Amplified Fragment Length Polymorphism Research Articles

RELATIONSHIP BETWEEN GENETIC AND ENVIRONMENTAL CHARACTERISTICS OF LITHUANIAN POPULATIONS OF PURPLE LOOSESTRIFE (LYTHRUM SALICARIA)

The present study evaluated genetic diversity of Lithuanian populations of Lythrum salicaria in relation to parameters of riparian environment. Growing along Nemunas, Seaside and Lielupė river basins, 15 populations were examined using amplified fragment length polymorphism markers. Molecular data were related to the river basins, type of land use and cover, natural vice versa regulated fragments of the rivers. Population mean genetic diversity parameters were as follows: percentage of polymorphic loci (57.2), expected heterozygosity (0.183), polymorphismc information content (0.218). Mantel test revealed correlation (R2 = 0.0986, p = 0.01) between genetic and geographic distance of populations. Greater genetic diversity within, rather than among populations (ΦPT = 0.213) was observed. According to the Bayesian clustering, studied populations are admixtures of two gene pools. Analysis of molecular variance revealed significant differentiation between populations belonging to distinct river basins, between populations from natural vs. regulated fragments of the rivers.

Strain Degeneration in White Button Mushroom (Agaricus bisporus) Using Amplified Fragment Length Polymorphism

Background: The growing population and limited agricultural land have pushed the demand for higher food production per land area. Edible mushrooms are a rich source of protein. Agaricus bisporus is the most common edible mushroom with high economic value. One of the most important steps in the production process of white button mushroom is the production of spawn. It is highly important to make pure high-quality mycelium during the production of spawn. Studies have shown that at this stage of production, a high level of morphological changes occurs in some mushrooms, which causes the formation of fluffy-shaped mycelium. This phenomenon is a kind of strain degeneration in edible mushrooms and is one of the biggest concerns of the producers of white button mushroom. Objectives: The purpose of this study was to investigate the nature of stroma (strain degeneration) in white button mushroom, and to identify the causes of genetic changes. Methods: In this experiment, ten successive subcultures were produced from the mother mycelium on potato dextrose agar medium (pH: 6.2), and the genetic changes of mycelium were examined after ten successive subcultures. DNA extraction was performed after sampling of the mother, normal, and fluffy mycelia. Six pairs of designed primers and propagated parts were loaded into six polyacrylamide gels. DNA fragments were visualized using silver staining. Genetic comparisons were made between the mother, normal, and fluffy mycelia by the amplified fragment length polymorphism (AFLP) method, and the results were evaluated using POPGENE software. Results: The AFLP marker produced 137 fragments for the H2 strain, of which 57 fragments had polymorphism between the mother, normal, and fluffy mycelia. For the 737 strain, 149 fragments were produced, of which 73 fragments had polymorphism between the mother, normal, and fluffy mycelia. On average, the mother, normal, and fluffy mycelia had about 42 and 49% polymorphism for the H2 and 737 strains, respectively. The degree of polymorphism among the samples confirmed genetic differences between the mother, normal, and fluffy mycelia. Conclusions: Based on the results of the present study, we concluded that strain degeneration, which manifests as the fluffy mycelium of white button mushroom, has a genetic origin. These genetic differences can be attributed to diverse factors, such as the phenomenon of heterokaryosis, asexual recombination, and the formation of mitosis crossing over. These factors and their effects require further evaluations.

The Centre–Periphery Model, a Possible Explanation for the Distribution of Some Pinus spp. in the Sierra Madre Occidental, Mexico

Genetic diversity is key to survival of species. In evolutionary ecology, the general centre–periphery theory suggests that populations of species located at the margins of their distribution areas display less genetic diversity and greater genetic differentiation than populations from central areas. The aim of this study was to evaluate the genetic diversity and differentiation in six of the main pine species of the Sierra Madre Occidental (northern Mexico). The species considered were Pinus arizonica, P. cembroides, P. durangensis, Pinus engelmannii, P. herrerae and P. leiophylla, which occur at the margins and centre of the geographic distribution. We sampled needles from 2799 individuals belonging to 80 populations of the six species. We analysed amplified fragment length polymorphisms (AFLPs) to estimate diversity and rarity indexes, applied Principal Coordinate Analysis (PCoA), and used the Kruskal–Wallis test to detect genetic differences. Finally, we calculated Spearman’s correlation for association between variables. The general centre–periphery model only explained the traits in P. herrerae. The elevation gradient was an important factor that influenced genetic diversity. However, for elevation as partitioning criterion, most populations showed a central distribution. This information may be useful for establishing seed collections of priority individuals for maintenance in germplasm banks and their subsequent sustainable use.

