Current advances in genomics and breeding of leaf chicory (Cichorium intybus L.)

This review gives an overview of agricultural topics on a non-model species, in other words, leaf chicory. Often classified as a minor crop, \u201cRadicchio\u201d, the Italian name of leaf chicory, is assuming a very important role at both a local and national level, as it characterizes a high proportion of the agricultural income of suited areas. Botanical classification along the genus Cichorium is reported and a detailed description of the most important cultivated biotypes typical of northern Italy is presented. A special consideration is reserved to breeding aspects, from molecular marker-assisted selection to the implementation of the first genome draft and leaf transcriptomes. Sexual barriers, for example, self-incompatibility or male-sterility, are described in great detail with the aim to be utilized for breeding purposes. The main aspects of seed production are also critically presented. In conclusion, the present work is a sort of handbook to better understand this orphan crop and it is mainly directed to breeders and seed producers dealing with leaf chicory

DNA barcoding as a molecular tool to track down mislabeling and food piracy

DNA barcoding is a molecular technology that allows the identification of any biological species by amplifying, sequencing and querying the information from genic and/or intergenic standardized target regions belonging to the extranuclear genomes. Although these sequences represent a small fraction of the total DNA of a cell, both chloroplast and mitochondrial barcodes chosen for identifying plant and animal species, respectively, have shown sufficient nucleotide diversity to assess the taxonomic identity of the vast majority of organisms used in agriculture. Consequently, cpDNA and mtDNA barcoding protocols are being used more and more in the food industry and food supply chains for food labeling, not only to support food safety but also to uncover food piracy in freshly commercialized and technologically processed products. Since the extranuclear genomes are present in many copies within each cell, this technology is being more easily exploited to recover information even in degraded samples or transformed materials deriving from crop varieties and livestock species. The strong standardization that characterizes protocols used worldwide for DNA barcoding makes this technology particularly suitable for routine analyses required by agencies to safeguard food safety and quality. Here we conduct a critical review of the potentials of DNA barcoding for food labeling along with the main findings in the area of food piracy, with particular reference to agrifood and livestock foodstuffs

cpDNA barcoding by combined End-Point and Real-Time PCR analyses to identify and quantify the main contaminants of oregano (Origanum vulgare L.) in commercial batches

Oregano (Origanum vulgare L.) is a flowering plant that belongs to the mint family (Lamiaceae). It is used as a culinary herb and is often commercialized as a fine powder or a mixture of small fragments of dried leaves, which makes morphological recognition difficult. Like other commercial preparations of drugs and spices, the contamination of oregano mixtures with vegetable matter of lower quality, or the use of generic misleading names, are frequent and stress the need to develop a molecular traceability system to easily, quickly, and cheaply unveil these scams. The DNA-based analytical approach known as cpDNA barcoding is particularly suited for fraud identification in crop plant species (fresh products and food derivatives), and it represents a promising traceability tool as an alternative or complement to traditional detection methods. In the present study, we used a combined approach based on both qualitative and quantitative cpDNA barcoding with end-point and real-time polymerase chain reaction (PCR) analyses to assess the type and degree of contamination in commercial batches of common oregano. In a preliminary qualitative screening, we amplified, cloned, and sequenced a number of universal trnH-psbA- and trnL-barcoded regions, to identify the main contaminants in the samples under investigation. On the basis of these findings, we then developed and validated a species-specific and sequence-targeted method of testing for the quantitative assessment of contaminants, using trnL gene intron assays. Surprisingly, the results obtained in our case study indicated an almost total absence of O. vulgare in the commercial batches analyzed, but a high presence of group I contaminants (Satureja pilosa Velen.), and a moderate presence of group II contaminants (Cistus lanidifer L./Cistus albidus)

Le nuove vie del miglioramento genetico delle piante agroalimentari: dalle tecnologie di breeding cisgenico a quelle di editing genomico.

Advances in genome sequencing have led to the rapid development of new biotechnologies for the genetic improvement of crop plants. Among the new biotechnological tools, the most promising are cisgenic breeding and genomic editing, which have great potentials for the intensification of sustainable agriculture systems and the implementation of food security, in response to the growing population and the climate change that we are witnessing. Contrary to what happens with transgenesis, the plant genetically modified by these new technologies has the peculiarity of not having exogenous coding DNA and therefore it is not distinguishable from plants improved using traditional plant breeding schemes. Our goal is to explain how these new bio- technologies work, highlighting the potential applications and drawbacks, compared to traditional plant breeding techniques

Genomics of flower identity in grapevine (Vitis vinifera L.)

