State of the Art of Genetic Engineering in Potato: From the First Report to Its Future Potential.

Potato (Solanum tuberosum L.) is a crop of world importance that produces tubers of high nutritional quality. It is considered one of the promising crops to overcome the challenges of poverty and hunger worldwide. However, it is exposed to different biotic and abiotic stresses that can cause significant losses in production. Thus, potato is a candidate of special relevance for improvements through conventional breeding and biotechnology. Since conventional breeding is time-consuming and challenging, genetic engineering provides the opportunity to introduce/switch-off genes of interest without altering the allelic combination that characterize successful commercial cultivars or to induce targeted sequence modifications by New Breeding Techniques. There is a variety of methods for potato improvement via genetic transformation. Most of them incorporate genes of interest into the nuclear genome; nevertheless, the development of plastid transformation protocols broadened the available approaches for potato breeding. Although all methods have their advantages and disadvantages, Agrobacterium-mediated transformation is the most used approach. Alternative methods such as particle bombardment, protoplast transfection with polyethylene glycol and microinjection are also effective. Independently of the DNA delivery approach, critical steps for a successful transformation are a rapid and efficient regeneration protocol and a selection system. Several critical factors affect the transformation efficiency: vector type, insert size, Agrobacterium strain, explant type, composition of the subculture media, selective agent, among others. Moreover, transient or stable transformation, constitutive or inducible promoters, antibiotic/herbicide resistance or marker-free strategies can be considered. Although great efforts have been made to optimize all the parameters, potato transformation protocols are highly genotype-dependent. Genome editing technologies provide promising tools in genetic engineering allowing precise modification of targeted sequences. Interestingly, transient expression of genome editing components in potato protoplasts was reported to generate edited plants without the integration of any foreign DNA, which is a valuable aspect from both a scientific and a regulatory perspective. In this review, current challenges and opportunities concerning potato genetic engineering strategies developed to date are discussed. We describe their critical parameters and constrains, and the potential application of the available tools for functional analyses or biotechnological purposes. Public concerns and safety issues are also addressed.

Identification of a New Cotton Disease Caused by an Atypical Cotton Leafroll Dwarf Virus in Argentina.

An outbreak of a new disease occurred in cotton (Gossypium hirsutum) fields in northwest Argentina starting in the 2009-10 growing season and is still spreading steadily. The characteristic symptoms of the disease included slight leaf rolling and a bushy phenotype in the upper part of the plant. In this study, we determined the complete nucleotide sequences of two independent virus genomes isolated from cotton blue disease (CBD)-resistant and -susceptible cotton varieties. This virus genome comprised 5,866 nucleotides with an organization similar to that of the genus Polerovirus and was closely related to cotton leafroll dwarf virus, with protein identity ranging from 88 to 98%. The virus was subsequently transmitted to a CBD-resistant cotton variety using Aphis gossypii and symptoms were successfully reproduced. To study the persistence of the virus, we analyzed symptomatic plants from CBD-resistant varieties from different cotton-growing fields between 2013 and 2015 and showed the presence of the same virus strain. In addition, a constructed full-length infectious cDNA clone from the virus caused disease symptoms in systemic leaves of CBD-resistant cotton plants. Altogether, the new leafroll disease in CBD-resistant cotton plants is caused by an atypical cotton leafroll dwarf virus.

Population structure and genetic diversity characterization of a sunflower association mapping population using SSR and SNP markers.

BACKGROUND: Argentina has a long tradition of sunflower breeding, and its germplasm is a valuable genetic resource worldwide. However, knowledge of the genetic constitution and variability levels of the Argentinean germplasm is still scarce, rendering the global map of cultivated sunflower diversity incomplete. In this study, 42 microsatellite loci and 384 single nucleotide polymorphisms (SNPs) were used to characterize the first association mapping population used for quantitative trait loci mapping in sunflower, along with a selection of allied open-pollinated and composite populations from the germplasm bank of the National Institute of Agricultural Technology of Argentina. The ability of different kinds of markers to assess genetic diversity and population structure was also evaluated. RESULTS: The analysis of polymorphism in the set of sunflower accessions studied here showed that both the microsatellites and SNP markers were informative for germplasm characterization, although to different extents. In general, the estimates of genetic variability were moderate. The average genetic diversity, as quantified by the expected heterozygosity, was 0.52 for SSR loci and 0.29 for SNPs. Within SSR markers, those derived from non-coding regions were able to capture higher levels of diversity than EST-SSR. A significant correlation was found between SSR and SNP- based genetic distances among accessions. Bayesian and multivariate methods were used to infer population structure. Evidence for the existence of three different genetic groups was found consistently across data sets (i.e., SSR, SNP and SSR + SNP), with the maintainer/restorer status being the most prevalent characteristic associated with group delimitation. CONCLUSION: The present study constitutes the first report comparing the performance of SSR and SNP markers for population genetics analysis in cultivated sunflower. We show that the SSR and SNP panels examined here, either used separately or in conjunction, allowed consistent estimations of genetic diversity and population structure in sunflower breeding materials. The generated knowledge about the levels of diversity and population structure of sunflower germplasm is an important contribution to this crop breeding and conservation.

