De novo transcriptome sequencing and SSR markers development for Cedrela balansae C.DC., a native tree species of northwest Argentina.

The endangered Cedrela balansae C.DC. (Meliaceae) is a high-value timber species with great potential for forest plantations that inhabits the tropical forests in Northwestern Argentina.Research on this species is scarce because of the limited genetic and genomic information available. Here, we explored the transcriptome of C. balansae using 454 GS FLX Titanium next-generation sequencing (NGS) technology. Following de novo assembling, we identified 27,111 non-redundant unigenes longer than 200 bp, and considered these transcripts for further downstream analysis. The functional annotation was performed searching the 27,111 unigenes against the NR-Protein and the Interproscan databases. This analysis revealed 26,977 genes with homology in at least one of the Database analyzed. Furthermore, 7,774 unigenes in 142 different active biological pathways in C. balansae were identified with the KEGG database. Moreover, after in silico analyses, we detected 2,663 simple sequence repeats (SSRs) markers. A subset of 70 SSRs related to important "stress tolerance" traits based on functional annotation evidence, were selected for wet PCR-validation in C. balansae and other Cedrela species inhabiting in northwest and northeast of Argentina (C. fissilis, C. saltensis and C. angustifolia). Successful transferability was between 77% and 93% and thanks to this study, 32 polymorphic functional SSRs for all analyzed Cedrela species are now available. The gene catalog and molecular markers obtained here represent a starting point for further research, which will assist genetic breeding programs in the Cedrela genus and will contribute to identifying key populations for its preservation.

Molecular evidence of hybrid zones of Cedrela (Meliaceae) in the Yungas of Northwestern Argentina.

In the Yungas of Northwestern Argentina, three endangered species of Cedrela (C. angustifolia, C. saltensis, and C. balansae) follow altitudinal gradients of distribution with contact zones between them. We sampled 210 individuals from 20 populations that spanned most of Cedrela's geographical range in the Yungas, and used Amplified Fragment Length Polymorphism (AFLP) markers and DNA sequences of the nuclear Internal Transcribed Spacer (ITS) to investigate hybrid zones. Data analyses employed an array of complementary methods, including principal coordinate analyses, Bayesian clustering analyses, maximum likelihood tree-building, and network techniques. Both nuclear molecular systems - AFLP and ITS - provided insights into the evolutionary history of Cedrela in the Yungas in a congruent manner. We uncovered strong support for the occurrence of natural hybridization between C. balansae and C. saltensis. Additionally, we identified hybrid zones in areas of sympatry (at both the Calilegua National Park and the San Andrés farm) and in transition zones from 820 to 1100meters above sea level (localities of Pintascayo and Acambuco). There was no evidence for hybridization of either C. balansae or C. saltensis with C. angustifolia. The role of hybrid populations in conservation and use of genetic resources in the Yungas were discussed.

Effect of latitudinal gradient and impact of logging on genetic diversity of Cedrela lilloi along the Argentine Yungas Rainforest.

Cedrela lilloi C. DC. (cedro coya, Meliaceae), an important south American timber species, has been historically overexploited through selective logging in Argentine Yungas Rainforest. Management and conservation programs of the species require knowledge of its genetic variation patterns; however, no information is available. Molecular genetic variability of the species was characterized to identify high-priority populations for conservation and domestication purposes. Fourteen native populations (160 individuals) along a latitudinal gradient and with different logging's intensities were assessed by 293 polymorphic AFLP (amplified fragment length polymorphism) markers. Genetic diversity was low (Ht = 0.135), according to marginal location of the species in Argentina. Most of the diversity was distributed within populations (87%). Northern populations showed significant higher genetic diversity (R(2)= 0.69) that agreed with latitudinal pattern of distribution of taxonomic diversity in the Yungas. Three clusters were identified by Bayesian analysis in correspondence with northern, central, and southern Yungas. An analysis of molecular variance (AMOVA) revealed significant genetic differences among latitudinal clusters even when logging (Φ(RT) = 0.07) and unlogging populations (Φ(PT) = 0.10) were separately analyzed. Loss of genetic diversity with increasing logging intensity was observed between neighboring populations with different disturbance (Φ(PT) = 0.03-0.10). Bottlenecks in disturbed populations are suggested as the main cause. Our results emphasize both: the necessity of maintaining the genetic diversity in protected areas that appear as possible long-term refuges of the species; and to rescue for the national system of protected areas some high genetic diversity populations that are on private fields.

Molecular identification of new varieties of Nierembergia linariaefolia (Graham), a native Argentinean ornamental plant.

Six Nierembergia linariaefolia clones were selected for ornamental traits during a native germplasm development program. For fingerprinting diagnosis, 13 anchored inter-simple sequence repeat (ISSR) primers and 6 amplified fragment length polymorphism (AFLP) primer-enzyme combinations were used. Both markers revealed high levels of polymorphism, enabling genetic discrimination of the accessions analyzed by using 443 informative ISSRs and 541 AFLP markers. Both molecular techniques are suitable for monitoring genetic diversity in Nierembergia linariaefolia and, under our experimental conditions, they showed correlation coefficients of 0.629 for similarity matrices and of 0.649 in the cophenetic matrices. These results suggest that ISSRs are a good choice for DNA analysis in N. linariaefolia when simple manipulation and a low budget are required.

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.