The identity of the four characteristic whorls of typical eudicots, namely, sepals, petals, stamens, and carpels, is specified by the overlapping action of homeotic genes, whose single and combined contributions have been described in detail in the so-called ABCDE model. Continuous species-specific refinements and translations resulted in this model providing the basis for understanding the genetic and molecular mechanisms of flower development in model organisms, such as Arabidopsis thaliana and other main plant species. Although grapevine (Vitis vinifera L.) represents an extremely important cultivated fruit crop globally, studies related to the genetic determinism of flower development are still rare, probably because of the limited interest in sexual reproduction in a plant that is predominantly propagated asexually. Nonetheless, several studies have identified and functionally characterized some ABCDE orthologs in grapevine. The present study is intended to provide a comprehensive screenshot of the transcriptional behavior of 18 representative grapevine ABCDE genes encoding MADS-box transcription factors in a developmental kinetic process, from preanthesis to the postfertilization stage and in different flower organs, namely, the calyx, calyptra, anthers, filaments, ovary, and embryos. The transcript levels found were compared with the proposed model for Arabidopsis to evaluate their biological consistency. With a few exceptions, the results confirmed the expression pattern expected based on the Arabidopsis data

Genetic segregation and genomic hybridization patterns support an allotetraploid structure and disomic inheritance for Salix species

The Salix alba L. (white willow)-Salix fragilis L. (crack willow) complex includes closely related polyploid species, mainly tetraploid (2n = 4x = 76), which are dioecious and hence obligate allogamous. Because little is known about the genome constitution and chromosome behavior of these pure willow trees, genetic analysis of their naturally occurring interspecific polyploid hybrids is still very difficult. A two-way pseudo-testcross strategy was exploited using single-dose AFLP markers in order to assess the main inheritance patterns of tetraploid biotypes (disomy vs. tetrasomy) in segregating populations stemmed from S. alba × S. fragilis crosses and reciprocals. In addition, a genomic in situ hybridization (GISH) technology was implemented in willow to shed some light on the genome structure of S. alba and S. fragilis species, and their hybrids (allopolyploidy vs. autopolyploidy). The frequency of S. alba-specific molecular markers was almost double compared to that of S. fragilis-specific ones, suggesting the phylogenetic hypothesis of S. fragilis as derivative species from S. alba-like progenitors. Cytogenetic observations at pro-metaphase revealed about half of the chromosome complements being less contracted than the remaining ones, supporting an allopolyploid origin of both S. alba and S. fragilis. Both genetic segregation and genomic hybridization data are consistent with an allotetraploid nature of the Salix species. In particular, the vast majority of the AFLP markers were inherited according to disomic patterns in S. alba × S. fragilis populations and reciprocals. Moreover, in all S. alba against S. fragilis hybridizations and reciprocals, GISH signals were observed only on the contracted chromosomes whereas the non-contracted chromosomes were never hybridized. In conclusion, half of the chromosomes of the pure species S. alba and S. fragilis are closely related and they could share a common diploid ancestor, while the rest of chromosomes are morphologically differentiated in either S. alba or S. fragilis and they should derive from distinct diploid ancestors.</p

The leaf transcriptome of fennel (Foeniculum vulgare Mill.) enables characterization of the t-anethole pathway and the discovery of microsatellites and single-nucleotide variants

Fennel is a plant species of both agronomic and pharmaceutical interest that is characterized by a shortage of genetic and molecular data. Taking advantage of NGS technology, we sequenced and annotated the first fennel leaf transcriptome using material from four different lines and two different bioinformatic approaches: de novo and genome-guided transcriptome assembly. A reference transcriptome for assembly was produced by combining these two approaches. Among the 79,263 transcripts obtained, 47,775 were annotated using BLASTX analysis performed against the NR protein database subset with 11,853 transcripts representing putative full-length CDS. Bioinformatic analyses revealed 1,011 transcripts encoding transcription factors, mainly from the BHLH, MYB-related, C2H2, MYB, and ERF families, and 6,411 EST-SSR regions. Single-nucleotide variants of SNPs and indels were identified among the 8 samples at a frequency of 0.5 and 0.04 variants per Kb, respectively. Finally, the assembled transcripts were screened to identify genes related to the biosynthesis of t-anethole, a compound well-known for its nutraceutical and medical properties. For each of the 11 genes encoding structural enzymes in the t-anethole biosynthetic pathway, we identified at least one transcript showing a significant match. Overall, our work represents a treasure trove of information exploitable both for marker-assisted breeding and for in-depth studies on thousands of genes, including those involved in t-anethole biosynthesis

Transcriptome pathways in leaf and root of grapevine genotypes with contrasting drought tolerance

