Genetic and comparative mapping of Lupinus luteus L. highlight syntenic regions with major orthologous genes controlling anthracnose resistance and flowering time.

Anthracnose susceptibility and ill-adapted flowering time severely affect Lupinus luteus yield, which has high seed protein content, is excellent for sustainable agriculture, but requires genetic improvement to fulfil its potential. This study aimed to (1) develop a genetic map; (2) define collinearity and regions of synteny with Lupinus angustifolius; and (3) map QTLs/candidate genes for anthracnose resistant and flowering time. A few linkage groups/genomic regions tended to be associated with segregation distortion, but did not affect the map. The developed map showed collinearity, and syntenic regions with L. angustifolius. Major QTLs were mapped in syntenic regions. Alleles from the wild parent and cultivar, explained 75% of the phenotypic variance for anthracnose resistance and 83% for early flowering, respectively. Marker sequences flanking the QTLs showed high homology with the Lanr1 gene and Flowering-locus-T of L. angustifolius. This suggests orthologous genes for both traits in the L. luteus genome. The findings are remarkable, revealing the potential to combine early flowering/anthracnose resistant in fulfilling yield capacity in L. luteus, and can be a major strategy in the genetic improvement and usage of this species for sustainable protein production. Allele sequences and PCR-marker tagging of these genes are being applied in marker assisted selection.

Development and characterization of microsatellite markers in Gaultheria pumila Lf. (Ericaceae).

BACKGROUND: Polymorphic microsatellite markers were developed for Gaultheria pumila (Ericaceae) to evaluate genetic diversity and population structure within its native range in Chile. This is a very important Ericaceae endemic to Chile with a large commercial potential. Its resistance to different abiotic conditions makes it a valuable target for genetic improvement. RESULTS: Ten polymorphic simple sequence repeat (SSR) loci were isolated from Gaultheria pumila using new-generation 454 FLX Titanium pyrosequencing technology. The mean number of alleles per locus ranged from 2 to 4. Observed and expected heterozygosity ranged from 0.00 to 1.0 and 0.00 to 0.64, respectively. CONCLUSIONS: From 10 SSR markers developed for G. pumila, 9 markers are promising candidates for analyzing genetic variation within or between natural populations of G. pumila and other species from the same genus.

Development and characterization of microsatellite markers in Gaultheria pumila Lf. (Ericaceae)

BACKGROUND: Polymorphic microsatellite markers were developed for Gaultheria pumila (Ericaceae) to evaluate genetic diversity and population structure within its native range in Chile. This is a very important Ericaceae endemic to Chile with a large commercial potential. Its resistance to different abiotic conditions makes it a valuable target for genetic improvement. RESULTS: Ten polymorphic simple sequence repeat (SSR) loci were isolated from Gaultheria pumila using new-generation 454 FLX Titanium pyrosequencing technology. The mean number of alleles per locus ranged from 2 to 4. Observed and expected heterozygosity ranged from 0.00 to 1.0 and 0.00 to 0.64, respectively. CONCLUSIONS: From 10 SSR markers developed for G. pumila, 9 markers are promising candidates for analyzing genetic variation within or between natural populations of G. pumila and other species from the same genus.

In vitro asymbiotic germination for micropropagation of the recalcitrant terrestrial orchid Chloraea crispa (Orchidaceae).

PREMISE OF THE STUDY: Chloraea crispa is a terrestrial Orchidaceae species native to Chile, characterized by a beautiful and showy inflorescence. The species has a great potential for commercial exploitation in the cut flower industry, but it is essential to improve propagation methods to avoid endangering its natural populations. Because this species is hard to propagate using traditional greenhouse techniques, in vitro techniques offer an effective tool for its large-scale production in terms of germination, growth, and propagation. METHODS: The current study evaluated the effect of the culture medium on the asymbiotic germination of C. crispa seeds, as well as the effects of the plant growth regulators 6-benzylaminopurine and indole-3-butyric acid. Different light regimes were also studied. RESULTS: A significant effect was observed for the interaction between culture media and light regime on the morphogenic response of the seeds. The highest rate of embryonic germination was obtained in Van Waes medium supplemented with 0.1 mg·L-1 of 6-benzylaminopurine. DISCUSSION: For the first time, asymbiotic culture of this species using biotechnology tools has been developed. Plantlets developed very well under in vitro conditions, allowing the possibility to propagate and store genetic material for conservation and domestication purposes.

