Plant Molecular Genetics and Functional Genomics

We have investigated the regulation by N-acetyl-glucosamine of the nag1 gene of the mycoparasitic biocontrol fungus Trichoderma atroviride (=T. harzianum P1), which encodes a 73-kDa N-acetyl-β-D-glucosaminidase. The use of translational fusions revealed that a 290-bp fragment of the 5′ regulatory region of nag1 is sufficient to confer inducibility on the Aspergillus niger goxA gene. The region between positions –150 and –290, upstream of the nag1 coding region, was investigated using in vivo methylation protection analysis and electrophoretic mobility shift assays (EMSAs). Two neighbouring regions that interacted with regulatory proteins were identified, and bases essential for these interactions were determined in vitro. These data reveal protein binding to a CCCCT element at –240, a CCAGN13CTGG motif at –284, and a CCAAT-box which is present in the spacer of the latter motif. Evidence for the binding of a Hap2/3/5 complex to this CCAAT motif is presented. Protein binding to all three motifs was constitutive, and no differences were observed between induced and non-induced cultures. Mutation of either the CCAGN13CTGG or the AGGGG motif resulted in loss of inducibility of nag1 expression by N-acetyl-D-glucosamine in vivo.

Biotechnology in the Agriculture sector covers indigenous plant and animal breeding to the choice of individuals and hybridization including technically complicated techniques of genetic engineering. The beneficiaries are farmers, manufacturers and consumers. Ethiopia has been regarded as one among the ancient countries of the world and also richer in natural resources and wide diversity in the crop has been recorded. Plant biotechnology portrays immense capacity for manipulation of crops to conclude with the use of least inputs, increased yield and productivity. It is a smart choice and very much necessary for the introduction of latest biotechnological tools to Ethiopian agricultural sector. Careful examination of crop varieties of Ethiopia through the usage of modern tools and techniques of biotechnology could certainly assist in unraveling genes that harness traits of colossal economic significance. Quite a few research and academic institutes of Ethiopia are conducting basic, advanced and related fields of research in biotechnology with limitations in tools, techniques and management. This review intends to update the information on status of research, opportunities and challenges in plant biotechnology and it will help the researchers, academicians, industrialists and students to know about the current status of Ethiopian plant biotechnology. Besides, it will be helpful for the policy maker to guide them further for plans and developments.

In silico derived properties on experimental validation revealed that hypothetical protein Alr2954 of Anabaena sp. PCC7120 is ADP-ribose pyrophosphatase, which belongs to nudix hydrolase superfamily. Presence of ADP-ribose binding site was attested by ADP-ribose pyrophosphatase activity (K m 44.71 ± 8.043 mM, V max 7.128 ± 0.417 μmol min −1 mg protein −1 , and K cat /K m 9.438 × 10 4 μM − 1 min −1). Besides ADP-ribose, the enzyme efficiently hydrolyzed various nucleoside phosphatases such as 8-oxo-dGDP, 8-oxo-dADP, 8-oxo-dGTP, 8-oxo-dATP, GDP-mannose, ADP-glucose, and NADH. qRT-PCR analysis of alr2954 showed significant expression under different abiotic stresses reconfirming its role in stress tolerance. Thus, Alr2954 qualifies to be a member of nudix hydrolase superfamily, which serves as ADP-ribose pyrophosphatase and assists in multiple abiotic stress tolerance.

Plant-parasitic nematodes secrete effector proteins to direct the endogenous molecular and physiological pathways of their hosts for their own benefit. The development of unique and highly specialized feeding cells within host roots is an important part of the infection process leading to a successful parasitic association. Yet, we still have a limited understanding of the exact mechanisms contributing to their differentiation. However, in the last decade, holistic molecular biology techniques, such as microarrays, combined with techniques for the isolation of RNA from particular plant cells, such as laser microdissection and microaspiration, have provided a detailed microanalysis of the transcriptional changes occurring in syncytia and giant-cells at early differentiation stages. These studies have revealed a dramatic dilution of feeding cell transcripts in excised infected root fragments uncovering important differences in transcriptional regulation in the feeding cells as compared to their surrounding tissues. The number of plant species analyzed is still limited. Thus, it is anticipated that next generation sequencing and proteomic analysis could open the possibility for cross species comparisons to identify conserved regulated genes and early protein changes in developing feeding cells. This knowledge is expected to be of value for its application to nematode control.

