Transcriptomic Approach for Global Distribution of SNP/Indel and Plant Genotyping.

Single Nucleotide Polymorphisms (SNPs) are the most common structural variants found in any genome. They have been used for different genetic studies, from the understanding of genetic structure of populations to the development of breeding selection markers. In this chapter we present the use of transcriptomic data obtained from contrasting phenotypes for a target trait, in searching of SNPs and insertions/deletions (InDels). This approach has the advantage that the identified markers are in or close to differentially expressed genes, and so they have higher chances to tag the genes underlying the phenotypic expression of a particular trait.

Phenotypic and genetic characterization of an Avena sativa L. germplasm collection of diverse origin: implications for food-oat breeding in Chile.

Oats are known for their nutritional value and also for their beneficial properties on human health, such as the reduction of cholesterol levels and risk of coronary heart disease; they are an important export product for Chile. During the last decade (2010-2022) over 90% of the oat cultivated area in Chile has been covered with Avena sativa L. cv. Supernova INIA. This lack of genetic diversity in a context of climate change could limit the long-term possibility of growing oats in Chile. The present study is a phenotypic and genetic analysis of 132 oat cultivars and pure lines of diverse origin that can be considered as potential breeding material. The germplasm was evaluated for 28 traits and analyzed with 14 SSR markers. The effects of genotypes on phenotype were significant over all traits (P ≤ 0.05). Most traits exhibited moderate to high broad-sense heritability with exceptions such as yield (H2 = 0.27) and hulls staining (H2 = 0.32). Significant undesirable correlations between traits were generally of small biological importance, which is auspicious for achieving breeding objectives. Some of the heritability data and correlations provided here have not been previously reported. The overall phenotypic diversity was high (H' = 0.68 ± 0.18). The germplasm was grouped into three phenotypic clusters, differing in their qualities for breeding. Twenty-six genotypes outperforming Supernova INIA were identified for breeding of conventional food-oats. The genetic diversity of the germplasm was moderate on average (He = 0.58 ± 0.03), varying between 0.32 (AM22) and 0.77 (AME178). Two genetic subpopulations supported by the Structure algorithm exhibited a genetic distance of 0.24, showing low divergence of the germplasm. The diversity and phenotypic values found in this collection of oat genotypes are promising with respect to obtaining genetic gain in the short term in breeding programs. However, the similar genetic diversity, higher phenotypic diversity, and better phenotypic performance of the germplasm created in Chile compared to foreign germplasm suggest that germplasm harboring new genetic diversity will be key to favor yield and quality in new oat cultivars in the long term.

Multiplex TaqMan Real-Time PCR Assay for Sensitive Detection of Two Weevil Species (Coleoptera: Curculionidae).

Rapid and cost-efficient identification of Naupactus species is becoming a key process for the exportation of citrus fruit from Chile and other countries, considering the quarantine regulations for some species of the cosmopolitan genus Naupactus. This study deals with the development of a fast and sensitive detection protocol for Naupactus cervinus (Coleoptera: Curculionidae) (Boheman) and Naupactus xanthographus (Coleoptera: Curculionidae) (Germar) based on multiplex TaqMan Real-time polymerase chain reaction. Both N. cervinus and N. xanthographus primer and probe sets achieved species-specific detection in a linear range from 1 pg/µl to 1 × 10-6 pg/µl, allowing detection of as few as 160 copies of template DNA. Non-target amplifications were not detected and a panel composed of 480 test samples had 100% coincidence with the respective morphological identification.

Identification of SNPs and InDels associated with berry size in table grapes integrating genetic and transcriptomic approaches.