Meristem culture and subsequent micropropagation of Chilean strawberry (Fragaria chiloensis (L.) Duch.).

BACKGROUND: Vegetative propagation of Fragaria sp. is traditionally carried out using stolons. This system of propagation, in addition to being slow, can spread plant diseases, particularly serious being viral. In vitro culture of meristems and the establishment of micropropagation protocols are important tools for solving these problems. In recent years, considerable effort has been made to develop in vitro propagation of the commercial strawberry in order to produce virus-free plants of high quality. These previous results can serve as the basis for developing in vitro-based propagation technologies in the less studied species Fragaria chiloensis. RESULTS: In this context, we studied the cultivation of meristems and establishment of a micropropagation protocol for F. chiloensis. The addition of polyvinylpyrrolidone (PVP) improved the meristem regeneration efficiency of F. chiloensis accessions. Similarly, the use of 6-benzylaminopurine (BAP) in the culture media increased the average rate of multiplication to 3-6 shoots per plant. In addition, the use of 6-benzylaminopurine (BAP), had low levels (near zero) of explant losses due to oxidation. However, plant height as well as number of leaves and roots were higher in media without growth regulators, with average values of 0.5 cm, 9 leaves and 4 roots per plant. CONCLUSIONS: For the first time in Chilean strawberry, meristem culture demonstrated to be an efficient tool for eliminating virus from infected plants, giving the possibility to produce disease free propagation material. Also, the addition of PVP into the basal MS medium improved the efficiency of plant recovery from isolated meristems. Farmers can now access to high quality plant material produced by biotech tools which will improve their technological practices.

Meristem culture and subsequent micropropagation of Chilean strawberry (Fragaria chiloensis (L) Duch. )

BACKGROUND: Vegetative propagation of Fragaria sp. is traditionally carried out using stolons. This system of propagation, in addition to being slow, can spread plant diseases, particularly serious being viral. In vitro culture of meristems and the establishment of micropropagation protocols are important tools for solving these problems. In recent years, considerable effort has been made to develop in vitro propagation of the commercial strawberry in order to produce virus-free plants of high quality. These previous results can serve as the basis for developing in vitro-based propagation technologies in the less studied species Fragaria chiloensis. RESULTS: In this context, we studied the cultivation of meristems and establishment of a micropropagation protocol for F. chiloensis. The addition of polyvinylpyrrolidone (PVP) improved the meristem regeneration efficiency of F. chiloensis accessions. Similarly, the use of 6-benzylaminopurine (BAP) in the culture media increased the average rate of multiplication to 3-6 shoots per plant. In addition, the use of 6-benzylaminopurine (BAP), had low levels (near zero) of explant losses due to oxidation. However, plant height as well as number of leaves and roots were higher in media without growth regulators, with average values of 0.5 cm, 9 leaves and 4 roots per plant. CONCLUSIONS: For the first time in Chilean strawberry, meristem culture demonstrated to be an efficient tool for eliminating virus from infected plants, giving the possibility to produce disease free propagation material. Also, the addition of PVP into the basal MS medium improved the efficiency of plant recovery from isolated meristems. Farmers can now access to high quality plant material produced by biotech tools which will improve their technological practices.

Morphometric and phytochemical characterization of chaura fruits (Gaultheria pumila): a native Chilean berry with commercial potential.