Bouea merupakan anggota dari famili Anacardiaceae yang terdistribusi luas di daerah Malesia. Malesia merupakan kawasan yang terbentang antara benua Asia dan Australia mencakup Filipina, Brunei, Papua Nugini, Indonesia, Malaysia, dan Singapura. Tanaman ini memiliki variasi fenotip karena keragaman genetik dan adaptasi di berbagai ragam kondisi lingkungan. Tujuan dari penelitian ini adalah mengklasifikasikan Bouea macrophylla Griffit berdasarkan variasi genetik menggunakan DNA kloroplas. Daerah TrnL-F intergenic spacer merupakan daerah nonkoding pada DNA kloroplas yang dapat digunakan sebagai penanda untuk melihat variasi genetik intraspesifik. Sampel diperoleh dari 5 tempat di Indonesia yaitu Ambon, Banten, Aceh, Sumatera Barat, dan Kalimantan Barat. Isolasi DNA menggunakan metode Doyle dan Doyle yang dimodifikasi, kemudian diamplifikasi menggunakan primer trnL-F forward dan primer trnL-F reverse. Produk PCR dengan panjang fragmen 350-400 bp kemudian disekuensing di 1st Base Singapura. Data hasil sekuensing dianalisis menggunakan software MEGA 6.06 untuk membangun pohon filogenetik menggunakan out group Mangifera indica dan Anacardium occidentale. Hasil dari peneltian ini menunjukkan bahwa daerah trnL-F pada DNA kloroplas adalah daerah dengan konservatif yang tinggi. Bouea macrophylla Griffit memiliki kedekatan dengan Anacardium occidentael. Analisis pohon filogenetik dengan menggunakan Software Mega 6.06 menunjukkan bahwa konsistensi spesies tersebut dari nenek moyang yang sama dan memisah dari spesies Mangifera indica dan Anacaardium occidentale sebagai out group.

The Y chromosome is one of the best genetic materials to explore the evolutionary history of human populations. Global analyses of Y chromosomal short tandem repeats (STRs) data can reveal very interesting world population structures and histories. However, previous Y-STR works tended to focus on small geographical ranges or only included limited sample sizes. In this study, we have investigated population structure and demographic history using 17 Y chromosomal STRs data of 979 males from 44 worldwide populations. The largest genetic distances have been observed between pairs of African and non-African populations. American populations with the lowest genetic diversities also showed large genetic distances and coancestry coefficients with other populations, whereas Eurasian populations displayed close genetic affinities. African populations tend to have the oldest time to the most recent common ancestors (TMRCAs), the largest effective population sizes and the earliest expansion times, whereas the American, Siberian, Melanesian, and isolated Atayal populations have the most recent TMRCAs and expansion times, and the smallest effective population sizes. This clear geographic pattern is well consistent with serial founder model for the origin of populations outside Africa. The Y-STR dataset presented here provides the most detailed view of worldwide population structure and human male demographic history, and additionally will be of great benefit to future forensic applications and population genetic studies.

The plant RNase T2 family is divided into two different subfamilies. S-RNases are involved in rejection of self-pollen during the establishment of self-incompatibility in three plant families. S-like RNases, on the other hand, are not involved in self-incompatibility, and although gene expression studies point to a role in plant defense and phosphate recycling, their biological roles are less well understood. Although S-RNases have been subjects of many phylogenetic studies, few have included an extensive analysis of S-like RNases, and genome-wide analyses to determine the number of S-like RNases in fully sequenced plant genomes are missing. We characterized the eight RNase T2 genes present in the Oryza sativa genome; and we also identified the full complement of RNase T2 genes present in other fully sequenced plant genomes. Phylogenetics and gene expression analyses identified two classes among the S-like RNase subfamily. Class I genes show tissue specificity and stress regulation. Inactivation of RNase activity has occurred repeatedly throughout evolution. On the other hand, Class II seems to have conserved more ancestral characteristics; and, unlike other S-like RNases, genes in this class are conserved in all plant species analyzed and most are constitutively expressed. Our results suggest that gene duplication resulted in high diversification of Class I genes. Many of these genes are differentially expressed in response to stress, and we propose that protein characteristics, such as the increase in basic residues can have a defense role independent of RNase activity. On the other hand, constitutive expression and phylogenetic conservation suggest that Class II S-like RNases may have a housekeeping role.