MaSTR™: an effective probabilistic genotyping tool for interpretation of STR mixtures associated with differentially degraded DNA.

The recently developed probabilistic genotyping software package MaSTR™ (SoftGenetics LLC) was used to develop statistical weight estimates for a variety of two-person STR mixture profiles with differentially degraded sources of DNA. A total of 864 analyses, on 144 two-person profiles, were performed. Mixture ratios ranged from 1:1 to 1:10, including pristine sources of DNA and various combinations of artificially degraded DNA (average size fragments of 150 or 250 bps). Quantities of DNA template were varied (0.1 to 0.5 ngs of total input) and MaSTR™ analysis was performed with eight chains of 10,000 or 40,000 iterations, with or without a conditioning profile to generate likelihood ratio (LR) values. Overall, the software performed as expected. The resulting log(LR) values for pristine mixture profiles were typically greater than 1030. Lower-quality mixture data associated with sources of DNA at ~ 0.05 ngs for each contributor resulted in peak imbalance and allelic dropout which reduced the weight in support of a contributor. This was exacerbated by higher levels of degradation, with some instances resulting in log(LR) values in support of an exclusion. These studies provide additional support for the use of probabilistic genotyping software solutions in forensic investigations, addressing concerns raised by the President's Council of Advisors on Science and Technology (PCAST).

SCClone: Accurate Clustering of Tumor Single-Cell DNA Sequencing Data.

Single-cell DNA sequencing (scDNA-seq) enables high-resolution profiling of genetic diversity among single cells and is especially useful for deciphering the intra-tumor heterogeneity and evolutionary history of tumor. Specific technical issues such as allele dropout, false-positive errors, and doublets make scDNA-seq data incomplete and error-prone, giving rise to a severe challenge of accurately inferring clonal architecture of tumor. To effectively address these issues, we introduce a new computational method called SCClone for reasoning subclones from single nucleotide variation (SNV) data of single cells. Specifically, SCClone leverages a probability mixture model for binary data to cluster single cells into distinct subclones. To accurately decipher underlying clonal composition, a novel model selection scheme based on inter-cluster variance is employed to find the optimal number of subclones. Extensive evaluations on various simulated datasets suggest SCClone has strong robustness against different technical noises in scDNA-seq data and achieves better performance than the state-of-the-art methods in reasoning clonal composition. Further evaluations of SCClone on three real scDNA-seq datasets show that it can effectively find the underlying subclones from severely disturbed data. The SCClone software is freely available at https://github.com/qasimyu/scclone.

CellPhy: accurate and fast probabilistic inference of single-cell phylogenies from scDNA-seq data

We introduce CellPhy, a maximum likelihood framework for inferring phylogenetic trees from somatic single-cell single-nucleotide variants. CellPhy leverages a finite-site Markov genotype model with 16 diploid states and considers amplification error and allelic dropout. We implement CellPhy into RAxML-NG, a widely used phylogenetic inference package that provides statistical confidence measurements and scales well on large datasets with hundreds or thousands of cells. Comprehensive simulations suggest that CellPhy is more robust to single-cell genomics errors and outperforms state-of-the-art methods under realistic scenarios, both in accuracy and speed. CellPhy is freely available at https://github.com/amkozlov/cellphy.