Agroinoculation of a full-length cDNA clone of cotton leafroll dwarf virus (CLRDV) results in systemic infection in cotton and the model plant Nicotiana benthamiana.

Cotton blue disease is the most important viral disease of cotton in the southern part of South America. Its etiological agent, cotton leafroll dwarf virus (CLRDV), is specifically transmitted to host plants by the aphid vector (Aphis gossypii) and any attempt to perform mechanical inoculations of this virus into its host has failed. This limitation has held back the study of this virus and the disease it causes. In this study, a full-length cDNA of CLRDV was constructed and expressed in vivo under the control of cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system for the cloned cDNA construct of CLRDV was developed. Northern and immunoblot analyses showed that after several weeks the replicon of CLRDV delivered by Agrobacterium tumefaciens in Gossypium hirsutum plants gave rise to a systemic infection and typical blue disease symptoms correlated to the presence of viral RNA and P3 capsid protein. We also demonstrated that the virus that accumulated in the agroinfected plants was transmissible by the vector A. gossypii. This result confirms the production of biologically active transmissible virions. In addition, the clone was infectious in Nicotiana benthamiana plants which developed interveinal chlorosis three weeks postinoculation and CLRDV was detected both in the inoculated and systemic leaves. Attempts to agroinfect Arabidopsis thaliana plants were irregularly successful. Although no symptoms were observed, the P3 capsid protein as well as the genomic and subgenomic RNAs were irregularly detected in systemic leaves of some agroinfiltrated plants. The inefficient infection rate infers that A. thaliana is a poor host for CLRDV. This is the first report on the construction of a biologically-active infectious full-length clone of a cotton RNA virus showing successful agroinfection of host and non-host plants. The system herein developed will be useful to study CLRDV viral functions and plant-virus interactions using a reverse genetic approach.

Selection strategy for a seedling seed orchard design based on trait selection index and genomic analysis by molecular markers: a case study for Eucalyptus dunnii.

Molecular genetic analysis was applied to 162 individuals of 37 half-sib selected families belonging to six provenances of Eucalyptus dunnii Maiden in a provenance/family trial. The individuals were selected by a trait selection index and genetic diversity criteria were later applied for designing seedling seed orchards. Genetic diversity and its distribution, as well as relationships among individuals, were assessed on the basis of nine microsatellite loci and 243 amplified fragment length polymorphism markers. High diversity was found with both kinds of markers. Clear-cut genomic patterns of identification (fingerprinting) were obtained for each individual. Genetic differentiation estimates consistently showed low differentiation among provenances (R(ST1) = 0.069, theta(P) = 0.026 and F(CT) = 0.035) and great differentiation among families (R(ST2) = 0.223, theta(S) = 0.174 and F(SC) = 0.164). A high proportion of the total variation was observed within families (around 80% by both marker analyses), suggesting that orchard design should be based on individual or family selection rather than on provenance selection, and that individual ranking by both trait selection index and molecular genetic diversity criteria should be considered. A selection procedure for a seedling seed orchard design is proposed.

Sequence and phylogenetic analysis of genome segments S1, S2, S3 and S6 of Mal de Río Cuarto virus, a newly accepted Fijivirus species.

Mal de Río Cuarto virus (MRCV) is a newly described species of the genus Fijivirus, family Reoviridae. The nucleotide sequence of four MRCV genome segments was determined. MRCV S1, S2, S3 and S6 were predicted to encode proteins of 168.4, 134.4, 141.7 and 90 kDa, respectively. MRCV S1 encodes a basic protein that contains conserved RNA-dependent RNA polymerase motifs, and is homologous to Rice black streaked dwarf virus (RBSDV), Fiji disease virus (FDV) and Nilaparvata lugens reovirus (NLRV) polymerases as well as to corresponding proteins of members of other genera of the Reoviridae. MRCV S2 codes for a protein with intermediate homology to the ones coded by RBSDV S4 and FDV S3 'B' spike, which is presumably the B-spike protein. MRCV S3 most probably encodes the major core protein and is highly homologous to corresponding proteins of RBSDV S2 and FDV S3. MRCV S6-encoded protein has low homology to the proteins of unknown function coded by RBSDV S6 and FDV S6. The identity levels between all analyzed MRCV coded proteins and their RBSDV counterparts varied between 84.5 and 44.8%. The analysis of the reported sequences allowed a phylogenetic comparison of MRCV with other reovirus and supported its taxonomic status within the genus.