Aluminum toxicity is the main factor limiting the productivity of crop plants in acid soils, particularly in the tropics and subtropics. In this study, a doubled-haploid population derived from the rice (Oryza sativa L.) breeding lines CT9993 and IR62266 was used to map genes controlling Al tolerance. A genetic linkage map consisting of 280 DNA markers (RFLP, AFLP and SSR) was constructed to determine the position and nature of quantitative trait loci (QTLs) affecting Al tolerance. Three characters – control root length (CRL), Al-stressed root length (SRL) and root length ratio (RR) – were evaluated for the DH lines and the parents at the seedling stage in nutrient solution. A total of 20 QTLs controlling root growth under Al stress and control conditions were detected and distributed over 10 of the 12 rice chromosomes, reflecting multigenic control of these traits. The two QTLs of largest effect, qALRR-1-1 and qALRR-8 for root length ratio (a measurement of Al tolerance) were localized on chromosomes 1 and 8, respectively. Three other QTLs in addition to qALRR-8 were apparently unique in the CT9993 × IR62266 mapping population, which may explain the high level of Al tolerance in CT9993. Comparative mapping identified a conserved genomic region on chromosome 1 associated with Al tolerance across three rice genetic backgrounds. This region provides an important starting point for isolating genes responsible for different mechanisms of aluminum tolerance and understanding the genetic nature of this trait in rice and other cereals.

Aging is one of the chief biomedical problems of the 21st century. After decades of basic research on biogerontology
(the science of aging), the aging process still remains an enigma. Although hundreds of "theories" on aging have
been formulated and many fundamental insights about age-related changes and genetic as well as environmental
interventions that change the pace of aging have been discovered, the actual why and how we age remain enigmatic.
In the post-genomic era there is an exponential increase in data. As a consequence it is a challenge to utilize all
information based on it and derive meaningful knowledge about biological phenomena. No individual scientist, no
group, nor consortium is capable of keeping up within their own field and are overwhelmed by the explosion of data
increase. Machine learning applied on biological data has the potential to solve this and cause a paradigm shift from
hypothesis-driven research (which predominates biological research including biogerontology) to data-driven
research.
This dissertation addresses this problem. In particular it proposes and executes the use of machine learning on
current existing data to predict drivers of aging (and therefore helps to distinguish causes from consequences),
interventions to counteract aging, and specific hypotheses to fill in research gaps that require experimental
validation.
The objective of this project is therefore to build computational models that are based on data relevant to the
phenomenon of aging and to predict as many of its aspects and dimensions as possible (thus elucidate their relations
to each other). For converting between and sorting within dimensions which are relevant to aging, different machine
learning models are evaluated. Ones models are built, it can be determined how much they can explain different
aspects of aging. Those models will also be capable of specifying which features are most relevant for prediction (in
both classification or regression). It is possible to train models that incorporate age-related changes based on
transcriptomic, proteomic, metabolomic, epigenomic as well as morphological data and their combinations. Machine
learning is further used to convert between and within them.
This work focuses on three types of predictors. Subsequently, discoveries are made with the statistical and learning
algorithms. The first model (lifespan predictor) is trained on predicting the lifespan based on genotype, environment
and combinations thereof. It is useful for predicting lifespan extending interventions on the population level. The
second model (age predictor) is trained on predicting the age given features measured on individuals. This is useful
for identifying biomarkers of aging and to determine the effects of interventions on the level of individuals. The third
model predicts functions/regulations of biological entities in regard to the aging process based on heterogeneous data
such as ontologies and diverse omics including time-series gene expression profiles (which can be visualized as
plots), and linked data. It is used to understand the role of genes and proteins as well as perhaps other entities such as
small molecules including lipids and other metabolites. Functions of proteins, which are still unknown, especially
those involved in yeast lipid metabolism and its regulation, can be predicted.
For this purpose we use primarily yeast as model organism as well as data on humans. Other biomedical model
organisms might be added if found beneficial.
The novel aspects of this research are for instance that 1) aging is investigated systematically in an unbiased
data-driven approach, 2) lifespan is predicted as continuous values, 3) age is predicted by combining multiple omics
data, 4) functions and regulations of biological entities like genes are predicted with high confidence from
heterogeneous data sources.
This thesis discovered that genetics is the most important feature of lifespan determination. Phenotypic features
related to lipid and membranes such as vacuolar morphology and autophagy activity are important for lifespan
determination according to the best performing models. A age predictor based on transcriptomics and proteomics can
highly accurately determine the age. It selected features are associated with both translation and lipid metabolism.
Among the top selected features are transcripts of genes when deleted exhibit abnormal vacuolar morphology as well
as targets of Opi1. Opi1 itself and its regulators were found to be differentially regulated post-transcriptional or
post-translational. Lastly, a function predictor for genes was created that achieved exceptional accuracy of
classifying aging genes. It learned for instance that piecemeal autophagy of the nucleus is strongly predictive for
aging-suppressor genes while cytoplasmic translation is strongly predictive for gerontogenes