A novel mutation at the AMEL primer binding region on the Y chromosome in AMELY negative male

Gender identification in forensic DNA typing is an important tool for criminal investigation as well as STR typing. Most methods are based on a size difference of amelogenin X and Y (AMELX and AMELY). There have been some reports that the method by amelogenin (AMEL) incorrectly typed some males as females because the AMELY allele is not detected. AMELY allele dropout is often caused by deletions encompassing AMELY on Yp11.2 and accompanied with Y-STR allele dropout (especially DYS458). However, an unusual deletion was found in our laboratory. The AMELY allele could be recovered by using another commercial kit and alternative AMEL primer sets, and the Y-STR markers resulted in a complete profile. Sequencing results showed that there was an 8bp deletion in AMELY at the position corresponding to 41-48bp downstream from the 3' end of the 6bp deletion site in AMELX. This is considered a novel mutation at a primer binding region.

Allelic dropout in PAH affecting the results of genetic diagnosis in phenylketonuria.

Phenylketonuria (PKU) is an inherited autosomal recessive disorder of phenylalanine metabolism. It is mainly caused by a deficiency in phenylalanine hydroxylase (PAH) and frequently diagnosed with Sanger sequencing. To some extent, allelic dropout can explain the inconsistency in genotype and phenotype. Three families were evaluated through DNA sequence analysis, multiplex ligation-dependent probe amplification (MLPA) and prenatal diagnosis technologies. The possibility of inconsistency in phenotype and genotype with c.331C>T variant was analysed. Through pedigree analysis, three mothers carried a homozygous c.331C>T variant, which was a false-positive result. New primers were used, and this error was caused by allelic dropout. In this case, c.158G>A was likely a benign variant. Sequence variants in primer-binding regions could cause allelic dropout, creating unpredictable errors in genotyping. Our results emphasised the need for careful measures to treat genotype-phenotype inconsistencies.

Constructing the wolfberry (Lycium spp.) genetic linkage map using AFLP and SSR markers

Genetic linkage maps are important for quantitative trait locus (QTL) and marker-assisted selection breeding. The wolfberry (Lycium spp.) is an important food and traditional medicine in China. However, few construction genetic linkage maps have been reported because of the lack of genomic and genetic resources. In this study, a population of 89 F1 seedings was derived from a cross between two heterozygous parents, L. chinense var. potaninii ‘BF-01’ (female) and L. barbarum var. auranticarpum ‘NH-01’ (male), in order to construct a genetic linkage map using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers based on the double pseudo-test cross mapping strategy. The resulting genetic map consisted of 165 markers (74 AFLPs and 91 SSRs) distributed across 12 linkage groups and spanned a total length of 557.6 cM with an average distance of 3.38 cM between adjacent markers. The 12 linkage groups contained 3 to 21 markers and ranged in length from 8.6 to 58.3 cM. Twenty-nine segregated markers distributed in the map were mainly located on LG4 and LG9 linkage groups at P<0.05. This is the first linkage map of Lycium species using SSR and AFLP markers, which can serve as basis for improving genes and selective breeding of the genome assembly.

Факторы риска формирования хронического полипозного риносинусита у детей с муковисцидозом

