Nicholas Kibet Korir

Deep sequencing discovery of novel and conserved microRNAs in trifoliate orange (Citrus trifoliata)

BackgroundMicroRNAs (miRNAs) play a critical role in post-transcriptional gene regulation and have been shown to control many genes involved in various biological and metabolic processes. There have been extensive studies to discover miRNAs and analyze their functions in model plant species, such as Arabidopsis and rice. Deep sequencing technologies have facilitated identification of species-specific or lowly expressed as well as conserved or highly expressed miRNAs in plants.ResultsIn this research, we used Solexa sequencing to discover new microRNAs in trifoliate orange (Citrus trifoliata) which is an important rootstock of citrus. A total of 13,106,753 reads representing 4,876,395 distinct sequences were obtained from a short RNA library generated from small RNA extracted from C. trifoliata flower and fruit tissues. Based on sequence similarity and hairpin structure prediction, we found that 156,639 reads representing 63 sequences from 42 highly conserved miRNA families, have perfect matches to known miRNAs. We also identified 10 novel miRNA candidates whose precursors were all potentially generated from citrus ESTs. In addition, five miRNA* sequences were also sequenced. These sequences had not been earlier described in other plant species and accumulation of the 10 novel miRNAs were confirmed by qRT-PCR analysis. Potential target genes were predicted for most conserved and novel miRNAs. Moreover, four target genes including one encoding IRX12 copper ion binding/oxidoreductase and three genes encoding NB-LRR disease resistance protein have been experimentally verified by detection of the miRNA-mediated mRNA cleavage in C. trifoliata.ConclusionDeep sequencing of short RNAs from C. trifoliata flowers and fruits identified 10 new potential miRNAs and 42 highly conserved miRNA families, indicating that specific miRNAs exist in C. trifoliata. These results show that regulatory miRNAs exist in agronomically important trifoliate orange and may play an important role in citrus growth, development, and response to disease.

Genome-wide Analysis of the AP2/ERF Superfamily in Peach (Prunus persica)

We identified 131 AP2/ERF (APETALA2/ethylene-responsive factor) genes in material from peach using the gene sequences of AP2/ERF amino acids of Arabidopsis thaliana (Brassicaceae) as probes. Based on the number of AP2/ERF domains and individual gene characteristics, the AP2/ERF superfamily gene in peach can be classified broadly into three families, ERF (ethylene-responsive factor), RAV (related to ABI3/VP1), and AP2 (APETALA2), containing 104, 5, and 21 members, respectively, along with a solo gene (ppa005376m). The 104 genes in the ERF family were further divided into 11 groups based on the group classification made for Arabidopsis. The scaffold localizations of the AP2/ERF genes indicated that 129 AP2/ERF genes were all located on scaffolds 1 to 8, except for two genes, which were on scaffolds 17 and 10. Although the primary structure varied among AP2/ERF superfamily proteins, their tertiary structures were similar. Most ERF family genes have no introns, while members of the AP2 family have more introns than genes in the ERF and RAV families. All sequences of AP2 family genes were disrupted by introns into several segments of varying sizes. The expression of the AP2/ERF superfamily genes was highest in the mesocarp; it was far higher than in the other seven tissues that we examined, implying that AP2/ERF superfamily genes play an important role in fruit growth and development in the peach. These results will be useful for selecting candidate genes from specific subgroups for functional analysis.

Plant variety and cultivar identification: advances and prospects

Plant variety and cultivar identification is one of the most important aspects in agricultural systems. The large number of varieties or landraces among crop plants has made it difficult to identify and characterize varieties solely on the basis of morphological characters because they are non stable and originate due to environmental and climatic conditions, and therefore phenotypic plasticity is an outcome of adaptation. To mitigate this, scientists have developed and employed molecular markers, statistical tests and software to identify and characterize the required plant cultivars or varieties for cultivation, breeding programs as well as for cultivar-right-protection. The establishment of genome and transcriptome sequencing projects for many crops has led to generation of a huge wealth of sequence information that could find much use in identification of plants and their varieties. We review the current status of plant variety and cultivar identification, where an attempt has been made to describe the different strategies available for plant identification. We have found that despite the availability of methods and suitable markers for a wide range of crops, there is dearth of simple ways of making both morphological descriptors and molecular markers easy, referable and practical to use although there are ongoing attempts at making this possible. Certain limitations present a number of challenges for the development and utilization of modern scientific methods in variety or cultivar identification in many important crops.

Characterization of grapevine microR164 and its target genes.