BACKGROUND: Berry size is considered as one of the main selection criteria in table grapes breeding programs, due to the consumer preferences. However, berry size is a complex quantitive trait under polygenic control, and its genetic determination of berry weight is not yet fully understood. The aim of this work was to perform marker discovery using a transcriptomic approach, in order to identify and characterize SNP and InDel markers associated with berry size in table grapes. We used an integrative analysis based on RNA-Seq, SNP/InDel search and validation on table grape segregants and varieties with different genetic backgrounds. RESULTS: Thirty SNPs and eight InDels were identified using a transcriptomic approach (RNA-Seq). These markers were selected from SNP/InDel found among segregants from a Ruby x Sultanina population with contrasting phenotypes for berry size. The set of 38 SNP and InDel markers was distributed in eight chromosomes. Genotype-phenotype association analyses were performed using a set of 13 RxS segregants and 41 table grapes varieties with different genetic backgrounds during three seasons. The results showed several degrees of association of these markers with berry size (10.2 to 30.7%) as other berry-related traits such as length and width. The co-localization of SNP and /or InDel markers and previously reported QTLs and candidate genes associated with berry size were analysed. CONCLUSIONS: We identified a set of informative and transferable SNP and InDel markers associated with berry size. Our results suggest the suitability of SNPs and InDels as candidate markers for berry weight in seedless table grape breeding. The identification of genomic regions associated with berry weight in chromosomes 8, 15 and 17 was achieved with supporting evidence derived from a transcriptome experiment focused on SNP/InDel search, as well as from a QTL-linkage mapping approach. New regions possibly associated with berry weight in chromosomes 3, 6, 9 and 14 were identified.

An Efficient Duplex PCR Method for Sex Identification of the European Grapevine Moth Lobesia botrana (Lepidoptera: Tortricidae) at Any Developmental Stage.

Many genetic studies in insects require sex identification of individuals in all developmental stages. The most common sex chromosome system in lepidopterans is WZ/ZZ; the W chromosome is present only in females. Based on two W chromosome-specific short sequences (CpW2 and CpW5) described in Cydia pomonella (L.) (Lepidoptera: Tortricidae), we identified homologous female-specific sequences in Lobesia botrana Den. & Schiff, a polyphagous and very harmful species present in Chile since 2008. From this starting point, we extended the sequence information using the inverse PCR method, identifying the first W-specific sequences described up to now for the moth. Finally, we developed a duplex PCR method for rapid and sensitive determination of sex in L. botrana from larva to adult. The method showed a detection limit of 1 pg of genomic DNA; a blind panel of samples exhibited exact correspondence with the morphological identification. These results will be very useful for studies requiring sex-specific analyses at any developmental stage, contributing also to the understanding of gene expression in the insect, as well as to the eventual development of control protocols against the moth, such as the development of genetic sexing strains for the implementation of the sterile insect technique.

Simple distinction of grapevine (Vitis vinifera L.) genotypes by direct ATR-FTIR.

The identification of grapevine (Vitis vinifera L.) genotypes is conventionally a laborious activity that must be carried out by specialized staff. In this work a novel and simple method for differentiation of grapevine genotypes is presented. Direct measurements of leaves by attenuated total reflectance Fourier-transform infrared spectroscopy (ATR-FTIR) combined with chemometric methods were used for classification of six genotypes (five varieties and a pair of clones), viz. Cinsault, Gewurztraminer (clone 643), Moscatel de Alejandría, País, Pinot Noir (French clone 777), Pinot Noir (local clone 'Valdivieso'). These were successfully classified and identified through supervised pattern recognition methods such as soft independent modeling of class analogy (SIMCA) and partial least square discriminant analysis (PLS-DA). The error rate for spectra classification of test sets by both models was 0.08. The results demonstrate the advantages of using ATR-FTIR as a rapid and non-destructive tool that achieves accurate grapevine genotype differentiation.

Transcriptomic study of pedicels from GA3-treated table grape genotypes with different susceptibility to berry drop reveals responses elicited in cell wall yield, primary growth and phenylpropanoids synthesis.