The investigation of the pathogenesis of chronic rhinosinusitis in patients with cystic fibrosis (CF) and analysis of molecular biomarkers can promote the development of tailored approaches to its treatment and prevention. Objective. To analyze associations between sinonasal polyposis and genetic polymorphisms of the xenobiotic metabolizing enzymes (XMEs) in CF patients. Patients and methods. We performed a retrospective comparative analysis of 145 CF patients from the Russian Federation (RF) and 82 patients from the Republic of Belarus (RB). Patients provided blood samples that were later used for DNA extraction; clinical information was collected for all study participants. The diagnosis was based on the guidelines of the European and Russian Consensus. This was a case-control study. The XME gene polymorphisms were assessed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) or amplified fragment length polymorphism (AFLP) analysis with special primers. The following genes and polymorphic variants were chosen for the study: phase I (CYP2C9*3 (rs1057910; c.1075A&gt;C; I359L), CYP2C9*2 (rs1799853; c.430C&gt;T; R144C), CYP2C19*2 (rs4244285; c.681G&gt;A), CYP2C19*3 (rs4986893; c.636G&gt;A; W212X), CYP2D6*4 (rs3892097; c.1846G&gt;A), CYP3A4*3 (rs4986910; M445T; c.1334T&gt;C), CYP3A4*1B (rs2740574; c.-392C&gt;T); phase II (deletions in GSTT1 and GSTM1; GSTP1 (c.313A&gt;G); GCLC (TNR(GAG), c.-129C&gt;T); GCLM (c.-588C&gt;T); NAT2 (c.191G&gt;A, c.282C&gt;T, c.341T&gt;C, c.434A&gt;C, c.481C&gt;T, c.590G&gt;A, c.803A&gt;G, c.845A&gt;C, c.857G&gt;A)). Results. Mean patient age was 12.69 ± 6.9 years (5.03–40.36) and did not vary between the RF and RB patients. The male to female ratio was 0.52:0.48 (118:109). Nasal polyposis was diagnosed in 61 patients (26.9%), while 166 children (73.1%) had no nasal polyps. We found that patients with chronic rhinosinusitis and nasal polyps were more likely to have ‘severe’ CFTR genotypes: 95.6% vs 4.4% of patients with a ‘mild’ genotype (р = 0.05). We observed no significant associations between nasal polyposis and analyzed XME gene polymorphisms, as well as glutathione metabolic genes. Conclusion. We found no associations between nasal polyposis and XME gene polymorphisms (phase 1 and 2), which suggests the need for further research aimed to identify other molecular mechanisms underlying the development of nasal polyps in CF patients. Key words: sinonasal polyposis, chronic rhinosinusitis, xenobiotic biotransformation enzymes, cystic fibrosis, glutathione, inflammation

Sequence Variations of 31 Υ-Chromosomal Short Tandem Repeats Analyzed by Massively Parallel Sequencing in Three U.S. Population Groups and Korean Population.

BackgroundRapidly mutating (RM) Y-chromosomal short tandem repeats (Y-STRs) have been demonstrated to increase the possibility of distinguishing between male relatives due to a higher mutation rate than conventional Y-STRs. Massively parallel sequencing (MPS) can be useful for forensic DNA typing as it allows the detection of sequence variants of many forensic markers. Here, we present sequence variations of 31 Y-STRs including nine RM Y-STRs (DYF387S1, DYF399S1, DYF404S1, DYS449, DYS518, DYS570, DYS576, DYS612, and DYS627), their frequencies, distribution, and the gain in the number of alleles using MPS.MethodsWe constructed a multiplex MPS assay capable of simultaneously amplifying 32 Y-chromosomal markers, producing amplicons ranging from 85–274 bp. Barcoded libraries from 220 unrelated males from four populations—African Americans, Caucasians, Hispanics, and Koreans—were generated via two-step polymerase chain reaction and sequenced on a MiSeq system. Genotype concordance between the capillary electrophoresis (CE) and MPS method and sequence variation of Y-STRs were investigated.ResultsIn total, 195 alleles were increased by MPS compared to CE-based alleles (261 to 456). The DYS518 marker showed the largest increase due to repeat region variation (a 3.69-fold increase). The highest increase in the number of alleles due to single nucleotide polymorphisms in the flanking region was found in DYF399S1. RM Y-STRs had more diverse sequences than conventional Y-STRs. Furthermore, null alleles were observed in DYS576 due to primer-binding site mutation, and allele drop-outs in DYS449 resulted from low marker coverage of less than the threshold.ConclusionThe results suggest that the expanded and discriminative MPS assay could provide more genetic information for Y-STRs, especially for RM Y-STRs, and could advance male individualization. Compiling sequence-based Y-STR data for worldwide populations would facilitate the application of MPS in the field of forensic genetics and could be applicable in solving male-related forensic cases.

Internal validation for analytical and stochastic thresholds in the GlobalFiler system

Interpretation of crime stain profiles is one of the challenging tasks of forensic scientists. Before using the probabilistic genotyping system, forensic scientists should interpret crime stain profiles using analytical and stochastic thresholds. According to the guidelines presented by the Scientific Working Group on DNA Analysis Methods, these thresholds shall be based on and supported by applicable internal validation studies. In this study, we performed the internal validation study of our DNA typing system to determine these thresholds. A total of 350 DNA samples and 11 negative controls were amplified using GlobalFilerTM PCR Amplification Kit with 30 cycles and PCR products were then analyzed on a SeqStudio Genetic Analyzer. As a result, the analytical threshold was set to 110 RFU, which has high sensitivity to obtain almost full profiles of 0.063 ng DNA, and has high specificity not to detect most of the pull-up and drop-in peaks. The stochastic threshold was set to 890 RFU allowing a 1% probability of allelic drop-out based on logistic regression. These thresholds are useful for interpreting crime stain profiles in our GlobalFiler system.

