Biotechnology applications in sugar beet breeding

The aim of this thesis was to identify molecular markers associated to tolerance to biotic and abiotic stresses in sugar beet. Sugar beet is one of the world’s most important crops currently supplying around 20% of the sugar consumed worldwide. The crop is damaged by many adverse environmental conditions and the development of varieties that require fewer technical inputs for cultivation is one of the main research demands. To achieve this, sugar beet breeding is focusing on genetic improvement programs assisted by molecular markers. These methods are making selection procedures more rapid, accurate and less expensive. The development of a large set of SNP markers can facilitate the identification and exploitation of genes affecting important traits, such as resistance to biotic and abiotic stresses. Several techniques are used to enable SNP marker discovery in plants. Among these, the Restriction-site Associated DNA (RAD) technique is widely used. The RAD technique is based on acquiring and characterizing the genomic regions adjacent to a set of specific restriction enzyme recognition sites. Bulk Segregant Analysis (BSA) is a method to identify DNA markers linked to genes or genomic regions of interest. DNA samples from individuals showing contrasting phenotype are compared with a large set of molecular markers to select those linked to the trait of interest. The first part of the thesis presents a panel of 192 SNPs for effective sugar beet genetic diversity assessment using a recently released platform (QuantStudio 12K Flex system coupled with Taqman OpenArray technology) that has key elements required for high-throughput SNP genotyping. In the second part, the 192 SNPs were used to assess the phylogenetic relationship between Rizor and Holly (Rz1) resistance sources. The molecular results demonstrate that the resistances to rhizomania used by farmers over the last 30 years derived from sea beet collected in the Po River Delta. Analysis of molecular variance and principal coordinate analysis confirmed that Rizor and Rz1 couldn’t be distinguished as separate sources of resistance. In the third part, a marker linked to the first nematode tolerance gene (HsBvm-1) from Beta vulgaris ssp. maritima valuable for high-throughput marker-assisted selection was identified and mapped on chromosome 5. The fourth and fifth parts focus on resistance to abiotic stresses that compromise sugar production. Premature flowering or bolting, due to cold temperatures in early spring, is an undesirable characteristic that causes severe sugar yield losses and interferes with harvesting. A new locus involved in the genetic determination of bolting tendency was studied and a SNP marker associated with bolting tendency was found on chromosome 6. SNP location on the sugar beet genome confirms the association with flowering since it was mapped in a matrix metalloproteinase gene that causes late flowering and early senescence in Arabidopsis thaliana. Given the close and positive relationships between yield and root morpho-physiological traits, a BSA was conducted to identify a SNP marker linked to root elongation rate in sugar beet. SNP10139 was mapped on the peptide transporter gene influencing root elongation in Arabidopsis thaliana. The result suggests that SNPs developed in these studies could serve as a source for genotyping of sugar beet parental lines and varieties, with relevant impact on breeding program decisions

Genetic approaches to exploit landraces for improvement of Triticum turgidum ssp. durum in the age of climate change

Addressing the challenges of climate change and durum wheat production is becoming an important driver for food and nutrition security in the Mediterranean area, where are located the major producing countries (Italy, Spain, France, Greece, Morocco, Algeria, Tunisia, Turkey, and Syria). One of the emergent strategies, to cope with durum wheat adaptation, is the exploration and exploitation of the existing genetic variability in landrace populations. In this context, this review aims to highlight the important role of durum wheat landraces as a useful genetic resource to improve the sustainability of Mediterranean agroecosystems, with a focus on adaptation to environmental stresses. We described the most recent molecular techniques and statistical approaches suitable for the identification of beneficial genes/alleles related to the most important traits in landraces and the development of molecular markers for marker-assisted selection. Finally, we outline the state of the art about landraces genetic diversity and signature of selection, already identified from these accessions, for adaptability to the environment

Genetic approaches to exploit landraces for improvement of Triticum turgidum ssp. durum in the age of climate change

Addressing the challenges of climate change and durum wheat production is becoming an important driver for food and nutrition security in the Mediterranean area, where are located the major producing countries (Italy, Spain, France, Greece, Morocco, Algeria, Tunisia, Turkey, and Syria). One of the emergent strategies, to cope with durum wheat adaptation, is the exploration and exploitation of the existing genetic variability in landrace populations. In this context, this review aims to highlight the important role of durum wheat landraces as a useful genetic resource to improve the sustainability of Mediterranean agroecosystems, with a focus on adaptation to environmental stresses. We described the most recent molecular techniques and statistical approaches suitable for the identification of beneficial genes/alleles related to the most important traits in landraces and the development of molecular markers for marker-assisted selection. Finally, we outline the state of the art about landraces genetic diversity and signature of selection, already identified from these accessions, for adaptability to the environment

Targeted next-generation sequencing identification of mutations in disease resistance gene analogs (RGAs) in wild and cultivated beets