BACKGROUND: Gibberellins (GA3) are the most sprayed growth regulator for table grape production worldwide, increasing berry size of seedless varieties through pericarp cell expansion. However, these treatments also exacerbate berry drop, which has a detrimental effect on the postharvest quality of commercialized clusters. Several studies have suggested that pedicel stiffening caused by GA3 would have a role in this disorder. Nevertheless, transcriptional and phenotypic information regarding pedicel responses to GA3 is minimal. RESULTS: Characterization of responses to GA3 treatments using the lines L23 and Thompson Seedless showed that the former was up to six times more susceptible to berry drop than the latter. GA3 also increased the diameter and dry matter percentage of the pedicel on both genotypes. Induction of lignin biosynthesis-related genes by GA3 has been reported, so the quantity of this polymer was measured. The acetyl bromide method detected a decreased concentration of lignin 7 days after GA3 treatment, due to a higher cell wall yield of the isolated fractions of GA3-treated pedicel samples which caused a dilution effect. Thus, an initial enrichment of primary cell wall components in response to GA3 was suggested, particularly in the L23 background. A transcriptomic profiling was performed to identify which genes were associated with these phenotypic changes. This analysis identified 1281 and 1787 genes differentially upregulated by GA3 in L23 and cv. Thompson Seedless, respectively. Concomitantly, 1202 and 1317 downregulated genes were detected in L23 and cv. Thompson Seedless (FDR < 0.05). Gene ontology analysis of upregulated genes showed enrichment in pathways including phenylpropanoids, cell wall metabolism, xylem development, photosynthesis and the cell cycle at 7 days post GA3 application. Twelve genes were characterized by qPCR and striking differences were observed between genotypes, mainly in genes related to cell wall synthesis. CONCLUSIONS: High levels of berry drop are related to an early strong response of primary cell wall synthesis in the pedicel promoted by GA3 treatment. Genetic backgrounds can produce similar phenotypic responses to GA3, although there is considerable variation in the regulation of genes in terms of which are expressed, and the extent of transcript levels achieved within the same time frame.

New microsatellites for the Atacama Desert endemic Balsamocarpon brevifolium (Fabaceae).

PREMISE: Algarrobilla (Balsamocarpon brevifolium, Fabaceae) is an endemic xerophytic shrub restricted to the Atacama Desert in northern Chile. Extensive utilization of the region for coal production has endangered this species. Conservation efforts are underway, with genetic diversity analyses being key to the restoration of these populations. METHODS AND RESULTS: Fifteen new microsatellite markers were developed for B. brevifolium and used to analyze three populations from the Atacama and Coquimbo regions in Chile. Microsatellites were highly polymorphic, with an average of 5.77 alleles per marker and an average level of expected heterozygosity of 0.72. These markers were evaluated and cross-amplified on two related species (Senna cumingii and Caesalpinia angulata) with partial success. CONCLUSIONS: The development of this set of markers permits an extensive study of B. brevifolium populations for conservation purposes.

Comparative Transcriptome Profiling in a Segregating Peach Population with Contrasting Juiciness Phenotypes.

Cold storage of fruit is one of the methods most commonly employed to extend the postharvest lifespan of peaches ( Prunus persica (L.) Batsch). However, fruit quality in this species is affected negatively by mealiness, a physiological disorder triggered by chilling injury after long periods of exposure to low temperatures during storage and manifested mainly as a lack of juiciness, which ultimately modifies the organoleptic properties of peach fruit. The aim of this study was to identify molecular components and metabolic processes underlying mealiness in susceptible and nonsusceptible segregants. Transcriptome and qRT-PCR profiling were applied to individuals with contrasting juiciness phenotypes in a segregating F2 population. Our results suggest that mealiness is a multiscale phenomenon, because juicy and mealy fruit display distinctive reprogramming processes affecting translational machinery and lipid, sugar, and oxidative metabolism. The candidate genes identified may be useful tools for further crop improvement.

Exogenous gibberellic acid application induces the overexpression of key genes for pedicel lignification and an increase in berry drop in table grape.

