Daniele Trebbi

Functional differentiation of the sugar beet root system as indicator of developmental phase change

Developmental phase transitions in the plant root system have not been well characterized. In this study we compared the dynamics of sucrose accumulation with changes in gene expression analyzed with cDNA-amplified fragment length polymorphism (AFLP) in the developing tap root of sugar beet (Beta vulgaris, L.) during the first 9 weeks after emergence (WAE). Although differences between lines were evident as soon as 9 WAE, sucrose showed a marked increase in the rate of accumulation between 4 and 6 WAE and a remarkable shift in gene expression was observed between 5 and 6 WAE. These changes were evident in two unrelated genetic backgrounds and suggest that physiological and gene expression changes represent a functional differentiation of the tap root. These changes were considered as indicators of a developmental change in the sugar beet root system. To identify genes and metabolic pathways involved in this developmental shift, a root cDNA library was hybridized with probes enriched for 3- and 7-WAE transcripts and differentially expressed transcripts were analyzed by cDNA microarray. Several genes involved in the regulation of tissue development were found to be differentially regulated. Genes involved in protein metabolism, disease-related and secretory system were upregulated before the functional differentiation transition, while genes under hormonal control were upregulated after the functional differentiation transition. This developmental phase change of the root system is important to understand plant developmental regulation at the whole-plant level and will likely be useful as early selection parameter in breeding programs.

Evaluation of genetic diversity and root traits of sea beet accessions of the Adriatic Sea coast

Thirty-nine sea beet [Beta vulgaris L. ssp. maritima (L.) Arcang.] accessions of the Adriatic coast were screened genetically and for their adaptive morpho-functional root traits in order to identify new sources of abiotic resistances for sugar beet breeding programs. Genetic diversity was evaluated with 21 microsatellites markers that identified 44 polymorphic alleles. Sea beets grouped into two main clusters: the West and the East Adriatic coast groups, with the latter showing higher genetic diversity. Among sea beet accessions with desirable root traits, four accessions have proved to be interesting for sugar beet [B. vulgaris (L.) ssp. vulgaris] breeding aimed to improve tolerance to nutritional stresses. Lastovo (ID 29) and Zut (ID 34) accessions were characterized by the highest values of RER, TRL, FRL and RSA still maintaining a high value of RTD, while Grado (ID 21) an Portic (ID 23) accessions were characterized by the highest RTD, but with low values of RER, TRL, FRL and RSA parameters.

Identification and Validation of a SNP Marker Linked to the Gene HsBvm-1 for Nematode Resistance in Sugar Beet

The beet-cyst nematode (Heterodera schachtii Schmidt) is one of the major pests of sugar beet. The identification of molecular markers associated with nematode tolerance would be helpful for developing tolerant varieties. The aim of this study was to identify single nucleotide polymorphism (SNP) markers linked to nematode tolerance from the Beta vulgaris ssp. maritima source WB242. A WB242-derived F2 population was phenotyped for host-plant nematode reaction revealing a 3:1 segregation ratio of the tolerant and susceptible phenotypes and suggesting the action of a gene designated as HsBvm-1. Bulked segregant analysis (BSA) was used. The most tolerant and susceptible individuals were pooled and subjected to restriction site associated DNA sequencing (RAD-Seq) analysis, which identified 7,241 SNPs. A subset of 384 candidate SNPs segregating between bulks were genotyped on the 20 most-tolerant and most-susceptible individuals, identifying a single marker (SNP192) showing complete association with nematode tolerance. Segregation of SNP192 confirmed the inheritance of tolerance by a single gene. This association was further validated on a set of 26 commercial tolerant and susceptible varieties, showing the presence of the SNP192 WB242-type allele only in the tolerant varieties. We identified and mapped on chromosome 5 the first nematode tolerance gene (HsBvm-1) from Beta vulgaris ssp. maritima and released information on SNP192, a linked marker valuable for high-throughput, marker-assisted breeding of nematode tolerance in sugar beet.

Sugar beet resistance to rhizomania: state of the art and perspectives.

