Identification of a Unique Genomic Region in Sweet Chestnut (Castanea sativa Mill.) That Controls Resistance to Asian Chestnut Gall Wasp Dryocosmus kuriphilus Yasumatsu.

The Asian chestnut gall wasp (ACGW) (Hymenoptera Dryocosmus kuriphilus Yasumatsu) is a severe pest of sweet chestnut (Castanea sativa Mill.) with a strong impact on growth and nut production. A comparative field trial in Central Italy, including provenances from Spain, Italy, and Greece, was screened for ACGW infestation over consecutive years. The Greek provenance Hortiatis expressed a high proportion of immune plants and was used to perform a genome-wide association study based on DNA pool sequencing (Pool-GWAS) by comparing two DNA pools from 25 susceptible and 25 resistant plants. DNA pools were sequenced with 50X coverage depth. Sequence reads were aligned to a C. mollissima reference genome and the pools were compared to identify SNPs associated with resistance. Twenty-one significant SNPs were identified and highlighted a small genomic region on pseudochromosome 3 (Chr 3), containing 12 candidate genes of three gene families: Cytochrome P450, UDP-glycosyltransferase, and Rac-like GTP-binding protein. Functional analyses revealed a putative metabolic gene cluster related to saccharide biosynthesis in the genomic regions associated with resistance that could be involved in the production of a toxic metabolite against parasites. The comparison with previous genetic studies confirmed the involvement of Chr 3 in the control of resistance to ACGW.

Overexpression of MADS-box Gene AGAMOUS-LIKE 12 Activates Root Development in Juglans sp. and Arabidopsis thaliana.

Until recently, the roles of plant MADS-box genes have mainly been characterized during inflorescence and flower differentiation. In order to precise the roles of AGAMOUS-LIKE 12, one of the few MADS-box genes preferentially expressed in roots, we placed its cDNA under the control of the double 35S CaMV promoter to produce transgenic walnut tree and Arabidopsis plants. In Juglans sp., transgenic somatic embryos showed significantly higher germination rates but abnormal development of their shoot apex prevented their conversion into plants. In addition, a wide range of developmental abnormalities corresponding to ectopic root-like structures affected the transgenic lines suggesting partial reorientations of the embryonic program toward root differentiation. In Arabidopsis, AtAGL12 overexpression lead to the production of faster growing plants presenting dramatically wider and shorter root phenotypes linked to increased meristematic cell numbers within the root apex. In the upper part of the roots, abnormal cell divisions patterns within the pericycle layer generated large ectopic cell masses that did not prevent plants to grow. Taken together, our results confirm in both species that AGL12 positively regulates root meristem cell division and promotes overall root vascular tissue formation. Genetic engineering of AGL12 expression levels could be useful to modulate root architecture and development.

Correction to: Long-term study of a subdioecious Populus ×canescens family reveals sex lability of females and reproduction behaviour of cosexual plants.

Table 4 in the original publication reports incomplete genotype names in the column "Cross" and wrong codes in the column "Generation".

Long-term study of a subdioecious Populus ×canescens family reveals sex lability of females and reproduction behaviour of cosexual plants.

KEY MESSAGE: Cosexual Populus ×canescens plants are inconstant females with life course plasticity of sex phenotype and can reproduce by selfing. Populus species are dioecious, but deviations from dioecy are reported in some cases. The objectives of this study were to investigate the phenotypic expression and the inheritance of subdioecy in a Populus ×canescens pedigree. The F1 progeny was monitored for sex during 14 years. Thirty per cent of individuals expressed deviations from dioecy and long-term plasticity of sex. Some plants started flowering as male, then became cosexual, and finally turned female. Two cosexual individuals were self-pollinated and generated a selfed progeny markedly impaired by inbreeding depression, but able to reproduce by outcrossing. Sex segregation of the F1 progeny statistically fitted the expected ratio 1:2:1 (female:male:cosexual). By analysis of DNA markers, the cosexual individuals were genetically clustered with the females. The segregation ratio and the genetic profile indicated that cosexual plants were female with altered sex phenotype. Linkage analysis identified a putative sex-determining region with suppressed recombination on chromosome 19 of the male Populus tremula parent. The male sex trait was linked to the pericentromeric region of the P. tremula chromosome 19, whereas the cosexual trait was linked to chromosome 19 of the female Populus alba parent. A genetic model is proposed to explain inheritance and phenotypic expression of sex.

