Potato miR828 Is Associated With Purple Tuber Skin and Flesh Color.

Anthocyanins are plant pigments responsible for the colors of many flowers, fruits and storage organs and have roles in abiotic and biotic stress resistance. Anthocyanins and polyphenols are bioactive compounds in plants including potato (Solanum tuberosum L.) which is the most important non-cereal crop in the world, cultivated for its tubers rich in starch and nutrients. The genetic regulation of the flavonoid biosynthetic pathway is relatively well known leading to the formation of anthocyanins. However, our knowledge of post-transcriptional regulation of anthocyanin biosynthesis is limited. There is increasing evidence that micro RNAs (miRNAs) and other small RNAs can regulate the expression level of key factors in anthocyanin production. In this study we have found strong associations between the high levels of miR828, TAS4 D4(-) and purple/red color of tuber skin and flesh. This was confirmed not only in different cultivars but in pigmented and non-pigmented sectors of the same tuber. Phytochemical analyses verified the levels of anthocyanins and polyphenols in different tissues. We showed that miR828 is able to direct cleavage of the RNA originating from Trans-acting siRNA gene 4 (TAS4) and initiate the production of phased small interfering RNAs (siRNAs) whose production depends on RNA-dependent RNA polymerase 6 (RDR6). MYB transcription factors were predicted as potential targets of miR828 and TAS4 D4(-) and their expression was characterized. MYB12 and R2R3-MYB genes showed decreased expression levels in purple skin and flesh in contrast with high levels of small RNAs in the same tissues. Moreover, we confirmed that R2R3-MYB and MYB-36284 are direct targets of the small RNAs. Overall, this study sheds light on the small RNA directed anthocyanin regulation in potato, which is an important member of the Solanaceae family.

Insight on Genes Affecting Tuber Development in Potato upon Potato spindle tuber viroid (PSTVd) Infection.

Potato (Solanum tuberosum L) is a natural host of Potato spindle tuber viroid (PSTVd) which can cause characteristic symptoms on developing plants including stunting phenotype and distortion of leaves and tubers. PSTVd is the type species of the family Pospiviroidae, and can replicate in the nucleus and move systemically throughout the plant. It is not well understood how the viroid can affect host genes for successful invasion and which genes show altered expression levels upon infection. Our primary focus in this study is the identification of genes which can affect tuber formation since viroid infection can strongly influence tuber development and especially tuber shape. In this study, we used a large-scale method to identify differentially expressed genes in potato. We have identified defence, stress and sugar metabolism related genes having altered expression levels upon infection. Additionally, hormone pathway related genes showed significant up- or down-regulation. DWARF1/DIMINUTO, Gibberellin 7-oxidase and BEL5 transcripts were identified and validated showing differential expression in viroid infected tissues. Our study suggests that gibberellin and brassinosteroid pathways have a possible role in tuber development upon PSTVd infection.

Variation in the interaction between alleles of HvAPETALA2 and microRNA172 determines the density of grains on the barley inflorescence.

Within the cereal grasses, variation in inflorescence architecture results in a conspicuous morphological diversity that in crop species influences the yield of cereal grains. Although significant progress has been made in identifying some of the genes underlying this variation in maize and rice, in the temperate cereals, a group that includes wheat, barley, and rye, only the dosage-dependent and highly pleiotropic Q locus in hexaploid wheat has been molecularly characterized. Here we show that the characteristic variation in the density of grains along the inflorescence, or spike, of modern cultivated barley (Hordeum vulgare) is largely the consequence of a perturbed interaction between microRNA172 and its corresponding binding site in the mRNA of an APELATA2 (AP2)-like transcription factor, HvAP2. We used genome-wide association and biparental mapping to identify HvAP2. By comparing inflorescence development and HvAP2 transcript abundance in an extreme dense-spike mutant and its nearly isogenic WT line, we show that HvAP2 turnover driven by microRNA 172 regulates the length of a critical developmental window that is required for elongation of the inflorescence internodes. Our data indicate that this heterochronic change, an altered timing of developmental events caused by specific temporal variation in the efficiency of HvAP2 turnover, leads to the striking differences in the size and shape of the barley spike.

INTERMEDIUM-C, a modifier of lateral spikelet fertility in barley, is an ortholog of the maize domestication gene TEOSINTE BRANCHED 1.

The domestication of cereals has involved common changes in morphological features, such as seed size, seed retention and modification of vegetative and inflorescence architecture that ultimately contributed to an increase in harvested yield. In barley, this process has resulted in two different cultivated types, two-rowed and six-rowed forms, both derived from the wild two-rowed ancestor, with archaeo-botanical evidence indicating the origin of six-rowed barley early in the domestication of the species, some 8,600-8,000 years ago. Variation at SIX-ROWED SPIKE 1 (VRS1) is sufficient to control this phenotype. However, phenotypes imposed by VRS1 alleles are modified by alleles at the INTERMEDIUM-C (INT-C) locus. Here we show that INT-C is an ortholog of the maize domestication gene TEOSINTE BRANCHED 1 (TB1) and identify 17 coding mutations in barley TB1 correlated with lateral spikelet fertility phenotypes.

Genetic dissection of barley morphology and development.

