Identification and verification of seed development related miRNAs in kernel almond by small RNA sequencing and qPCR.

Many studies have investigated the role of miRNAs on the yield of various plants, but so far, no report is available on the identification and role of miRNAs in fruit and seed development of almonds. In this study, preliminary analysis by high-throughput sequencing of short RNAs of kernels from the crosses between almond cultivars 'Sefid' × 'Mamaee' (with small and large kernels, respectively) and 'Sefid' × 'P. orientalis' (with small kernels) showed that the expressions of several miRNAs such as Pdu-miR395a-3p, Pdu-miR8123-5p, Pdu-miR482f, Pdu-miR6285, and Pdu-miR396a were significantly different. These miRNAs targeted genes encoding different proteins such as NYFB-3, SPX1, PGSIP3 (GUX2), GH3.9, and BEN1. The result of RT-qPCR revealed that the expression of these genes showed significant differences between the crosses and developmental stages of the seeds, suggesting that these genes might be involved in controlling kernel size because the presence of these miRNAs had a negative effect on their target genes. Pollen source can influence kernel size by affecting hormonal signaling and metabolic pathways through related miRNAs, a phenomenon known as xenia.

A flowering inhibitor of the temperature-dependent pathway in Crocus sativus L.

Saffron is the world highest-priced spice because its production requires intensive hand labour. Reduce saffron production costs require containerised plant production under controlled conditions and expand the flowering period. Controlling the flowering process and identify the factors involved in saffron flowering is crucial to introduce technical improvements. The research carried out so far in saffron has allowed an extensive knowledge of the influence of temperature on the flower induction, but the molecular mechanisms controlling flowering induction processes are largely unknown. The present study is the first conducted to isolate and characterize a regulator gene of saffron floral induction the Short Vegetative Phase (SVP) gene, which represses the floral initiation genes in the temperature response pathway, which involved in saffron flower induction. The results obtained from both phylogenetic analysis and T-coffee alignment confirms that the isolated sequence belongs to the SVP gene clades of MADS-box gene family. Gene expression analysis in different developmental stages revealed the highest expression of SVP transcript (CsSVP) during the dormancy and the vegetative stages, but decrease when flower development initiated and it was the least in late September when flower primordia are developed. Furthermore, its expression increased in the apical bud when corms are storage at 9-10 ºC, thus inhibiting flower induction. Additionally, comparison of the CsSVP transcript in apical buds from big and small corms, differing in their flowering capacity, indicates that the CsSVP transcript is present only in vegetative buds. Taken together, these results suggested inhibitory role of the SVP gene.

The effect of drought stress on polyphenolic compounds and expression of flavonoid biosynthesis related genes in Achillea pachycephala Rech.f.

This study investigated the effect of drought stress on the amount of phenolic and flavonoid compounds as well as H2O2 and malondialdehyde (MDA) in Achillea pachycephala. The expression patterns of the key genes and their molecular mechanisms in the phenylpropanoid pathway (PAL, CHS, CHI, F3H, F3'H, F3'5'H, FLS) were also assessed during drought stress using quantitative real-time polymerase chain reaction (qRT-PCR). The samples were harvested at 0, 7, 14, 21 and 28 days after exposure to drought stress. High-performance liquid chromatography (HPLC) analysis was performed to determine the changes of phenolic and flavonoid compounds - chlorogenic acid, caffeic acid, rutin, luteolin-7-O-glycoside, 1,3-dicaffeoylquinic acid, apigenin-7-O-glycoside, luteolin, apigenin and kaempferol - during stress conditions. Concentrations of most of the compounds increased with increasing drought stress duration. Most of the phenolic acids continued to accumulate with increasing duration of stress, while flavonoids dramatically decreased at day 28 of stress. Chlorogenic acid was the most abundant phenolic acid (4.97 mg/100 g dry weight [DW]) at the beginning of the experiment, while it decreased at day 7 and increased again at day 21. However, different trends were observed for some flavonoids, such as luteolin and apigenin. At the beginning of stress treatment, high accumulation of free radicals (H2O2) and lipid peroxidation (MDA) led to elevated expression of most of the flavonoid genes. MDA increased from 22.66 to 43.28 µmol g-1 DW at day 28. CHS gene expression was elevated at day 7, while chi gene expression remained unchanged. At the end of the stress period, most of the flavonoid concentrations and expression of the relevant genes also increased. The results can facilitate selection of appropriate drought conditions to obtain the highest levels of flavonoids such as luteolin and apigenin and phenolic compounds such as chlorogenic acid for improved health benefits.