MicroRNAs (miRNAs) are an extensive class of newly identified small RNAs that regulate gene expression at post-transcription level by mRNA cleavage or translation. In our study, we used qRT-PCR and found that Vv-miR164 is expression in grapevine leaves, stems, tendrils, inflorescences, flowers and fruits. In addition, two potential target genes for Vv-miR164 were also found and verified by PPM-RACE and RLM-RACE. The results not only maps the cleavage site of the target mRNA but allowed for detection the expression pattern of cleaved fragments that can indicate the regulatory function of this miRNA on its target genes. These target genes were explored by qRT-PCR where some exhibited different expression patterns from their corresponding miRNA, indicating the cleavage mode of the miRNA on its target genes. The efficient and powerful approach used in this study can help in further understanding of how miRNAs cleaved their target mRNAs. Results from this study prove the importance of Vv-miR164 in regulating development and growth of grapes, and adds to the existing knowledge of small RNA-mediated regulation in grapes.

Cloning and expression of genes related to the sucrose-metabolizing enzymes and carbohydrate changes in peach

To shed light on the relationship between sucrose metabolism and expression of genes related to sucrose-metabolizing enzymes, six genes encoding sucrose-metabolizing enzymes were isolated, and the levels of four main carbohydrates and related enzyme activities as well as the expression of these six genes were determined in fruits, leaves and phloem-enriched fraction throughout peach fruit development. Sucrose content in mature fruit ranked first followed by glucose, fructose and sorbitol in that order, while sorbitol was the highest and sucrose lowest in phloem-enriched fraction and leaves. Glucose and fructose had similar change patterns throughout fruit development. Cloning results reveal that the nucleotide sequences of the six genes have high similarity to corresponding genes isolated from other plants. In addition, the expression of these genes and the levels of related enzyme activities varied with tissue and stage of fruit development, suggesting a complexity in relationships between carbohydrates, enzymes activities and related gene expression. Sucrose phosphate synthase maybe a key enzyme involved in sucrose synthesis while sucrose synthase may mainly be responsible for sucrose synthesis in peach fruits at later stages of development. Further studies are needed to genetically and physiologically characterize these genes and enzymes in peach and to gain a better understanding of their functions and relationship with carbohydrate metabolism.

Genome-wide analysis of the homeodomain-leucine zipper (HD-ZIP) gene family in peach (Prunus persica).

In this study, 33 homeodomain-leucine zipper (HD-ZIP) genes were identified in peach using the HD-ZIP amino acid sequences of Arabidopsis thaliana as a probe. Based on the phylogenetic analysis and the individual gene or protein characteristics, the HD-ZIP gene family in peach can be classified into 4 subfamilies, HD-ZIP I, II, III, and IV, containing 14, 7, 4, and 8 members, respectively. The most closely related peach HD-ZIP members within the same subfamilies shared very similar gene structure in terms of either intron/exon numbers or lengths. Almost all members of the same subfamily shared common motif compositions, thereby implying that the HD-ZIP proteins within the same subfamily may have functional similarity. The 33 peach HD-ZIP genes were distributed across scaffolds 1 to 7. Although the primary structure varied among HD-ZIP family proteins, their tertiary structures were similar. The results from this study will be useful in selecting candidate genes from specific subfamilies for functional analysis.

Identification of Known and Novel microRNAs and Their Targets in Peach (Prunus persica) Fruit by High-Throughput Sequencing

MicroRNAs (miRNAs) are a group of non-coding RNAs that have functions in post-transcriptional gene regulation in plants. Although the most important economic component of peach trees (Prunus persica) is the fruit, not much is known about miRNAs in this organ. In this study, miRNAs and their targets were identified and characterized from libraries of small RNAs of peach fruit through Solexa based-sequencing and bioinformatics approaches. A total of 557 known peach miRNAs belonging to 34 miRNA families were identified, and some of these miRNAs were found to be highly conserved in at least four other plant species. Using the most current criteria for miRNA annotation, 275 putative novel miRNAs were predicted, and the sequencing frequencies of these novel miRNAs were less than those of the conserved miRNAs. In total, 3959 and 1614 target genes for 349 known and 193 novel miRNAs, respectively, were predicted with the criteria that a single target gene can be targeted by different miRNAs and that a single miRNA can also have a large number of target genes. Three targets were even found to be targeted by 13 novel miRNAs that contained the same complete miRNA sequence at different locations and had different scaffolds. The proteins predicted to be targeted by the miRNAs identified in this study encompass a wide range of transcription factors and are involved in many biological processes and pathways, including development, metabolism, stress responses and signal transduction. A total of 115 and 101 target genes were identified to be cleaved by 60 known miRNAs and 27 novel miRNAs through degradome sequencing, respectively. These miRNAs induce cleavage of their targets precisely at the position between nucleotides 10 and 11 of the miRNA sequences from the 5’ to the 3’ end. Thirty conserved miRNAs and 19 novel miRNAs exhibited differential expression profiles in the peach, and the expression patterns of some miRNAs appeared to be tissue- or developmental stage-specific. The findings of this study provide an important basis for the analysis of miRNAs, their targets and the functions of these targets in peach fruit.