BACKGROUND: For the first time, a morphometric characterization of chaura (Gaultheria pumila) fruits has been conducted between natural populations growing in the Villarrica National Park, Araucania Region, Chile. Chaura is a native Ericaceae from Chile that produces aromatic and tasty fruits which could be of agricultural interest. RESULTS: To influence the decision for a further domestication of G. pumila, both the fruit sizes (indicator of productivity) and the nutritional properties of the fruits have been determined from different subpopulations. Samples were a total of 74 plants and 15 fruits per plant which were randomly harvested following its natural distribution around the Villarrica volcano. Altogether, fresh weight, shape, color, diameter in the pole and the equatorial dimensions were determined as phenotypic traits of the G. pumila fruits. Meanwhile the total soluble solids, anthocyanin and pectin contents were calculated as nutritional traits of the Chaura fruits. Results showed a high phenotypic diversity between the sampled population with three main fruit shapes and three predominant colors. The round shapes were the most abundant, whereas a significant correlation was found among fruit size with weight and color. The highest fresh weight (597.3 mg), pole diameter (7.1 mm) and equatorial diameter (6.5 mm) were estimated in the pink color fruits. CONCLUSIONS: The total amount of anthocyanin was higher in red fruits, while the maximum pectin content was obtained in the round white fruits. Overall results must pave the way for a further domestication and introduction of the Chaura species in the agro-productive system in Chile.

Chlorophyll, anthocyanin, and gas exchange changes assessed by spectroradiometry in Fragaria chiloensis under salt stress.

Chlorophyll and anthocyanin contents provide a valuable indicator of the status of a plant's physiology, but to be more widely utilized it needs to be assessed easily and non-destructively. This is particularly evident in terms of assessing and exploiting germplasm for plant-breeding programs. We report, for the first time, experiments with Fragaria chiloensis (L.) Duch. and the estimation of the effects of response to salinity stress (0, 30, and 60 mmol NaCl/L) in terms of these pigments content and gas exchange. It is shown that both pigments (which interestingly, themselves show a high correlation) give a good indication of stress response. Both pigments can be accurately predicted using spectral reflectance indices (SRI); however, the accuracy of the predictions was slightly improved using multilinear regression analysis models and genetic algorithm analysis. Specifically for chlorophyll content, unlike other species, the use of published SRI gave better indications of stress response than Normalized Difference Vegetation Index. The effect of salt on gas exchange is only evident at the highest concentration and some SRI gave better prediction performance than the known Photochemical Reflectance Index. This information will therefore be useful for identifying tolerant genotypes to salt stress for incorporation in breeding programs.

Genetic structure of the aphid, Chaetosiphon fragaefolii, and its role as a vector of the Strawberry Yellow Edge Virus to a native strawberry, Fragaria chiloensis in Chile.

The monoecious anholocyclical aphid, Chaetosiphon fragaefolii (Cockerell) (Homoptera: Aphididae), was collected on a native strawberry, Fragaria chiloensis (L.) Duchesne (Rosales: Rosaceae) from different sites in Chile. The presence of this aphid was recorded during two consecutive years. F. chiloensis plants were collected from seven natural and cultivated growing areas in central and southern Chile. Aphids were genotyped by cross-species amplification of four microsatellite loci from other aphid species. In addition, the aphid borne virus Strawberry mild yellow edge virus was confirmed in F. chiloensis plants by double-antibody sandwich ELISA and RT-PCR. Genetic variability and structure of the aphid populations was assessed from the geo-referenced individuals through AMOVA and a Bayesian assignment test. The presence of C. fragaefolii, during the two-year study was detected in only four of the seven sites (Curepto, Contulmo, Chilián and Cucao). Genetic variation among these populations reached 19% of the total variance. When assigning the individuals to groups, these were separated in three genetic clusters geographically disjunct. Of the seven sampled sites, six were positive for the virus by RT-PCR, and five by double-antibody sandwich ELISA . The incidence of the virus ranged from 0-100%. Presence of the virus corresponded with the presence of the aphid in all but two sites (Chilian and Vilches). The greatest incidence of Strawberry mild yellow edge virus was related to the abundance of aphids. On the other hand, sequences of the coat protein gene of the different virus samples did not show correspondence with either the genetic groups of the aphids or the sampling sites. The genetic structure of aphids could suggest that dispersal is mainly through human activities, and the spread to natural areas has not yet occurred on a great scale.