Expression pattern of salt tolerance-related genes in Aegilops cylindrica.

Aegilops cylindrica, a salt-tolerant gene pool of wheat, is a useful plant model for understanding mechanism of salt tolerance. A salt-tolerant USL26 and a salt-sensitive K44 genotypes of A. cylindrica, originating from Uremia Salt Lake shores in Northwest Iran and a non-saline Kurdestan province in West Iran, respectively, were identified based on screening evaluation and used for this work. The objective of the current study was to investigate the expression patterns of four genes related to ion homeostasis in this species. Under treatment of 400 mM NaCl, USL26 showed significantly higher root and shoot dry matter levels and K+ concentrations, together with lower Na+ concentrations than K44 genotype. A. cylindrica HKT1;5 (AecHKT1;5), SOS1 (AecSOS1), NHX1 (AecNHX1) and VP1 (AecVP1) were partially sequenced to design each gene specific primer. Quantitative real-time PCR showed a differential expression pattern of these genes between the two genotypes and between the root and shoot tissues. Expressions of AecHKT1;5 and AecSOS1 was greater in the roots than in the shoots of USL26 while AecNHX1 and AecVP1 were equally expressed in both tissues of USL26 and K44. The higher transcripts of AecHKT1;5 in the roots versus the shoots could explain both the lower Na+ in the shoots and the much lower Na+ and higher K+ concentrations in the roots/shoots of USL26 compared to K44. Therefore, the involvement of AecHKT1;5 in shoot-to-root handover of Na+ in possible combination with the exclusion of excessive Na+ from the root in the salt-tolerant genotype are suggested.

Proteomics analysis of Medicago truncatula response to infection by the phytopathogenic bacterium Ralstonia solanacearum points to jasmonate and salicylate defence pathways.

The infection of the model legume Medicago truncatula with Ralstonia solanacearum GMI1000 gives rise to bacterial wilt disease via colonisation of roots. The root and leaf responses to early infection (1 and 3 days post infection) were characterised to investigate the molecular mechanisms of plant resistance or susceptibility. A proteomics approach based on pools of susceptible and resistant recombinant inbred lines was used to specifically target the mechanisms for tolerance. Differential abundances were evidenced for proteins involved in defence (e.g., PR5, PR10, or Kunitz protease inhibitors) and signalling pathways (such as cyclophilin). R. solanacearum inoculation modifies expression levels of those genes, either in both genotypes (AOS1, LOX4, and proteinase inhibitors) or specifically in the resistant line (PR proteins). Exogenous application of salicylic acid (SA) enhanced tolerance to the bacteria, whereas methyl jasmonate (MeJA) enhanced short-term tolerance then promoted disease in the susceptible ecotype, suggesting that they may mediate defence responses. Conversely, proteomics-identified genes were also shown to be SA or MeJA responsive. This is the first description of differential response to R. solanacearum in M. truncatula. Our results suggest that root basal defence is activated at 1 dpi, together with the JA pathway. Specific resistance is then evidenced at three dpi, with the up-regulation of SA-dependent PR proteins.

Recombinant production, purification and characterization of vessel dilator in E. coli.

Vessel dilator is a 3.9-KDa potent anticancer peptide and a valuable candidate in the treatment of conditions such as congestive heart failure and acute renal failure amongst others. Here we report the recombinant production of vessel dilator in Escherichia coli. Three different synthetic ORF's dubbed VDI, VDII and VDIII, each encoding a trimmer of the vessel dilator peptide attached to a His tag sequence at their C- terminal, were synthesized and placed in pET21c expression vectors. The highest yield, following expression in E. coli BL21 (DE3), was recorded with VDII that carried the shortest fusion partner. Subsequent to the initial capture of the fusion protein by a Ni affinity column, the vessel dilator monomers were cleaved by trypsin treatment, and further purified to at least 90% homogeneity by anion exchange chromatography. De-novo sequencing and in vivo anticancer activity tests were used to verify the peptide sequence and its biological activity, respectively. The final yield was estimated to be approximately 15 mg of the purified vessel dilator per gram wet weight of the bacterial cells.

