Pasquale Piccirillo

A PCR based SNPs marker for specific characterization of English walnut (Juglans regia L.) cultivars

English walnut (Juglans regia L.) is the most economically important species from all the 21 species belonging to the genus Juglans and is an important and healthy food as well as base material for timber industry. The aim of this study was to develop a simple technique for specific characterization of English walnut using DNA method. The first and second internal transcribed spacers (ITS1 and ITS2) as well as the intervening 5.8S coding region of the rRNA gene for 18 cultivars of J. regia L. isolated from different geographic origins were characterized. The size of the spacers sequences ranged from 257 to 263 bases for ITS1 and from 217 to 219 bases for ITS2. Variation of GC contents has also been observed and scored as 55–56.7 and 57.1–58.9% for ITS1 and ITS2, respectively. This data exhibited the presence of polymorphism among cultivars. Alignment of the ITS1-5.8S-ITS2 sequences from 18 walnut cultivars showed that there were 244 single nucleotide polymorphisms (SNPs) and 1 short insertion–deletion (indel) at 5′ end ITS1. Amplification refractory mutation system strategy was successfully applied to the SNP markers of the ITS1 and ITS2 sequences for the fingerprinting analysis of 17 on 18 walnut cultivars. The prediction of ITS1 and ITS2 RNA secondary structure from each cultivar was improved by detecting key functional elements shared by all sequences in the alignments. Phylogenetic analysis of the ITS1-5.8S-ITS2 region clearly separated the isolated sequences into two clusters. The results showed that ITS1 and ITS2 region could be used to discriminate these walnut cultivars.

Analysis of different European hazelnut (Corylus avellana L.) cultivars: authentication, phenotypic features, and phenolic profiles.

Hazelnuts exhibit functional properties due to their content in fatty acids and phenolic compounds that could positively affect human health. The food industry requires precise traits for morphological, chemical, and physical kernel features so that some cultivars could be more suitable for specific industrial processing. In this study, agronomical and morphological features of 29 hazelnut cultivars were evaluated and a detailed structural characterization of kernel polyphenols was performed, confirming the presence of protocatechuic acid, flavan-3-ols such as catechin, procyanidin B2, six procyanidin oligomers, flavonols, and one dihydrochalcone in all the analyzed cultivars. In addition, an innovative methodology based on the MALDI-TOF mass spectrometric analysis of peptide/protein components extracted from kernels was developed for the authentication of the most valuable cultivars. The proposed method is rapid, simple, and reliable and holds the potential to be applied in quality control processes. These results could be useful in hazelnut cultivar evaluation and choice for growers, breeders, and food industry.

Use of Nuclear and Mitochondrial Single Nucleotide Polymorphisms to Characterize English Walnut ( Juglans regia L.) Genotypes

English walnut (Juglans regia L.) is the most economically important species, for both food and timber, of the 21 species belonging to the genus Juglans. This study was undertaken to analyze and compare DNA sequences of the mitochondrial cytochrome oxidase subunit II (COX2) and ribosomal DNA (rDNA) genes in the molecular characterization of 30 English walnut genotypes. rDNA sequences revealed the presence of 402 variations, including 101 in 3′ ends of 18S, 21 in internal transcribed spacer 1(ITS1), 170 in ITS2, 30 in 5.8S, and 80 in 5′ ends of 28S regions. Cox2 intron I sequences showed 769 variable positions and GG insertion/deletion at 3′ end regions. Based on single nucleotide polymorphism markers of rDNA and cox2 intron I sequences, an amplification refractory mutation system was used to fingerprint 18 out of 30 walnut genotypes. The findings revealed that the cox2 intron I region, either alone or in conjunction with rDNA, could be used effectively in identifying these walnut genotypes.

Transcription Factors and Environmental Stresses in Plants

Plants are exposed to environmental changes, which are perceived as stresses when they are quick and extreme. Drought, salt, and extreme temperatures, in particular, limit agricultural crop productivity, affecting all stages of plant growth and reproduction, and therefore strongly decreasing crop yield. Worldwide estimates show that most yield loss (70%) can be directly due to abiotic stresses. Moreover, the increasing phenomenon of enthronization and the incorrect use of agricultural land have strongly contributed to land degradation. A large number of abiotic stress-responsive genes have been reported in a variety of plants including Arabidopsis and major crops such as barley, maize, rice, and wheat. Transcriptional control of the expression of these genes is a crucial part of plant response to abiotic stresses. Therefore, recently the transcriptional mechanisms involved in the response to several abiotic stresses have been the subject of intense research, which have been productive in identifying transcription factors (TFs) as important “key or master regulators” of gene expression under stress. An increasing number of TFs have been recently described and essential transcription factor binding regions have been identified for many genes. In fact, these systems of regulation work, thanks to specific cis-elements located in the promoter regions of target genes, which are called regulons. The main regulons that respond to abiotic stresses are DREB1-CBF (dehydration-responsive element binding protein 1/C-repeat binding factor), which is involved in the cold stress response, and DREB2, which acts in ABA-independent gene expression for response to heat and osmotic stress, whereas the ABA-responsive element (ABRE) binding protein (AREB)/ABRE binding factor (ABF) regulon operates in gene expression depending on ABA under osmotic stress. Other regulons, such as MYB/MYC and NAC, induce or repress the expression of genes involved in abiotic stress response. In the last few years, several studies have shown that TFs are powerful tools to engineer enhanced stress tolerance in plants. Therefore, in this chapter, we will summarize the major TFs involved in crop plants’ abiotic stress signaling and responses, and the relative plants’ adaptive mechanisms at the molecular level. A major finding on molecular mechanisms that occur in stress conditions is the one-way pass for the improvement of stress tolerance in crop plants.