Most of the world’s wine-producing regions are subjected to seasonal drought,and,in the light of the dramatic climate-change events occurring in recent years, the selection of resistant rootstocks is becoming a crucial factor for the development of sustainable agricultural models to ensure optimal grape berry development and ripening. In this study, roots and leaves of 101.14 (drought-susceptible) and M4 (drought-tolerant) rootstocks were sampled in progressive drought and mRNA-seq profiles were evaluated. Physiological characterization indicated that only M4 was able to maintain high leaf transpiration and net assimilation rates under severe stress conditions. Statistical analyses, carried out on mRNA-seq data, highlighted that “treatment” (water stress) and “genotype” (rootstock-genotype) seem to be the main variables explaining differential gene expression in roots and leaves tissues, respectively. Upon water-stress, roots and leaves of the tolerant genotype M4 exhibit a higher induction of stilbenes (i.e., STS) and flavonoids (e.g., CHS, F3H, FLS) biosynthetic genes. Moreover, the higher expression of STS genes in M4 is coupled with an up-regulation of WRKYs transcription factors. STS genes promoter regions, extracted from whole genome of M4 and 101.14, highlighted a higher number of WBOX cis elements (binding site for WRKYs) in the tolerant genotype

MIK2 is a candidate gene of the S-locus for sporophytic self-incompatibility (SSI) in chicory (Cichorium intybus, Asteraceae)

The Cichorium genus offers a unique opportunity to study the sporophytic self incompatibility (SSI) system, being composed of species characterized by highly efficient SI (C. intybus) and complete self compatibility (C. endivia). The chicory genome was used to map 7 previously identified SSI locus-associated markers. The region containing the S locus was restricted to an 4 M bp window on chromosome 5. Among the genes predicted in this region, MDIS1 INTERACTING RECEPTOR LIKE KINASE 2 (MIK2) was promising as a candidate for SSI. Its ortholog in Arabidopsis is involved in pollen stigma recognition reactions, and its protein structure is similar to that of S-receptor kinase (SRK), a key component of the SSI in the Brassica genus. The sequencing of MIK2 in chicory and endive accessions revealed two contrasting scenarios. In C. endivia, MIK2 was fully conserved even comparing different botanical varieties (smooth and curly). In C. intybus, 387 SNPs and 3 INDELs were identified when comparing accessions of different biotypes from the same botanical variety (radicchio). The SNP distribution throughout the gene was uneven, with hypervariable domains preferentially localized in the LRR-rich extracellular region, putatively identified as the receptor domain. The gene was hypothesized to be under positive selection, as the nonsynonymous mutations were more than double the synonymous ones (dN / dS = 2.17). An analogous situation was observed analyzing the first 500 bp of the MIK2 promoter: no SNPs were observed among the endive samples, whereas 44 SNPs and 6 INDELs were detected among the chicory samples. Further analyses are needed to confirm the role of MIK2 in SSI and to demonstrate whether the 23 species-specific nonsynonymous SNPs in the CDS and/or the species-specific 10 bp INDEL found in a CCAAT box region of the promoter are responsible for the contrasting sexual behaviors of the two species

Heterosis in horticultural crop breeding: combining old theoretical bases with modern genomic views

Heterosis in plants has been among the challenging topics for plant scientists worldwide. The production of F1 hybrid varieties of seed-propagated horticultural species is one of the most successful applications of plant breeding techniques. The exploitation of the heterosis phenomenon promotes homogeneity and maximizes crop yields and is a way for breeders to legally control and protect their commercial products. In the past heterosis has been largely studied and explored in cereal crop systems, considering maize as a model for understanding the genetic bases of this phenomenon. To date, crossbreeding in horticultural vegetables has also rapidly progressed. F1 hybrid varieties are available for many horticultural crops, including both allogamous and autogamous species. Several genetic and nongenetic mechanisms have been applied to facilitate the large-scale production of F1 hybrid seeds in vegetable crops to prevent undesirable selfing. Although the development and commercialization of F1 hybrids is currently common in agriculture, this phenomenon is still being investigated at different levels. With the rapid accumulation of knowledge on plant genome structures and gene activities and the advancement of new genomics platforms and methodologies, significant progress has been achieved in recent years in the study of the genetic and molecular bases of heterosis. This paper provides a brief overview of current theoretical advances and practical predictions of the molecular mechanisms underlying heterosis in plants. The aim is to carefully summarize the fundamental mechanisms of heterosis in plants, focusing on horticultural plant breeding, to improve the existing knowledge in this research area. We describe the quantitative genetic model of phenotypic variation and combine evolutionary, phenotypic and molecular genetic views to explain the origin and manifestation of heterosis and its significance for breeding F1 hybrid varieties in horticultural crops. The principles of genomic prediction and its applications in genomic selection are then covered