Phylogeography and ecological differentiation of strictly Mediterranean taxa: the case of the Iberian endemic Odontites recordonii.

Ecological drivers for genetic differentiation in Mediterranean climates are still underexplored. We have used the strictly Mediterranean endemic Odontites recordonii as a model species to address this question. This species is one of the three Iberian representatives of the O. vernus group, which are morphologically similar. Thus, it was additionally necessary to clarify their phylogenetic relationships. We used amplified fragment length polymorphisms to reveal phylogenetic relationships within O. vernus group, and to reconstruct the phylogeographic patterns within O. recordonii. Additionally, ecological niche models were generated to detect refugia along the Quaternary climatic oscillations. And finally, alleles under natural selection were identified, and correlations between allele presences and environmental variables were calculated in order to shed light on the ecological drivers promoting differentiation. The three species from the O. vernus group were recovered as distinct species. Three genetic groups were found within O. recordonii and a putative refugium was detected for each one. Eighty-one alleles could be under diversifying selection, and 58 alleles showed significant correlations with environmental variables, especially with temperature and precipitation seasonality and summer drought. The three Iberian species of the O. vernus group are reciprocal monophyletic taxa. The three genetic groups of O. recordonii could have been restricted to narrow refugia during the Quaternary and displayed present distributions in accordance with bioclimatic conditions. Temperature and precipitation seasonality and the intensity of summer drought are definitory climatic parameters of Mediterranean-type climates, and they could have acted as drivers of genetic differentiation on O. recordonii.

Banana somaclonal variation assessed by Amplified Fragment Length Polymorphism profiles at early cycles of in vitro culture

Banana micropropagation for obtaining free-virus plants frequently provokes somaclonal variation that could increase useful genetic variability in this asexually propagated crop. Both exploring the cycle of in vitro culture in which somaclonal variation occurs and the amount of generated polymorphism, are necessary. In this work, preliminary results of somaclonal variation during early cycles of banana in vitro culture are reported. Four randomly selected regenerated plants from the fifth cycle and two samples from the mother plant were analyzed. A total of 36 AFLP primer combinations were assayed, and 24 of them produced amplicons varying among 50- 500 bp. The mother plant presented a total of 125 different amplicons while the regenerated plants jointly showed 131 different amplicons with a mean of 119.75 ± 3.97 per individual. High level of DNA polymorphism (24.43 %) was found among micropropagated plants and, additionally, the occurrence of somaclonal variation at earlier cycles was suggested by multivariate analysis of Principal Coordinates. In this study, somaclonal variation at early cycles of banana micropropagation was validated and the adequacy of AFLP technique to assess it at the molecular level was verified. The phenotypic effects of the detected somaclonal variations remain to be evaluated.

Molecular data confirms the existence of distinct lineages within Lumbricus friendi (Cognetti 1904) and related “friends”

Lumbricus friendi is a lumbricid earthworm mainly found in western Europe, considerably less well studied than its close relative L. terrestris and until now, has not been a subject of taxonomical debate. However, its disjointed geographical distribution suggesting striking similarities to the Celtic fringes and of the so-called “Lusitanian” species merits further investigation. Our aim was to examine the genetic variation of this species and other related taxa within the genus (L. terrestris, L. rubellus, L. rubellus friendoides and L. friendi bouchei) to test for the existence of cryptic lineages that might explain its current distribution. Using mitochondrial (16S and COI) and nuclear (Amplified Fragment Length Polymorphism, AFLP) markers, we provide the first genetic basis not only to support the recent claim that L. friendi bouchei should be elevated to species rank (L. bouchei Zicsi and Csuzdi 1999), but also to conclude that L. rubellus friendoides is a valid species too, deserving a new name (L. heracleus stat. nov.). In addition, the AFLP results indicated the existence of a high cryptic diversity within L. friendi populations, which lacked geographic structure, resulting in the French samples being genetically closer to those from Ireland and Wales than to the Spanish ones. Our findings further highlight the likelihood that L. friendi and L. bouchei might have been overlooked or confounded with L. terrestris/L. herculeus and question the reliability of L. friendi records reported in the literature and those deposited in museums and sequence libraries (we provide evidence that this seem to be the case with two examples). We therefore advocate for a better link between morphological diagnostic characters and molecular sequences and the taxonomical validation of museums’ collections and sequence repositories.