The maxicircle control region [also termed divergent region (DR)] composed of various repeat elements remains the most poorly studied part of the kinetoplast genome. Only three extensive DR sequences demonstrating no significant similarity were available for trypanosomatids (Leishmania tarentolae, Crithidia oncopelti, Trypanosoma brucei). Recently, extensive DR sequences have been obtained for Leishmania major and Trypanosoma cruzi. In this work we have sequenced DR fragments of Leishmania turanica, Leishmania mexicana, Leishmania chagasi and two monogenetic trypanosomatids Leptomonas seymouri and Leptomonas collosoma. With the emergence of the additional extensive sequences some conserved features of DR structure become evident. A conserved palindromic sequence has been revealed in the DRs of the studied Leishmania species, L. seymouri, and T. cruzi. The overall DR structure appears to be similar in all the Leishmania species, their relative L. seymouri, and T. brucei: long relatively GC-rich repeats are interspersed with clusters of short AT-rich repeats. C. oncopelti, L. collosoma, and T. cruzi have a completely different DR structure. Identification of conserved sequences and invariable structural features of the DR may further our understanding of the functioning of this important genome fragment.

There is an immediate need for a high-density genetic map of cotton anchored with fiber genes to facilitate marker-assisted selection (MAS) for improved fiber traits. With this goal in mind, genetic mapping with a new set of microsatellite markers [comprising both simple (SSR) and complex (CSR) sequence repeat markers] was performed on 183 recombinant inbred lines (RILs) developed from the progeny of the interspecific cross Gossypium hirsutum L. cv. TM1 × Gossypium barbadense L. Pima 3-79. Microsatellite markers were developed using 1557 ESTs-containing SSRs (≥10 bp) and 5794 EST-containing CSRs (≥12 bp) obtained from ~14,000 consensus sequences derived from fiber ESTs generated from the cultivated diploid species Gossypium arboreum L. cv AKA8401. From a total of 1232 EST-derived SSR (MUSS) and CSR (MUCS) primer-pairs, 1019 (83%) successfully amplified PCR products from a survey panel of six Gossypium species; 202 (19.8%) were polymorphic between the G. hirsutum L. and G. barbadense L. parents of the interspecific mapping population. Among these polymorphic markers, only 86 (42.6%) showed significant sequence homology to annotated genes with known function. The chromosomal locations of 36 microsatellites were associated with 14 chromosomes and/or 13 chromosome arms of the cotton genome by hypoaneuploid deficiency analysis, enabling us to assign genetic linkage groups (LG) to specific chromosomes. The resulting genetic map consists of 193 loci, including 121 new fiber loci not previously mapped. These fiber loci were mapped to 19 chromosomes and 11 LG spanning 1277 cM, providing approximately 27% genome coverage. Preliminary quantitative trait loci analysis suggested that chromosomes 2, 3, 15, and 18 may harbor genes for traits related to fiber quality. These new PCR-based microsatellite markers derived from cotton fiber ESTs will facilitate the development of a high-resolution integrated genetic map of cotton for structural and functional study of fiber genes and MAS of genes that enhance fiber quality.

We applied ancient DNA methods to shed light on the origin of ancient Hungarians and their relation to modern populations. Hungarians moved into the Carpathian Basin from the Eurasian Pontic steppes in the year 895 AD as a confederation of seven tribes, but their further origin remains obscure. Here, we present 17 mtDNA haplotypes and four Y-chromosome haplogroups, which portray the genetic composition of an entire small cemetery of the first generation Hungarians. Using novel algorithms to compare these mitochondrial DNA haplogroups with other ancient and modern Eurasian data, we revealed that a significant portion of the Hungarians probably originated from a long ago consolidated gene pool in Central Asia-South Siberia, which still persists in modern Hungarians. Another genetic layer of the early Hungarians was obtained during their westward migrations by admixing with various populations of European origin, and an important component of these was derived from the Caucasus region....