Production of haploids and doubled haploids in oil palm.

BACKGROUND: Oil palm is the world's most productive oil-food crop despite yielding well below its theoretical maximum. This maximum could be approached with the introduction of elite F1 varieties. The development of such elite lines has thus far been prevented by difficulties in generating homozygous parental types for F1 generation. RESULTS: Here we present the first high-throughput screen to identify spontaneously-formed haploid (H) and doubled haploid (DH) palms. We secured over 1,000 Hs and one DH from genetically diverse material and derived further DH/mixoploid palms from Hs using colchicine. We demonstrated viability of pollen from H plants and expect to generate 100% homogeneous F1 seed from intercrosses between DH/mixoploids once they develop female inflorescences. CONCLUSIONS: This study has generated genetically diverse H/DH palms from which parental clones can be selected in sufficient numbers to enable the commercial-scale breeding of F1 varieties. The anticipated step increase in productivity may help to relieve pressure to extend palm cultivation, and limit further expansion into biodiverse rainforest.

Comparison of transcriptional profiles of flavonoid genes and anthocyanin contents during fruit development of two botanical forms of Fragaria chiloensis ssp. chiloensis.

Difference in fruit pigmentation observed between two botanical forms of Fragaria chiloensis ssp. chiloensis (form chiloensis and form patagonica) was studied through transcriptional and chemical approaches. The proportion of different anthocyanins was demonstrated to be characteristic of each botanical form, with pelargonidin 3-glucoside being the most abundant in f. patagonica fruit and cyaniding 3-glucoside as the major one in f. chiloensis fruit. Partial gene sequences of the phenylpropanoid and flavonoid biosynthesis pathways were isolated from the native Chilean strawberry fruits, and used to design gene-specific primers in order to perform transcriptional analyses by qRT-PCR. These genes showed spatial, developmental, and genotypic associated patterns. The red fruit of f. patagonica exhibited higher transcript levels of anthocyanin-related genes and higher levels of anthocyanins compared to the barely pigmented fruit of f. chiloensis. The anthocyanin accumulation in F. chiloensis ssp. chiloensis fruits was concomitant with the particular progress of the transcriptional activity of genes involved in the biosynthesis of flavonoid pigments. The differences in anthocyanin contents, both in terms of type and quantity, between the two botanical forms of F. chiloensis ssp. chiloensis were coincident with the differential transcriptional patterns found in the anthocyanin-related genes.

A simple, high throughput method to locate single copy sequences from Bacterial Artificial Chromosome (BAC) libraries using High Resolution Melt analysis.

BACKGROUND: The high-throughput anchoring of genetic markers into contigs is required for many ongoing physical mapping projects. Multidimentional BAC pooling strategies for PCR-based screening of large insert libraries is a widely used alternative to high density filter hybridisation of bacterial colonies. To date, concerns over reliability have led most if not all groups engaged in high throughput physical mapping projects to favour BAC DNA isolation prior to amplification by conventional PCR. RESULTS: Here, we report the first combined use of Multiplex Tandem PCR (MT-PCR) and High Resolution Melt (HRM) analysis on bacterial stocks of BAC library superpools as a means of rapidly anchoring markers to BAC colonies and thereby to integrate genetic and physical maps. We exemplify the approach using a BAC library of the model plant Arabidopsis thaliana. Super pools of twenty five 384-well plates and two-dimension matrix pools of the BAC library were prepared for marker screening. The entire procedure only requires around 3 h to anchor one marker. CONCLUSIONS: A pre-amplification step during MT-PCR allows high multiplexing and increases the sensitivity and reliability of subsequent HRM discrimination. This simple gel-free protocol is more reliable, faster and far less costly than conventional PCR screening. The option to screen in parallel 3 genetic markers in one MT-PCR-HRM reaction using templates from directly pooled bacterial stocks of BAC-containing bacteria further reduces time for anchoring markers in physical maps of species with large genomes.

BAC-HAPPY mapping (BAP mapping): a new and efficient protocol for physical mapping.