Amplified Fragment Length Polymorphism (AFLP) analysis in Cyamopsis spp. for Genetic Diversity Study

The aim of present work is to study genetic diversity and phylogenetic relationship among 13 guar varieties from different germplasm conservation centers of India. A total of 64 primer combinations were tested and 24 primer combinations producing best DNA fingerprints were selected for final analysis. The AFLP marker gives keen amplification with 1,671 polymorphic bands and 62 unique bands ranging from 55 bp to 350 bp. Maximum numbers of unique bands were produced with E-ACT/M-CAG primer combination. This analysis allows determining association with an undefined feature and helps in identification of these genotypes. The phylogenetic relationship was explained by a dendrogram generated with UPGMA method. These guar genotypes may be used to develop new varieties with significant improvement in quality and quantity of gum.

Seed Oil Quality and Cultivation of Sambucus williamsii Hance as a New Oil Crop.

Natural edible oil derived from wild non-cultivated oil crops contributed to human daily nutritional diversity and disease prevention. It was important to investigate the nutritional value of these oils and the feasibility of crop cultivation. The present study focused on the assessment of seed oil quality of Sambucus williamsii Hance (SWH) and its molecular breeding. Wild SWH seed oil was extracted by supercritical CO2 technology and the composition of the oil was determined by using gas chromatography mass spectrometry (GC-MS) analysis. The oil content of SWH seeds reaches around 40%. Its seed oil was found to be rich in unsaturated fatty acids, such as 24.24% of linolenic acid and 50.56% of linoleic acid, and vitamin E (25.92 mg kg−1). The cytotoxicity and heavy metal analysis showed SWH seed oil was safe for consumption. In addition, the SWH strains with excellent characteristics were screened out for cultivation according to genetic diversity and morphological analysis. Amplified fragment length polymorphism (AFLP) markers were used to evaluate the genetic diversity of 28 accessions of wild SWH seeds and 5 accessions were selected to cultivate. Among them, two strains of SWH (sample 3 and 6) with high yielding (275.7 and 266.8 area yield kg−1) were suitable for dense planting and could be used to establish the raw material forest of SWH seed oil. The results of this study indicated the potential of development of selected SWH as novel oil crops and their wide cultivation.

Clariant & Air Liquide present MegaZonE: Novel catalyst layering technology for enhanced methanol synthesis performance

The systematic evaluation of the molecular diversity encompassed in Aconitum kongboense L. inbreds or parental lines offers an efficient means of exploiting the heterosis in A. kongboense as well as for management of biodiversity. An excellent and novel DNA-based molecular, amplified fragment length polymorphisms (AFLPs) markers, was firstly used to analysis the genetic diversity in A. kongboense genotypes. Out of 256 primers screened, a total of ten combinations successfully produced scorable, clear, reproducible and relatively high polymorphism bands, 64.12% of which were polymorphic. The values of number of polymorphic loci (NPL), percentage of polymorphic loci (PPB), observed number of alleles (Na) and effective number of alleles (Ne) were highest in population 3 (NPL = 77, PPB = 68.75%, Na = 1.688 ± 0.466, Ne = 1.412 ± 0.397) and lowest in population 2 (NPL = 57, PPB = 50.89%, Na = 1.509 ± 0.502, Ne = 1.273 ± 0.340). In addition, Jaccard's similarity coefficients among different populations varied from 0.45 to 1.00 with an average of 0.55. The data collected will contribute to identification, rational exploitation and conservation of germplasms of A. kongboense, and potentially useful to aid its breeding.

