De Novo Transcriptome Assembly and SNP Discovery for the Development of dCAPS Markers in Oat

Cultivated oat (Avena sativa L.) is an important cereal crop that has captured interest worldwide due to its nutritional properties and associated health benefits. Despite this interest, oat has lagged behind other cereal crops in genome studies and the development of DNA markers due to its large and complex genome. RNA-Seq technology has been widely used for transcriptome analysis, functional gene study, and DNA marker development. In this study, we performed the transcriptome sequencing of 10 oat varieties at the seedling stage using the Illumina platform for the development of DNA markers. In total, 31,187,392~41,304,176 trimmed reads (an average of 34,322,925) were generated from 10 oat varieties. All of the trimmed reads of these varieties were assembled and generated, yielding a total of 128,244 assembled unigenes with an average length of 1071.7 bp and N50 of 1752 bp. According to gene ontology (GO) analysis, 30.7% of unigenes were assigned to the “catalytic activity” of the parent term in the molecular function category. Of the 1273 dCAPS markers developed using 491 genotype-specific SNPs, 30 markers exhibiting polymorphism in 28 oat varieties were finally selected. The transcriptome data of oat varieties could be used for functional studies about the seedling stage of oat and information about sequence variations in DNA marker development. These 30 dCAPS markers will be utilized for oat genetic analysis, cultivar identification, and breeders’ rights protection.

Real-time Screening of Foods Using Repetitive Element PCR Reveals a DNA Marker Characteristic for Enterotoxigenic Bacillus Species

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables, and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic, including not just food-associated gastrointestinal toxicoinfections, but human endophthalmitis as well. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Repetitive element PCR was used to compare the banding patterns of each sample against B. cereus ATCC 14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe and were capable of causing gastroenteritis in consumers.

Designing a Genetic Screen to Identify Factors Required for Meiotic Nuclear Rejuvenation

During the natural cycle of life, most eukaryotic organisms grow old, age, and die. A common natural mechanism by which organisms “reset” their lifespan is through sexual reproduction; however, how this rejuvenation takes place remains unknown. My lab has found that meiosis in budding yeast, the developmental program that forms sex cells, eliminates age-induced damage. This involves the formation of a novel nuclear compartment, the Gametogenesis Uninherited Nuclear Compartment (GUNC), which acts as a trash can for accumulated age-induced damage. To understand the molecular details of this process, I worked on designing a screen for genes involved in GUNC formation. My mentor and I fused three different proteins targeted to the GUNC and a protein that is able to bind to a drug-resistance plasmid, in order to couple the inheritance of a selectable DNA marker with the elimination of age-induced damage. Initial testing of these three fusion proteins suggested that they were unable to successfully target the plasmid to the GUNC; as such, testing of additional candidate proteins is necessary. We plan to eventually use this system to identify mutations that disrupt GUNC formation and cause inheritance of the drug-resistance plasmid. By identifying and perturbing proteins involved in GUNC formation, we are hoping to be able to drive the inheritance of specific types of age-induced damage, allowing for the determination of what a symptom versus a cause of aging is.

The Evolution of Wahlenbergia (Campanulaceae) in Australasia

<p>Wahlenbergia is a large genus of flowering plants within the family Campanulaceae. In this thesis the first molecular phylogeny of Wahlenbergia was reconstructed from approximately 20% of the genus, based on the nuclear ribosomal ITS (nrITS) DNA marker and the chloroplast trnL-F DNA marker, with samples from South Africa, Europe, Australia and New Zealand. Additionally a large phylogeny with increased within-species sampling focusing on addressing taxonomic questions among the 45 Australasian species of Wahlenbergia was also reconstructed based on nrITS and trnL-F, plus an additional chloroplast DNA marker, trnK. Relationships and species limits of the New Zealand species of Wahlenbergia were further analysed using amplified fragment length polymorphisms (AFLPs). Wahlenbergia was found to be polyphyletic, though most of the species form a clade. Tree topologies and molecular dating analysis showed that the genus originated in South Africa about 16.2 million years ago (mya), then dispersed to Australasia before radiating there about 3.7 mya, thus refuting the hypothesis of Gondwanan vicariance for the Australasian species. Two dispersals from Australia to New Zealand are hypothesised, one leading to a radiation of species with the rhizomatous growth from about 1.0 mya and the other leading to a radiation of species with the radicate growth form 0.49 mya, although the radicate species might not form a clade. Low levels of genetic variation among individuals from Australia and New Zealand was revealed with all markers, and the phylogenies were poorly resolved as a result. The low genetic diversity is probably due to rapid and recent evolution during a period of geological and climatic change, coupled with incomplete lineage sorting and hybridisation. Phylogenies reconstructed using AFLPs were also poorly resolved, although AFLPs were found to be useful for species delimitation, as has been shown in studies of other plant groups. Despite the poor resolution, several morphological species and subspecies were recovered as monophyletic with DNA sequence data, notably the morphologically distinctive New Zealand W. cartilaginea, W. matthewsii and W. congesta subsp. congesta. Further research into species boundaries within the W. albomarginata/W. pygmaea complex is needed. Members of the New Zealand lowland radicate W. gracilis complex may all belong to the same morphologically variable species, although further research is needed to justify such a taxonomic change. The other New Zealand radicate species, W. vernicosa, is probably a separately evolving lineage, and is not conspecific with the Australian W. littoricola.</p>