Resistance gene analogs (RGAs) were searched bioinformatically in the sugar beet (Beta vulgaris L.) genome as potential candidates for improving resistance against different diseases. In the present study, Ion Torrent sequencing technology was used to identify mutations in 21 RGAs. The DNA samples of ninety-six individuals from six sea beets (Beta vulgaris L. subsp. maritima) and six sugar beet pollinators (eight individuals each) were used for the discovery of single-nucleotide polymorphisms (SNPs). Target amplicons of about 200 bp in length were designed with the Ion AmpliSeq Designer system in order to cover the DNA sequences of the RGAs. The number of SNPs ranged from 0 in four individuals to 278 in the pollinator R740 (which is resistant to rhizomania infection). Among different groups of beets, cytoplasmic male sterile lines had the highest number of SNPs (132) whereas the lowest number of SNPs belonged to O-types (95). The principal coordinates analysis (PCoA) showed that the polymorphisms inside the gene Bv8_184910_pkon (including the CCCTCC sequence) can effectively differentiate wild from cultivated beets, pointing at a possible mutation associated to rhizomania resistance that originated directly from cultivated beets. This is unlike other resistance sources that are introgressed from wild beets. This gene belongs to the receptor-like kinase (RLK) class of RGAs, and is associated to a hypothetical protein. In conclusion, this first report of using Ion Torrent sequencing technology in beet germplasm suggests that the identified sequence CCCTCC can be used in marker-assisted programs to differentiate wild from domestic beets and to identify other unknown disease resistance genes in beet

Transcriptional and Physiological Analyses to Assess the Effects of a Novel Biostimulant in Tomato

: This work aimed to study the effects in tomato (Solanum lycopersicum L.) of foliar applications of a novel calcium-based biostimulant (SOB01) using an omics approach involving transcriptomics and physiological profiling. A calcium-chloride fertilizer (SOB02) was used as a product reference standard. Plants were grown under well-watered (WW) and water stress (WS) conditions in a growth chamber. We firstly compared the transcriptome profile of treated and untreated tomato plants using the software RStudio. Totally, 968 and 1,657 differentially expressed genes (DEGs) (adj-p-value < 0.1 and |log2(fold change)| ≥ 1) were identified after SOB01 and SOB02 leaf treatments, respectively. Expression patterns of 9 DEGs involved in nutrient metabolism and osmotic stress tolerance were validated by real-time quantitative reverse transcription PCR (RT-qPCR) analysis. Principal component analysis (PCA) on RT-qPCR results highlighted that the gene expression profiles after SOB01 treatment in different water regimes were clustering together, suggesting that the expression pattern of the analyzed genes in well water and water stress plants was similar in the presence of SOB01 treatment. Physiological analyses demonstrated that the biostimulant application increased the photosynthetic rate and the chlorophyll content under water deficiency compared to the standard fertilizer and led to a higher yield in terms of fruit dry matter and a reduction in the number of cracked fruits. In conclusion, transcriptome and physiological profiling provided comprehensive information on the biostimulant effects highlighting that SOB01 applications improved the ability of the tomato plants to mitigate the negative effects of water stress

Innovative approaches to evaluate sugar beet responses to changes in sulfate availability

In this study, a system based on omics profiling was set-up for sugar beet (Beta vulgaris L. subsp. vulgaris) evaluation after changes in sulfate availability. Seedlings were grown on sulfate-deprived Hoagland solution. Six days after germination, 100 \ub5M MgSO4was added to the solution. Root samples were collected 36 h after treatments. WinRHIZO root-scanning approach was used for the automated image analysis of plant root morphology. Inductively Coupled Plasma Spectrometry (ICP-OES) and quadrupole-time-of-flight mass spectrometry (Q-TOF) were used for ionomic and metabolic analysis, respectively. Nanofluidic real-time PCR (OpenArray system) was used for molecular profiling. OpenArray chips were designed with TaqMan probes for 53 sugar beet genes putatively involved in sulfate nutrition. At morphological level treated seedlings showed significantly higher values (P < 0.01) than untreated plants for root traits related to soil exploration and nutrient uptake, such as total root length, fine roots length and root tips number. ICP-OES, Q-TOF and transcriptomic data revealed changes due to sulfate availability in sugar beet samples. Two key results are highlighted in sulfate-supplied roots and leaves. Firstly, high expression levels of auxin efflux carrier component 1 (PIN) and 5-phosphoribosyl-anthranilate, precursor of tryptophan and auxin synthesis, were observed in roots. Secondly, high levels of 2-Cys peroxiredoxin BAS1, chloroplastic, thioredoxin reductase (NADPH) and cysteine synthase, chloroplastic/chromoplastic, O-acetylserine sulfhydrylase, involved in protection against oxidative stress and cysteine synthase activity, respectively, were observed in leaves. Based on our findings, the combination of evaluated omics approaches could become a key system for the evaluation of the nutritional status of sugar beet under different nutrient availability conditions