Phenotypic plasticity, QTL mapping and genomic characterization of bud set in black poplar.

BACKGROUND: The genetic control of important adaptive traits, such as bud set, is still poorly understood in most forest trees species. Poplar is an ideal model tree to study bud set because of its indeterminate shoot growth. Thus, a full-sib family derived from an intraspecific cross of P. nigra with 162 clonally replicated progeny was used to assess the phenotypic plasticity and genetic variation of bud set in two sites of contrasting environmental conditions. RESULTS: Six crucial phenological stages of bud set were scored. Night length appeared to be the most important signal triggering the onset of growth cessation. Nevertheless, the effect of other environmental factors, such as temperature, increased during the process. Moreover, a considerable role of genotype × environment (G × E) interaction was found in all phenological stages with the lowest temperature appearing to influence the sensitivity of the most plastic genotypes.Descriptors of growth cessation and bud onset explained the largest part of phenotypic variation of the entire process. Quantitative trait loci (QTL) for these traits were detected. For the four selected traits (the onset of growth cessation (date2.5), the transition from shoot to bud (date1.5), the duration of bud formation (subproc1) and bud maturation (subproc2)) eight and sixteen QTL were mapped on the maternal and paternal map, respectively. The identified QTL, each one characterized by small or modest effect, highlighted the complex nature of traits involved in bud set process. Comparison between map location of QTL and P. trichocarpa genome sequence allowed the identification of 13 gene models, 67 bud set-related expressional and six functional candidate genes (CGs). These CGs are functionally related to relevant biological processes, environmental sensing, signaling, and cell growth and development. Some strong QTL had no obvious CGs, and hold great promise to identify unknown genes that affect bud set. CONCLUSIONS: This study provides a better understanding of the physiological and genetic dissection of bud set in poplar. The putative QTL identified will be tested for associations in P. nigra natural populations. The identified QTL and CGs will also serve as useful targets for poplar breeding.

Comparative study of transcriptional and physiological responses to salinity stress in two contrasting Populus alba L. genotypes.

Soil salinity is an important limiting factor to tree growth and productivity. Populus alba L. is a moderately salt-tolerant species and its natural populations are adapted to contrasting environments, thus providing genetic resources to identify key genes for tolerance to abiotic stress, such as salinity. To elucidate the molecular and genetic basis of variation for salinity tolerance in P. alba, we analyzed the short-term ecophysiological and transcriptome response to salinity. Two contrasting genotypes, 6K3, salt sensitive, and 14P11, salt tolerant, originating from North and South Italy, respectively, were challenged with salt stress (200 mM NaCl). Sodium accumulated in the leaves of salt-treated plants and its concentration increased with time. The net photosynthesis was strongly reduced by salinity in both genotypes, with 6K3 being significantly more affected than 14P11. The transcriptional changes in leaves were analyzed using cDNA microarrays containing about 7000 stress-related poplar expressed sequence tags (EST). A microarray experiment based on RNA pooling showed a number of salinity--regulated transcripts that markedly increased from 3 h to 3 days of salinity treatment. Thus, a detailed analysis was performed on replicated plants collected at 3 days, when ~20% of transcripts showed significant change induced by salinity. In 6K3, there were more genes with decreased expression than genes with increased expression, whereas such a difference was not found in 14P11. Most transcripts with decreased expression were shared between the two genotypes, whereas transcripts with increased expression were mostly regulated in a genotype-specific manner. The commonly decreased transcripts (71 genes) were functionally related to carbohydrate metabolism, energy metabolism and photosynthesis. These biological processes were consistent with the strong inhibition of photosynthesis, caused by salinity. The commonly increased transcripts (13 genes) were functionally related to primary metabolism and biosynthesis of proteins and macromolecules. The salinity-increased transcripts discriminated the molecular response of the two genotypes. In 14P11, the 21 genes specifically salinity-induced were related to stress response, cell development, cell death and catabolism. In 6K3, the 15 genes with salinity-increased expression were involved in protein biosynthesis, metabolism of macromolecules and cell organization and biogenesis. The difference in transcriptome response between the two genotypes could address the molecular basis of intra-specific variation of salinity tolerance in P. alba.