Molecular polymorphism in Pistacia vera L. using non-coding regions of chloroplast DNA.

The present study describes plastid DNA polymorphism and reports a comparative analysis of two non-coding cpDNA regions (trnC-trnD and atpB-rbcL) in pistachio. Seventeen different genotypes of domestic and wild pistachio from Iran, Syria, Turkey and America were sampled. Total genomic DNA was extracted and amplified with trnC-trnD and atpB-rbcL specific primers and then were sequenced. Phylogenetic relationships and depiction of phylogenetic trees were conducted. Cultivated genotypes of Pistacia vera were classified in a group regardless of their geographic location. P. vera was isolated from Sarakhs but they placed in the two close groups. Among cultivated genotypes, Jalab was separated from other cultivated genotypes. Pistacia Khinjuk was classified with Pistacia atlantica subsp. mutica. The findings confirm the common splitting hypothesis for commercial pistachio genotypes of the P. vera wild-type and also indicated the direct impact of Iranian genotypes in the evolutionary process of cultivated pistachios in other parts of the world. In conclusion it can be inferred that cultivated varieties of pistachio and P. vera var. sarakhs have the same origin, moreover genomic chloroplast could appropriately identify the interspecies relationships of pistachios.

Spectroscopic, biological, and molecular modeling studies on the interactions of [Fe(III)-meloxicam] with G-quadruplex DNA and investigation of its release from bovine serum albumin (BSA) nanoparticles.

The guanine-rich sequence, specifically in DNA, telomeric DNA, is a potential target of anticancer drugs. In this work, a mononuclear Fe(III) complex containing two meloxicam ligands was synthesized as a G-quadruplex stabilizer. The interaction between the Fe(III) complex and G-quadruplex with sequence of 5'-G3(T2AG3)3-3' (HTG21) was investigated using spectroscopic methods, molecular modeling, and polymerase chain reaction (PCR) assays. The spectroscopic methods of UV-vis, fluorescence, and circular dichroism showed that the metal complex can effectively induce and stabilize G-quadruplex structure in the G-rich 21-mer sequence. Also, the binding constant between the Fe(III) complex and G-quadruplex was measured by these methods and it was found to be 4.53(±0.30) × 10(5) M(-1)). The PCR stop assay indicated that the Fe(III) complex inhibits DNA amplification. The cell viability assay showed that the complex has significant antitumor activities against Hela cells. According to the UV-vis results, the interaction of the Fe(III) complex with duplex DNA is an order of magnitude lower than G-quadruplex. Furthermore, the release of the complex incorporated in bovine serum albumin nanoparticles was also investigated in physiological conditions. The release of the complex followed a bi-phasic release pattern with high and low releasing rates at the first and second phases, respectively. Also, in order to obtain the binding mode of the Fe(III) complex with G-quadruplex, molecular modeling was performed. The molecular docking results showed that the Fe(III) complex was docked to the end-stacked of the G-quadruplex with a π-π interaction, created between the meloxicam ligand and the guanine bases of the G-quadruplex.

Functional Analysis of a Pomegranate (Punica granatum L.) MYB Transcription Factor Involved in the Regulation of Anthocyanin Biosynthesis.

BACKGROUND: Pomegranate fruit (Punica granatum L.) is a rich source of anthocyanin pigments resulting in vibrant colours and anti-oxidant contents. Although the intensity and pattern of anthocyanin biosynthesis in fruit are strongly influenced by R2R3-MYB transcription factors, little is known about the regulation and role of MYB in anthocyanin pathway of pomegranate. OBJECTIVES: The present study was conducted to elucidate the relationship between the expression of MYB transcription factor and the anthocyanin accumulation during the colour development phase of pomegranate fruits. MATERIALS AND METHODS: In this work, R2R3-MYB transcription factor (PgMYB) was isolated and characterized from pomegranate skin through RACE-PCR. The expression of PgMYB gene was monitored in three distinct pomegranate accessions with distinctive skin colour and pattern by semi-quantitative RT-PCR. RESULTS: The results indicated a strong association between skin colour in mature pomegranate fruits with the PgMYB transcripts. The highest expression level of PgMYB gene was observed in Poost Siyah Yazd (dark purple skin) throughout the ripening process. Furthermore, comparison of PgMYB amino acid sequences with those of R2R3-MYB family in grapevine, eucalyptus, peach, cacao, populus and Arabidopsis demonstrated that this protein shares high similarity (75-85% amino acid identity) with their conserved MYB domain. Computational structure prediction of PgMYB showed that the three conserved amino acids (Asn, Lys and Lys) are present in the same position of the MYB domain. CONCLUSIONS: It is speculated that PgMYB gene influences the fruit colour and could be used to improve the accumula-tion of anthocyanin pigments in the pomegranate fruit.