Physical and linkage mapping underpin efforts to sequence and characterize the genomes of eukaryotic organisms by providing a skeleton framework for whole genome assembly. Hitherto, linkage and physical "contig" maps were generated independently prior to merging. Here, we develop a new and easy method, BAC HAPPY MAPPING (BAP mapping), that utilizes BAC library pools as a HAPPY mapping panel together with an Mbp-sized DNA panel to integrate the linkage and physical mapping efforts into one pipeline. Using Arabidopsis thaliana as an exemplar, a set of 40 Sequence Tagged Site (STS) markers spanning approximately 10% of chromosome 4 were simultaneously assembled onto a BAP map compiled using both a series of BAC pools each comprising 0.7x genome coverage and dilute (0.7x genome) samples of sheared genomic DNA. The resultant BAP map overcomes the need for polymorphic loci to separate genetic loci by recombination and allows physical mapping in segments of suppressed recombination that are difficult to analyze using traditional mapping techniques. Even virtual "BAC-HAPPY-mapping" to convert BAC landing data into BAC linkage contigs is possible.

Isolation and characterization of microsatellite loci from the woolly apple aphid Eriosoma lanigerum (Hemiptera: Aphididae: Eriosomatinae).

Eight novel microsatellite primer pairs are presented for Eriosoma lanigerum, representing the first microsatellite markers available for this genus. Loci were characterized for 27 individuals from one single orchard in Central Chile. All loci were polymorphic within E. lanigerum (three to 11 alleles per locus; observed heterozygosity ranging from 0.41 to 0.93), and are therefore useful for population genetic studies within the species.

High-resolution melt analysis to identify and map sequence-tagged site anchor points onto linkage maps: a white lupin (Lupinus albus) map as an exemplar.

* The provision of sequence-tagged site (STS) anchor points allows meaningful comparisons between mapping studies but can be a time-consuming process for nonmodel species or orphan crops. * Here, the first use of high-resolution melt analysis (HRM) to generate STS markers for use in linkage mapping is described. This strategy is rapid and low-cost, and circumvents the need for labelled primers or amplicon fractionation. * Using white lupin (Lupinus albus, x = 25) as a case study, HRM analysis was applied to identify 91 polymorphic markers from expressed sequence tag (EST)-derived and genomic libraries. Of these, 77 generated STS anchor points in the first fully resolved linkage map of the species. The map also included 230 amplified fragment length polymorphisms (AFLP) loci, spanned 1916 cM (84.2% coverage) and divided into the expected 25 linkage groups. * Quantitative trait loci (QTL) analyses performed on the population revealed genomic regions associated with several traits, including the agronomically important time to flowering (tf), alkaloid synthesis and stem height (Ph). Use of HRM-STS markers also allowed us to make direct comparisons between our map and that of the related crop, Lupinus angustifolius, based on the conversion of RFLP, microsatellite and single nucleotide polymorphism (SNP) markers into HRM markers.

E-cinnamic acid derivatives and phenolics from Chilean strawberry fruits, Fragaria chiloensis ssp. chiloensis.

Three E-cinnamic acid glycosides, tryptophan, and cyanidin-3-O-beta-D-glucopyranoside were isolated from ripe fruits of the Chilean strawberry Fragaria chiloensis ssp. chiloensis. 1-O-E-Cinnamoyl-beta-D-xylopyranoside, 1-O-E-cinnamoyl-beta-D-rhamnopyranoside, and 1-O-E-cinnamoyl-alpha-xylofuranosyl-(1-->6)-beta-D-glucopyranose are reported for the first time. The cinnamic acid glycosides and aromatic compound patterns in F. chiloensis fruits were determined by high-performance liquid chromatography (HPLC). HPLC analyses of extracts showed that cyanidin-3-O-beta-D-glucopyranoside and free ellagic acid are present in achenes while the E-cinnamoyl derivatives and tryptophan were identified only in the thalamus. The free radical scavenging effect of the fruit extract can be associated with the anthocyanin content.