Most table grape (Vitis vinifera L.) varieties require gibberellic acid (GA3) applications to obtain an adequate berry size in order to satisfy market requirements. However, GA3 treatments also produce severe berry drop in some cultivars, which occurs mainly after a cold storage period during post-harvest. Berry drop in bunches treated with GA3 has been related to the hardening and thickening of the pedicel produced by the over-accumulation of cellulose and its lignification. The main goal of this study was to compare the morphology and gene expression in pedicel samples of genotypes contrasting for berry drop susceptibility. These genotypes are Thompson Seedless, which exhibits a low incidence of berry drop, and a genetic line (Line #23) of INIA's breeding program that is very susceptible to berry drop at harvest and after storage in bunches sprayed with GA3. The parameters measured to study this phenomenon during fruit growth and post-harvest storage included fruit detachment force (FDF), hardness and thickness of the pedicel and berry drop frequency. Histological analyses of pedicel structures at harvest showed an increase in cell size and deposition of lignin in the cortex zone in both contrasting genotypes treated with GA3. The expression profile in both genotypes of the key lignin biosynthesis genes Vv4CL4, VvCCR1L and VvCAD1 analyzed by quantitative real time PCR (qPCR) revealed evident changes in response to GA3 treatments. In particular, gene VvCAD1 is overexpressed (100X) in pedicels of line #23 treated with GA3 after 30 and 45 days in cold storage compared to control. Moreover, the frequency of berry drop was higher for Line #23 treated with GA3 than for the control (23% vs. 1%). Our results suggest that gibberellic acid regulates the expression of the biosynthesis of lignin genes, generating changes in cell wall composition and pedicel structure that result in an increase in berry drop.

Differences in Vvufgt and VvmybA1 Gene Expression Levels and Phenolic Composition in Table Grape (Vitis vinifera L.) 'Red Globe' and Its Somaclonal Variant 'Pink Globe'.

A novel 'Red Globe' (RG)-derived grape variety, 'Pink Globe' (PG), was described and registered as a new genotype, with earlier ripening and sweeter taste than those of RG. Microsatellite analysis revealed that PG and RG are undifferentiable; however, the PG VvmybA1c contains six single-nucleotide polymorphisms within the coding and noncoding region, possibly related to the reduced VvmybA1 expression levels. Conversely, HPLC-DAD-ESI-MS/MS analysis showed significantly lower anthocyanin content in PG skin than in RG skin, and PG had no detectable trihydroxylated anthocyanins. Total flavonols did not differ between the variants, although some quercetin derivate concentrations were lower in PG. HPLC-FLD analysis revealed slightly higher concentrations of epicatechin and a procyanidin dimer in PG seeds, although the antioxidant capacity of crude extracts from either variety did not differ significantly. These differences, particularly in monomeric anthocyanin content, can be attributed to altered activity of a MYB-type transcription factor, reducing Vvufgt expression.

Transcriptome profiling of grapevine seedless segregants during berry development reveals candidate genes associated with berry weight.

BACKGROUND: Berry size is considered as one of the main selection criteria in table grape breeding programs. However, this is a quantitative and polygenic trait, and its genetic determination is still poorly understood. Considering its economic importance, it is relevant to determine its genetic architecture and elucidate the mechanisms involved in its expression. To approach this issue, an RNA-Seq experiment based on Illumina platform was performed (14 libraries), including seedless segregants with contrasting phenotypes for berry weight at fruit setting (FST) and 6-8 mm berries (B68) phenological stages. RESULTS: A group of 526 differentially expressed (DE) genes were identified, by comparing seedless segregants with contrasting phenotypes for berry weight: 101 genes from the FST stage and 463 from the B68 stage. Also, we integrated differential expression, principal components analysis (PCA), correlations and network co-expression analyses to characterize the transcriptome profiling observed in segregants with contrasting phenotypes for berry weight. After this, 68 DE genes were selected as candidate genes, and seven candidate genes were validated by real time-PCR, confirming their expression profiles. CONCLUSIONS: We have carried out the first transcriptome analysis focused on table grape seedless segregants with contrasting phenotypes for berry weight. Our findings contributed to the understanding of the mechanisms involved in berry weight determination. Also, this comparative transcriptome profiling revealed candidate genes for berry weight which could be evaluated as selection tools in table grape breeding programs.

Construction of High Density Sweet Cherry (Prunus avium L.) Linkage Maps Using Microsatellite Markers and SNPs Detected by Genotyping-by-Sequencing (GBS).