Intraspecific variation of physiological and molecular response to cadmium stress in Populus nigra L.

Little is known about the variability of response to heavy metal stress within tree species, although it could be a key for a better understanding of tolerance mechanisms and for breeding. The aim of the present study was to characterize the natural variation of response to cadmium (Cd) in Populus nigra L. in order to understand the mechanisms of Cd tolerance. For that, two P. nigra genotypes, originating from contrasting environments in northern (genotype 58-861) and southern (genotype Poli) Italy, were exposed to Cd stress in hydroponics for 3 weeks. The effect of stress was estimated by measuring biomass production, photosynthetic performance and accumulation and translocation of Cd at the end of the experiment. To better understand the mechanisms of Cd tolerance, the expression of some candidate genes involved in the ascorbate-glutathione cycle (ascorbate peroxidase, glutathione reductase, glutathione S-transferase) and in metal sequestration (metallothioneins) was analyzed in leaves. Biomass production and photosynthesis were affected by the treatment in both clones but the southern clone was markedly more tolerant to Cd stress than the other. Nevertheless, the Cd content in leaves was not significantly different between the two clones and was quite low compared to other species. The content of thiols and phytochelatins (PCs), associated with the transcription profile of the glutathione S-transferase gene, indicated relevant differences in the use of the PCs pathway under Cd stress, which could explain the different tolerance to Cd. The northern clone accumulated thiols but down-regulated the GST gene, whereas the southern clone accumulated PCs and up-regulated the GST gene, which can be useful to complex and detoxify Cd. These results suggest that the glutathione pathway is involved in the differential Cd tolerance of the two genotypes. The natural germplasm of P. nigra represents a valuable resource for understanding tolerance to Cd and for selection of plant material for phytoremediation.

Bud set in poplar--genetic dissection of a complex trait in natural and hybrid populations.

• The seasonal timing of growth events is crucial to tree distribution and conservation. The seasonal growth cycle is strongly adapted to the local climate that is changing because of global warming. We studied bud set as one cornerstone of the seasonal growth cycle in an integrative approach. • Bud set was dissected at the phenotypic level into several components, and phenotypic components with most genetic variation were identified. While phenotypic variation resided in the timing of growth cessation, and even so more in the duration from growth cessation to bud set, the timing of growth cessation had a stronger genetic component in both natural and hybrid populations. • Quantitative trait loci (QTL) were identified for the most discriminative phenotypic bud-set components across four poplar pedigrees. The QTL from different pedigrees were recurrently detected in six regions of the poplar genome. • These regions of 1.83-4.25 Mbp in size, containing between 202 and 394 genes, form the basis for further molecular-genetic dissection of bud set.

Allele-specific PCR in SNP genotyping.

The increasing need for large-scale genotyping applications of single nucleotide polymorphisms (SNPs) in model and nonmodel organisms requires the development of low-cost technologies accessible to minimally equipped laboratories. The method presented here allows efficient discrimination of SNPs by allele-specific PCR in a single reaction with standard PCR conditions. A common reverse primer and two forward allele-specific primers with different tails amplify two allele-specific PCR products of different lengths, which are further separated by agarose gel electrophoresis. PCR specificity is improved by the introduction of a destabilizing mismatch within the 30 end of the allele-specific primers. This is a simple and inexpensive method for SNP detection that does not require PCR optimization.