The productivity of sugar beet is strongly limited by several biotic stresses, among them rhizomania (induced by Beet necrotic yellow vein virus, BNYVV) which causes yield losses of 20–50% or more. The only way to control this disease is the use of resistant varieties. Sources of resistances have been found in the ancestor of the cultivated beets Beta vulgaris L. ssp. maritima (L.) Arcang. Rz1 is the major resistance gene present within commercial sugar beet varieties. This resistance was recognized as monogenic and dominant. Experimental evidence highlighted that Rz1 originated from sea beets belonging to Munerati’s genetic pool. The development of molecular markers linked to Rz1 allows the use of marker assisted selection (MAS) to introgress this gene into pollinator lines, to assess the trueness of hybridity and to remove off-type individuals. MAS speeds variety development and reduces production costs guaranteeing at the same time a high qualitative standard in variety development. Recent studies have shown an emergence of new BNYVV strains with increased virulence that could overcome Rz1 resistance. Therefore, exploitation of new genetic sources of resistance and the pyramiding of several resistance genes into new breeding lines is becoming a main priority for sugar beet breeding companies to maintain an adequate resistance level to rhizomania.

Effect of sulfate availability on root traits and microRNA395 expression in sugar beet

Nutritional stress is one of the main limits to sugar beet yield. This study evaluated morphological and molecular responses of sugar beet to changes in sulfate availability. Morphological characteristics of the root system and the accumulation of microRNA395 (miR395) were examined in sulfate(S)-supplemented and S-deprived seedlings under hydroponic conditions. We also investigated the functional role of miR395 in regulating the expression of APS1 gene coding for ATP-sulfurylase in roots and leaves. The S-deprived seedlings showed a significant increase in the number of root tips, in the miR395 expression in leaves but not in roots, and in the expression of APS1 gene. Our results indicate that miR395 may be a useful biomarker for sulfate status in sugar beet.

In-vitro physical mutagenesis of giant reed (Arundo donax L.)

Giant reed (Arundo donax L.) is a C3 perennial, warm‐season, rhizomatous grass of emerging interest for bioenergy and biomass derivatives production, and for phytoremediation. It only propagates vegetatively and very little genetic variation is found among ecotypes, basically precluding breeding efforts. With the objective to increase the genetic variation in this species, we developed and applied a mutagenesis protocol based on γ‐irradiation of in vitro cell cultures from which regenerants were obtained. Based on a radiosensitivity test, the irradiation dose reducing to 50% the number of regenerants per callus (RD50) was estimated at 35 Gy. A large mutagenic experiment was carried out by irradiating a total of 3120 calli with approx. 1×, 1.5× and 2× RD50. A total of 1004 regenerants from irradiated calli were hardened in pots and transplanted to the field. Initial phenotypic characterization of the collection showed correlated responses of biomass‐related quantitative traits to irradiation doses. Approx. 10% of field‐grown clones showed remarkable morphological aberrations including dwarfism, altered tillering, abnormal inflorescence, leaf variegation and others, which were tested for stability over generations. Clone lethality reached 0.4%. Our results show for the first time that physical mutagenesis can efficiently induce new genetic and phenotypic variation of agronomic and prospective industrial value in giant reed. The methodology and the plant materials described here may contribute to the domestication and the genetic improvement of this important biomass species.

Single nucleotide polymorphism markers linked to root elongation rate in sugar beet

The aim of this study was to identify single nucleotide polymorphism (SNP) markers genetically linked to root elongation rate (RER) in sugar beet (Beta vulgaris L.). A population of 244 F3 individuals, obtained from the cross between lines L01 (a low RER) and L18 (a high RER), was phenotyped by measuring RER of 11-d-old seedlings grown in a hydroponic culture. Two DNA bulks of 50 F3 individuals with extreme phenotypes were used for bulk segregant analysis by restriction-associated DNA sequencing. A total of 20 376 SNPs were identified. Single nucleotide polymorphisms were filtered to reduce the number of the false positive and mapped on candidate chromosomal regions of the B. vulgaris reference genome. One of the total of SNPs selected, SNP10139, was strongly linked to RER (P < 0.01). The pattern of association between the SNP10139 genotype and RER was also evaluated on a breeding line panel comprising 40 low and 40 high RER individuals with different allele frequencies between groups (P < 0.01). The SNP10139 sequence was mapped on the B. vulgaris peptide transporter (PTR) gene, a carrier that influences root elongation in Arabidopsis thaliana. Our results suggest that SNP10139 influence RER in sugar beet, and sequence information can be used in marker-assisted selection programs.