Advances in identification and validation of plant microRNAs and their target genes.

Developments in the field of molecular biology and genetics, such as microarray, gene transfer and discovery of small regulatory RNAs, have led to significant advances in plant biotechnology. Among the small RNAs, microRNAs (miRNAs) have elicited much interest as key post-transcriptional regulators in eukaryotic gene expression. Advances in genome and transcriptome sequencing of plants have facilitated the generation of a huge wealth of sequence information that can find much use in the discovery of novel miRNAs and their target genes. In this review, we present an overview of the developments in the strategies and methods used to identify and study miRNAs, their target genes and the mechanisms by which these miRNAs interact with their target genes since the discovery of the first miRNA. The approaches discussed include both reverse and forward genetics. We observed that despite the availability of advanced methods, certain limitations ranging from the cost of materials, equipment and personnel to the availability of genome sequences for many plant species present a number of challenges for the development and utilization of modern scientific methods for the elucidation and development of miRNAs in many important plant species.

Characterization and expression profiling of selected microRNAs in tomato (Solanum lycopersicon) ‘Jiangshu14’

Presence of selected tomato (Solanum lycopersicon) microRNAs (sly-miRNAs) was validated and their expression profiles established in roots, stems, leaves, flowers and fruits of tomato variety Jiangshu14 by quantitative RT-PCR (qRT-PCR). In addition conservation characteristics these sly-miRNAs were analyzed and target genes predicted bioinformatically. Results indicate that some of these miRNAs are specific to tomato while most are conserved in other plant species. Predicted sly-miRNA targets genes were shown to be targeted by either by a single or more miRNAs and are involved in diverse processes in tomato plant growth and development. All the 36 miRNAs were present in the cDNA of mixed tissues and qRT-PCR revealed that some of these sly-miRNAs are ubiquitous in tomato while others have tissue-specific expression. The experimental validation and expression profiling as well target gene prediction of these miRNAs in tomato as done in this study can add to the knowledge on the important roles played by these sly-miRNAs in the growth and development, environmental stress tolerance as well as pest and disease resistance in tomatoes and related species. In addition these findings broaden the knowledge of small RNA-mediated regulation in S. lycopersicon. It is recommended that experimental validation of the target genes be done so as to give a much more comprehensive information package on these miRNAs in tomato and specifically in the selected variety.

Whole genome identification and analysis of FK506-binding protein family genes in grapevine (Vitis vinifera L.).

In plant and animal species FK506-binding protein (FKBP) family genes are important conserved genes and it is defined as the receptors of FK506 and rapamycin, where they work as PPIase and protein folding chaperones. FKBP have been isolated from Arabidopsis thaliana, Oryza sativa, and Zea mays. In grape, twenty-three genes containing the FK506-binding domain (FKBP_C) were first time identified by HMMER and blast research, they were classified into three groups and 17 out of the 23 genes were located on 11 chromosomes (Chr1, 3, 5, 7, 8, 14, 15, 16, 17, 18, and 19). The predicted gene expression pattern and semi-quantitative RT-PCR results revealed that five VvFKBPs were expressed in all tissues, while seven VvFKBPs were expressed only in some of the tissues, and the remaining VvFKBPs were not expressed in leaf, stem, inflorescences, flowers, and a mixture of fruit tissues (small, medium and big-sized fruits). Most of the VvFKBPs in grapevine ‘Summer Black’ were similar to those predicted one in ‘Pinot Noir’ except for VvFKBP16-4 and VvFKBPa. VvFKBP12, FaFKBP12 and PpFKBP12 were cloned from ‘Summer Black’, ‘Sweet Charlie’ and ‘Xiahui 6’. Protein structure analysis confirmed that homologous genes have some differences during the process of protein structure construction. In this study, we characterized and verified 23 FKBP family genes in grapevine (Vitis vinifera L.) as well as their sub-cellular and chromosome location. The successful cloning of CDS regions and protein structural analysis of VvFKBP12, FaFKBP12, and PpFKBP12 can provide useful information for further study.