More than 2,000 genome-wide barley single nucleotide polymorphisms (SNPs) were developed by resequencing unigene fragments from eight diverse accessions. The average genome-wide SNP frequency observed in 877 unigenes was 1 SNP per 200 bp. However, SNP frequency was highly variable with the least number of SNP and SNP haplotypes observed within European cultivated germplasm reflecting effects of breeding history on genetic diversity. More than 300 SNP loci were mapped genetically in three experimental mapping populations which allowed the construction of an integrated SNP map incorporating a large number of RFLP, AFLP and SSR markers (1,237 loci in total). The genes used for SNP discovery were selected based on their transcriptional response to a variety of abiotic stresses. A set of known barley abiotic stress QTL was positioned on the linkage map, while the available sequence and gene expression information facilitated the identification of genes potentially associated with these traits. Comparison of the sequenced SNP loci to the rice genome sequence identified several regions of highly conserved gene order providing a framework for marker saturation in barley genomic regions of interest. The integration of genome-wide SNP and expression data with available genetic and phenotypic information will facilitate the identification of gene function in barley and other non-model organisms.

As a new anticancer treatment option, vaccinia virus (VACV) has shown remarkable antitumor activities (oncolysis) in preclinical studies, but potential infection of other organs remains a safety concern. We present here genome comparisons between the de novo sequence of GLV-1h68, a recombinant VACV, and other VACVs. The identified differences in open reading frames (ORFs) include genes encoding host-range selection, virulence and immune modulation proteins, e.g., ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. Phylogenetic analyses indicate that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. The reduced pathogenicity of GLV-1h68 is confirmed in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. The contribution of foreign gene expression cassettes in the F14.5L, J2R and A56R loci is analyzed, in particular the contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence of GLV-1h68 with its F14.5L-null and revertant viruses. GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism.

The genetics and biochemistry of anthocyanins and flavonol biosynthesis and their role in plant organ pigmentation is well established in model species. However, the genetic basis of colour variation is species specific and understanding this variation is very relevant in many fruit and flower crop species. Among grape cultivars, there is a wide genetic variation for berry colour ranging from yellow-green (“white” cultivars) to dark blue berries. Berry colour results from the synthesis and accumulation of anthocyanins in the berry skin, which in plants is commonly regulated by transcription factors belonging to the MYB and bHLH families. In this work, we aimed to identify the major genetic determinants of berry colour variation in a large collection of table grape cultivars and somatic variants. The genetic analyses of berry colour in a few grape segregating progenies had previously identified a single locus on linkage group 2 responsible for colour variation. Furthermore, somatic variation for berry skin colour in cultivar Italia had been associated with the presence of a Gret1 retrotransposon in the promoter region of VvmybA1, a Myb gene whose expression is associated to skin colouration. The results show that VvmybA1 is the gene underlying the mapped locus controlling berry colour in grape. Additionally, the molecular analyses indicate that genetic and somatic berry colour variation can be associated to molecular variation at VvmybA1 in more than 95% of the analyzed cultivars. Thus, VvmybA1 is a major determinant of berry colour variation in table grape and its instability is the major cause of somatic variation for this trait.

The review is devoted to genetic mechanisms of regulation of plant embryonic development. Numerous families of genes
identified to date that control this phase of plant ontogenesis are presented in detail; their key role in the formation and
development of plant seed is described. Data concerning important role of different classes of phytohormones such as
auxins, cytokinins, gibberellic acid, brassinosteroids, abscisic acid, ethylene and jasmonic acid in the regulation of plant
growth and development during embryogenesis are resulted.
Keywords: Plant Seed; Plant Embryonic Genes; Differentiation and Specialization of Embryonic Cells; Seed Growth and
Development; Plant Hormones; Auxins; Cytokinins; Gibberellic Acid; Brassinosteroids; Abscisic Acid; Ethylene; Jasmonic
Acid; Developmental and Phytohormonal Regulation of Plant Embryonic Genes.

We investigated how over-expression of a cDNA for human ornithine decarboxylase (odc) affects the polyamine pools in transgenic rice. We further investigated tissue-specific expression patterns and product accumulation levels of the transgene driven by either constitutive or seed-specific promoters. Our results indicate that: (1) whereas the expression of a heterologous arginine decarboxylase (adc) cDNA in rice resulted in increased putrescine