Since the early 20th century, barley (Hordeum vulgare) has been a model for investigating the effects of physical and chemical mutagens and for exploring the potential of mutation breeding in crop improvement. As a consequence, extensive and well-characterized collections of morphological and developmental mutants have been assembled that represent a valuable resource for exploring a wide range of complex and fundamental biological processes. We constructed a collection of 881 backcrossed lines containing mutant alleles that induce a majority of the morphological and developmental variation described in this species. After genotyping these lines with up to 3,072 single nucleotide polymorphisms, comparison to their recurrent parent defined the genetic location of 426 mutant alleles to chromosomal segments, each representing on average <3% of the barley genetic map. We show how the gene content in these segments can be predicted through conservation of synteny with model cereal genomes, providing a route to rapid gene identification.

Exploiting induced variation to dissect quantitative traits in barley.

The identification of genes underlying complex quantitative traits such as grain yield by means of conventional genetic analysis (positional cloning) requires the development of several large mapping populations. However, it is possible that phenotypically related, but more extreme, allelic variants generated by mutational studies could provide a means for more efficient cloning of QTLs (quantitative trait loci). In barley (Hordeum vulgare), with the development of high-throughput genome analysis tools, efficient genome-wide identification of genetic loci harbouring mutant alleles has recently become possible. Genotypic data from NILs (near-isogenic lines) that carry induced or natural variants of genes that control aspects of plant development can be compared with the location of QTLs to potentially identify candidate genes for development--related traits such as grain yield. As yield itself can be divided into a number of allometric component traits such as tillers per plant, kernels per spike and kernel size, mutant alleles that both affect these traits and are located within the confidence intervals for major yield QTLs may represent extreme variants of the underlying genes. In addition, the development of detailed comparative genomic models based on the alignment of a high-density barley gene map with the rice and sorghum physical maps, has enabled an informed prioritization of 'known function' genes as candidates for both QTLs and induced mutant genes.

Towards systems genetic analyses in barley: Integration of phenotypic, expression and genotype data into GeneNetwork.

BACKGROUND: A typical genetical genomics experiment results in four separate data sets; genotype, gene expression, higher-order phenotypic data and metadata that describe the protocols, processing and the array platform. Used in concert, these data sets provide the opportunity to perform genetic analysis at a systems level. Their predictive power is largely determined by the gene expression dataset where tens of millions of data points can be generated using currently available mRNA profiling technologies. Such large, multidimensional data sets often have value beyond that extracted during their initial analysis and interpretation, particularly if conducted on widely distributed reference genetic materials. Besides quality and scale, access to the data is of primary importance as accessibility potentially allows the extraction of considerable added value from the same primary dataset by the wider research community. Although the number of genetical genomics experiments in different plant species is rapidly increasing, none to date has been presented in a form that allows quick and efficient on-line testing for possible associations between genes, loci and traits of interest by an entire research community. DESCRIPTION: Using a reference population of 150 recombinant doubled haploid barley lines we generated novel phenotypic, mRNA abundance and SNP-based genotyping data sets, added them to a considerable volume of legacy trait data and entered them into the GeneNetwork http://www.genenetwork.org. GeneNetwork is a unified on-line analytical environment that enables the user to test genetic hypotheses about how component traits, such as mRNA abundance, may interact to condition more complex biological phenotypes (higher-order traits). Here we describe these barley data sets and demonstrate some of the functionalities GeneNetwork provides as an easily accessible and integrated analytical environment for exploring them. CONCLUSION: By integrating barley genotypic, phenotypic and mRNA abundance data sets directly within GeneNetwork's analytical environment we provide simple web access to the data for the research community. In this environment, a combination of correlation analysis and linkage mapping provides the potential to identify and substantiate gene targets for saturation mapping and positional cloning. By integrating datasets from an unsequenced crop plant (barley) in a database that has been designed for an animal model species (mouse) with a well established genome sequence, we prove the importance of the concept and practice of modular development and interoperability of software engineering for biological data sets.

Exploiting regulatory variation to identify genes underlying quantitative resistance to the wheat stem rust pathogen Puccinia graminis f. sp. tritici in barley.

We previously mapped mRNA transcript abundance traits (expression-QTL or eQTL) using the Barley1 Affymetrix array and 'whole plant' tissue from 139 progeny of the Steptoe x Morex (St/Mx) reference barley mapping population. Of the 22,840 probesets (genes) on the array, 15,987 reported transcript abundance signals that were suitable for eQTL analysis, and this revealed a genome-wide distribution of 23,738 significant eQTLs. Here we have explored the potential of using these mRNA abundance eQTL traits as surrogates for the identification of candidate genes underlying the interaction between barley and the wheat stem rust fungus Puccinia graminis f. sp. tritici. We re-analysed quantitative 'resistance phenotype' data collected on this population in 1990/1991 and identified six loci associated with barley's reaction to stem rust. One of these coincided with the major stem rust resistance locus Rpg1, that we had previously positionally cloned using this population. Correlation analysis between phenotype values for rust infection and mRNA abundance values reported by the 22,840 GeneChip probe sets placed Rpg1, which is on the Barley1 GeneChip, in the top five candidate genes for the major QTL on chromosome 7H corresponding to the location of Rpg1. A second co-located with the rpg4/Rpg5 stem rust resistance locus that has been mapped in a different population and the remaining four were novel. Correlation analyses identified candidate genes for the rpg4/Rpg5 locus on chromosome 5H. By combining our data with additional published mRNA profiling data sets, we identify a putative sensory transduction histidine kinase as a strong candidate for a novel resistance locus on chromosome 2H and compile candidate gene lists for the other three loci.