The Evolution of Wahlenbergia (Campanulaceae) in Australasia

<p>Wahlenbergia is a large genus of flowering plants within the family Campanulaceae. In this thesis the first molecular phylogeny of Wahlenbergia was reconstructed from approximately 20% of the genus, based on the nuclear ribosomal ITS (nrITS) DNA marker and the chloroplast trnL-F DNA marker, with samples from South Africa, Europe, Australia and New Zealand. Additionally a large phylogeny with increased within-species sampling focusing on addressing taxonomic questions among the 45 Australasian species of Wahlenbergia was also reconstructed based on nrITS and trnL-F, plus an additional chloroplast DNA marker, trnK. Relationships and species limits of the New Zealand species of Wahlenbergia were further analysed using amplified fragment length polymorphisms (AFLPs). Wahlenbergia was found to be polyphyletic, though most of the species form a clade. Tree topologies and molecular dating analysis showed that the genus originated in South Africa about 16.2 million years ago (mya), then dispersed to Australasia before radiating there about 3.7 mya, thus refuting the hypothesis of Gondwanan vicariance for the Australasian species. Two dispersals from Australia to New Zealand are hypothesised, one leading to a radiation of species with the rhizomatous growth from about 1.0 mya and the other leading to a radiation of species with the radicate growth form 0.49 mya, although the radicate species might not form a clade. Low levels of genetic variation among individuals from Australia and New Zealand was revealed with all markers, and the phylogenies were poorly resolved as a result. The low genetic diversity is probably due to rapid and recent evolution during a period of geological and climatic change, coupled with incomplete lineage sorting and hybridisation. Phylogenies reconstructed using AFLPs were also poorly resolved, although AFLPs were found to be useful for species delimitation, as has been shown in studies of other plant groups. Despite the poor resolution, several morphological species and subspecies were recovered as monophyletic with DNA sequence data, notably the morphologically distinctive New Zealand W. cartilaginea, W. matthewsii and W. congesta subsp. congesta. Further research into species boundaries within the W. albomarginata/W. pygmaea complex is needed. Members of the New Zealand lowland radicate W. gracilis complex may all belong to the same morphologically variable species, although further research is needed to justify such a taxonomic change. The other New Zealand radicate species, W. vernicosa, is probably a separately evolving lineage, and is not conspecific with the Australian W. littoricola.</p>

Genotyping of potato samples from the GenAgro ICG SB RAS collection using DNA markers of genes conferring resistance to phytopathogens

Wart (a disease caused by Synchytrium endobioticum) and golden cyst potato nematode (Globodera rostochiensis), which parasitize the roots of the host plant, cause significant damage to potato crop. Both of these disease factors are quarantined in the Russian Federation, and each registered variety is tested for resistance to their most common races and pathotypes. The main method of opposing such diseases is by the development of resistant varieties. An important step in this process is the selection of resistant genotypes from the population and the estimation of the resistance of hybrids obtained by crosses during the breeding process. Conducting a permanent phenotypic evaluation is associated with difficulties, for example, it is not always possible to work with pathogens, and phenotypic evaluation is very costly and time consuming. However, the use of DNA markers linked to resistance genes can significantly speed up and reduce the cost of the breeding process. The aim of the study was to screen the GenAgro potato collection of ICG SB RAS using known diagnostic PCR markers linked to golden potato cyst nematode and wart resistance. Genotyping was carried out on 73 potato samples using three DNA markers 57R, CP113, Gro1-4 associated with nematode resistance and one marker, NL25, associated with wart resistance. The genotyping data were compared with the data on the resistance of the collection samples. Only the 57R marker had a high level of correlation (Spearman R = 0.722008, p = 0.000000, p < 0.05) between resistance and the presence of a diagnostic fragment. The diagnostic efficiency of the 57R marker was 86.11 %. This marker can be successfully used for screening a collection, searching for resistant genotypes and marker-assisted selection. The other markers showed a low correlation between the presence of the DNA marker and resistance. The diagnostic efficiency of the CP113 marker was only 44.44 %. Spearman’s correlation coefficient (Spearman R = –0.109218, p = 0.361104, p < 0.05) did not show significant correlation between resistance and the DNA marker. The diagnostic efficiency of the NL25 marker was 61.11 %. No significant correlation was found between the NL25 marker and resistance (Spearman R = –0.017946, p = 0.881061, p < 0.05). The use of these markers for the search for resistant samples is not advisable.

Construction of DNA ladder for determination of small size DNA fragments

DNA marker is commonly used to determine the size of DNA fragments by electrophoresis in routine molecular biology laboratories. In this study, we report a new procedure to prepare recombinant plasmids pSY-60 which was partially digested by one restriction enzyme for generating DNA markers of 7 fragments from 60 to 420 bp. The procedure included a synthesis of 60 bp DNA fragment with EcoRI sites at both ends using PCR extension, self-ligation of the 60 bp fragments and subcloning the ligated product into plasmid, generating recombinant pSY-60. Once being cloned, 500 ng of 420 bp fragment purified from 100 µL PCR product was incompletely digested by EcoRI, sufficiently producing to 50 acrylamide gels. Our procedure for production of DNA markers could be simple, time saving and inexpensive in comparison with current ones widely used in most laboratories.