’ and ‘Fireside’ and is common in derived apple germplasm. Haplotypes at the LG16 QTL could not be adequately characterized due to variation between years combined with effects of this QTL being of relatively smaller size and being most evident in individuals that carry two copies of the deleterious haplotype at the LG2 QTL. These results suggest that limiting homozygosity of the deleterious haplotype at the LG2 QTL through marker-assisted breeding would be a valid strategy to limit soft scald and soggy breakdown incidences in apple seedling populations.