Studying genetic variability of pomegranate (Punica granatum L.) based on chloroplast DNA and barcode genes.

Chloroplast DNA has been used extensively to analyze plant phylogenies at different taxonomic levels because of its size, organization and sequence conservation. In the present research, two chloroplastic regions, petA­psaJ, trnC­trnD and four DNA barcodes (trnH­psbA, ITS, rbcL, matK), were used to introduce suitable regions for the assessment of genetic diversity among P. granatum L. genotypes. Analysis of psbE­petL in petA­psaJ region revealed 1,300 nucleotides with 4.29 % genetic diversity among genotypes, while trnC­petN in trnC­trnD region showed 1.8 % genetic diversity. Therefore, despite the results obtained from the study of other plants, the trnC­trnD region had a low potential for the evaluation of diversity among pomegranate genotypes. Analysis of DNA barcodes in pomegranate showed that trnH­psbA (genetic diversity 2.91 %) provides the highest intra-species variation, followed by ITS (genetic diversity 0.44 %). Eighteen genotypes from different geographical origins of Iran were used to investigate psbE­petL and trnH­psbA potential as novel barcodes to determine genetic polymorphism and characterize pomegranate genotypes. The results suggested that two regions, psbE­petL and trnH­psbA, were more suitable for determining intra-species relationships of pomegranate.

Assessment of genetic diversity of Bermudagrass (Cynodon dactylon) using ISSR markers.

Bermudagrass (Cynodon spp.) is a major turfgrass for home lawns, public parks, golf courses and sport fields and is known to have originated in the Middle East. Morphological and physiological characteristics are not sufficient to differentiate some bermudagrass genotypes because the differences between them are often subtle and subjected to environmental influences. In this study, twenty seven bermudagrass accessions and introductions, mostly from different parts of Iran, were assayed by inter-simple sequence repeat (ISSR) markers to differentiate and explore their genetic relationships. Fourteen ISSR primers amplified 389 fragments of which 313 (80.5%) were polymorphic. The average polymorphism information content (PIC) was 0.328, which shows that the majority of primers are informative. Cluster analysis using the un-weighted paired group method with arithmetic average (UPGMA) method and Jaccard's similarity coefficient (r = 0.828) grouped the accessions into six main clusters according to some degree to geographical origin, their chromosome number and some morphological characteristics. It can be concluded that there exists a wide genetic base of bermudograss in Iran and that ISSR markers are effective in determining genetic diversity and relationships among them.

Mapping QTL for grain yield, yield components, and spike features in a doubled haploid population of bread wheat.

A doubled haploid (DH) population derived from a cross between the Japanese cultivar 'Fukuho-kumogi' and the Israeli wheat line 'Oligoculm' was used to map genome regions involved in the expression of grain yield, yield components, and spike features in wheat (Triticum aestivum L). A total of 371 markers (RAPD, SSR, RFLP, AFLP, and two morphological traits) were used to construct the linkage map that covered 4190 cM of wheat genome including 28 linkage groups. The results of composite interval mapping for all studied traits showed that some of the quantitative trait loci (QTL) were stable over experiments conducted in 2004 and 2005. The major QTL located in the Hair-Xpsp2999 interval on chromosome 1A controlled the expression of grains/spike (R(2) = 12.9% in 2004 and 22.4% in 2005), grain weight/spike (R(2) = 21.4% in 2004 and 15.8% in 2005), and spike number (R(2) = 15.6% in 2004 and 5.4% in 2005). The QTL for grain yield located on chromosomes 6A, 6B, and 6D totally accounted for 27.2% and 31.7% of total variation in this trait in 2004 and 2005, respectively. Alleles inherited from 'Oligoculm' increased the length of spikes and had decreasing effects on spike number. According to the data obtained in 2005, locus Xgwm261 was associated with a highly significant spike length QTL (R(2) = 42.33%) and also the major QTL for spikelet compactness (R(2) = 26.1%).