Chromosome Doubling in Genetically Diverse Bilberry (Vaccinium myrtillus L.) Accessions and Evaluation of Tetraploids in Terms of Phenotype and Ability to Cross with Highbush Blueberry (V. corymbosum L.)

To expand the gene pool and introduce new traits to the tetraploid cultivars of Vaccinium corymbosum from wild diploid species V. myrtillus, it is necessary to double the chromosome number in diploid species in order to overcome a post zygotic crossing barrier and a strong triploid block, existing within the genus Vaccinium. Five genetically diverse bilberry genotypes were selected from 21 accessions taken from the breeding collection of the National Institute of Horticultural Research (Skierniewice, Poland) for this study. The bilberry genotypes were derived from the Polish locations of Bolimów Landscape Park, Budy Grabskie and forest complex Zwierzyniec (Łódź Province), and habitats in Norway. The selection of genotypes was made based on the analysis of amplified fragment length polymorphism (AFLP-PCR). Analysis of the Jaccard similarity indexes and the UPGMA method revealed that the examined accessions formed two main groups on the dendrogram. The first group consisted of accessions from Norway, while the second group agglomerated Polish accessions. A further two classes were distinguished in the Polish group: the first included accessions from Budy Grabskie and the second from Zwierzyniec, located ca. 9 km from Budy Grabskie. In order to obtain plant material for in vitro polyploidisation, in vitro shoot cultures of the selected accessions were initiated and multiplied. Both antimitotics used, colchicine and APM, induced tetraploids for all of the accessions. The obtained tetraploids were multiplied, rooted ex vitro and grown in a greenhouse and then in a field. The first flowering was observed in 1.5-year-old plants, either diploid or tetraploid. Diploids bloomed slightly earlier and more profusely than tetraploid plants. Compared to diploids, autotetraploids had significantly larger flowers by ca. 64% and larger pollen tetrads by ca. 35%. The germination capacity of pollen tetrads was high in tetraploids (87.8%), although slightly lower than in diploids (94.3%). After pollinating the flowers of three highbush blueberry cultivars with pollen from the bilberry tetraploid accession, J-4-4x, the plants formed fruits, some of which contained properly formed seeds. The effectiveness of interspecific crossing between V. corymbosum and tetraploid V. myrtillus, defined as the percentage of obtained seedlings in relation to the number of pollinated flowers, was highest (53.3%) in the blueberry ‘Liberty’, and lower in ‘Bluecrop’ and ‘Northland’, 14.8% and 10.0%, respectively. Before using the seedlings for further breeding, their hybridity will be confirmed by molecular markers and the phenotype will be evaluated.

Development of SSR Databases Available for Both NGS and Capillary Electrophoresis in Apple, Pear and Tea.

Developing new varieties in fruit and tea breeding programs is very costly and labor-intensive. Thus, establishing a variety discrimination system is important for protecting breeders’ rights and producers’ profits. Simple sequence repeat (SSR) databases that can be utilized for both next-generation sequencing (SSR-GBS) and polymerase chain reaction–capillary electrophoresis (PCR-CE) would be very useful in variety discrimination. In the present study, SSRs with tri-, tetra- and pentanucleotide repeats were examined in apple, pear and tea. Out of 37 SSRs that showed clear results in PCR-CE, 27 were suitable for SSR-GBS. Among the remaining markers, there was allele dropout for some markers that caused differences between the results of PCR-CE and SSR-GBS. For the selected 27 markers, the alleles detected by SSR-GBS were comparable to those detected by PCR-CE. Furthermore, we developed a computational pipeline for automated genotyping using SSR-GBS by setting a value “α” for each marker, a criterion whether a genotype is homozygous or heterozygous based on allele frequency. The set of 27 markers contains 10, 8 and 9 SSRs for apple, pear and tea, respectively, that are useful for both PCR-CE and SSR-GBS and suitable for automation. The databases help researchers discriminate varieties in various ways depending on sample size, markers and methods.