Meiotic behaviour of tetraploid wheats (Triticum turgidum L.) and their synthetic hexaploid wheat derivates influenced by meiotic restitution and heat stress.

Meiotic restitution is considered to be a common mechanism of polyploidization in plants and hence is one of the most important processes in plant speciation. Meiotic behaviour of plant chromosomes is influenced by both genetic and environmental factors. In this study, the meiotic behaviour of cereal crops was investigated, which includes tetraploid wheat genotypes (with and without the meiotic restitution trait) and their derivates (synthetic hexaploid wheats and a doubled haploid (DH) line), grown at two planting dates in the field. In addition, two local landraces of emmer wheat (Triticum turgidum ssp. dicoccum), one wheat cultivar (Chinese spring), one DH triticale cultivar (Eleanor) and one rye accession were included. Immature spikes of mid-autumn and end-winter sowing plants were collected in April and May 2008, respectively, fixed in Carnoy's solution and stained with hematoxylin. Pollen mother cells (PMCs) from anthers at different stages of meiotic process were analysed for their chromosomal behaviour and irregularities. Meiotic aberrations such as laggards, chromosome bridges, micronuclei, abnormal cytokines, chromatin pulling and meiotic restitution were observed and the studied genotypes were accordingly ranked as follows: triticale > synthetic hexaploid wheats > tetraploid wheats possessing meiotic restitution > tetraploid wheats lacking meiotic restitution > rye. The results indicated that the samples that had been planted in the autumn, thus experiencing an optimum temperature level at the flowering stage, exhibited less meiotic irregularities than winter planting samples that encountered heat stress at the flowering period.

Conversion of barley SNPs into PCR-based markers using dCAPS method.

Molecular genetic research relies heavily on the ability to detect polymorphisms in DNA. Single nucleotide polymorphisms (SNPs) are the most frequent form of DNA variation in the genome. In combination with a PCR assay, the corresponding SNP can be analyzed as a derived cleaved amplified polymorphic sequence (dCAPS) marker. The dCAPS method exploits the well-known specificity of a restriction endonuclease for its recognition site and can be used to virtually detect any SNP. Here, we describe the use of the dCAPS method for detecting single-nucleotide changes by means of a barley EST, CK569932, PCR-based marker.

Conversion of barley SNPs into PCR-based markers using dCAPS method

Molecular genetic research relies heavily on the ability to detect polymorphisms in DNA. Single nucleotide polymorphisms (SNPs) are the most frequent form of DNA variation in the genome. In combination with a PCR assay, the corresponding SNP can be analyzed as a derived cleaved amplified polymorphic sequence (dCAPS) marker. The dCAPS method exploits the well-known specificity of a restriction endonuclease for its recognition site and can be used to virtually detect any SNP. Here, we describe the use of the dCAPS method for detecting single-nucleotide changes by means of a barley EST, CK569932, PCR-based marker.

AFLP targeting of the 1-cM region conferring the vrs1 gene for six-rowed spike in barley, Hordeum vulgare L.

Spike morphology is a key characteristic in the study of barley domestication, yield, and use. Multiple alleles at the vrs1 locus control the development and fertility of the lateral spikelets of barley. We developed five amplified fragment length polymorphism (AFLP) markers tightly linked to the vrs1 locus using well-characterized near-isogenic lines as plant materials. The AFLP markers were integrated into three different maps, in which 'Azumamugi' was used as the maternal parent. Of the three maps, Hordeum vulgare L. 'Azumamugi' x H. vulgare 'Golden Promise' showed recombination of the AFLP markers and the vrs1 locus (closest, 0.05 cM), providing the best mapping population for positional cloning of alleles at the vrs1 locus. Conversion of AFLP bands into polymorphic sequence-tagged sites (STSs) is necessary for further high-throughput genotype scoring and for bacterial artificial chromosome (BAC) library screening. We cloned and sequenced the five AFLP bands and synthesized primer pairs. PCR amplification generated DNAs of the same size from all four parental lines for each marker. Restriction endonuclease treatment of e40m36-1110/AccIII, e34m13-260/Psp1406I, e52m32-270/FokI, and e31m26-520/MnlI revealed fragment length polymorphisms between 'Azumamugi' and all the two-rowed parents. Allelism between the AFLPs and corresponding STS markers was confirmed genetically, indicating the usefulness of the STSs as genetic markers.