Linkage maps are valuable tools in genetic and genomic studies. For sweet cherry, linkage maps have been constructed using mainly microsatellite markers (SSRs) and, recently, using single nucleotide polymorphism markers (SNPs) from a cherry 6K SNP array. Genotyping-by-sequencing (GBS), a new methodology based on high-throughput sequencing, holds great promise for identification of high number of SNPs and construction of high density linkage maps. In this study, GBS was used to identify SNPs from an intra-specific sweet cherry cross. A total of 8,476 high quality SNPs were selected for mapping. The physical position for each SNP was determined using the peach genome, Peach v1.0, as reference, and a homogeneous distribution of markers along the eight peach scaffolds was obtained. On average, 65.6% of the SNPs were present in genic regions and 49.8% were located in exonic regions. In addition to the SNPs, a group of SSRs was also used for construction of linkage maps. Parental and consensus high density maps were constructed by genotyping 166 siblings from a 'Rainier' x 'Rivedel' (Ra x Ri) cross. Using Ra x Ri population, 462, 489 and 985 markers were mapped into eight linkage groups in 'Rainier', 'Rivedel' and the Ra x Ri map, respectively, with 80% of mapped SNPs located in genic regions. Obtained maps spanned 549.5, 582.6 and 731.3 cM for 'Rainier', 'Rivedel' and consensus maps, respectively, with an average distance of 1.2 cM between adjacent markers for both 'Rainier' and 'Rivedel' maps and of 0.7 cM for Ra x Ri map. High synteny and co-linearity was observed between obtained maps and with Peach v1.0. These new high density linkage maps provide valuable information on the sweet cherry genome, and serve as the basis for identification of QTLs and genes relevant for the breeding of the species.

Whole genome comparison between table and wine grapes reveals a comprehensive catalog of structural variants.

BACKGROUND: Grapevine (Vitis vinifera L.) is the most important Mediterranean fruit crop, used to produce both wine and spirits as well as table grape and raisins. Wine and table grape cultivars represent two divergent germplasm pools with different origins and domestication history, as well as differential characteristics for berry size, cluster architecture and berry chemical profile, among others. 'Sultanina' plays a pivotal role in modern table grape breeding providing the main source of seedlessness. This cultivar is also one of the most planted for fresh consumption and raisins production. Given its importance, we sequenced it and implemented a novel strategy for the de novo assembly of its highly heterozygous genome. RESULTS: Our approach produced a draft genome of 466 Mb, recovering 82% of the genes present in the grapevine reference genome; in addition, we identified 240 novel genes. A large number of structural variants and SNPs were identified. Among them, 45 (21 SNPs and 24 INDELs) were experimentally confirmed in 'Sultanina' and six SNPs in other 23 table grape varieties. Transposable elements corresponded to ca. 80% of the repetitive sequences involved in structural variants and more than 2,000 genes were affected in their structure by these variants. Some of these genes are likely involved in embryo development, suggesting that they may contribute to seedlessness, a key trait for table grapes. CONCLUSIONS: This work produced the first structural variants and SNPs catalog for grapevine, constituting a novel and very powerful tool for genomic studies in this key fruit crop, particularly useful to support marker assisted breeding in table grapes.

Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data.

BACKGROUND: Data normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. However, the transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the performance of the target gene(s). This issue has been scarcely approached in woody species such as grapevine. RESULTS: A statistical criterion was applied to select a sub-set of 19 candidate reference genes from a total of 242 non-differentially expressed (NDE) genes derived from a RNA-Seq experiment comprising ca. 500 million reads obtained from 14 table-grape genotypes sampled at four phenological stages. From the 19 candidate reference genes, VvAIG1 (AvrRpt2-induced gene) and VvTCPB (T-complex 1 beta-like protein) were found to be the most stable ones after comparing the complete set of genotypes and phenological stages studied. This result was further validated by qPCR and geNorm analyses. CONCLUSIONS: Based on the evidence presented in this work, we propose to use the grapevine genes VvAIG1 or VvTCPB or both as a reference tool to normalize RNA expression in qPCR assays or other quantitative method intended to measure gene expression in berries and other tissues of this fruit crop, sampled at different developmental stages and physiological conditions.

Analysis of hydroxycinnamic acids derivatives in calafate (Berberis microphylla G. Forst) berries by liquid chromatography with photodiode array and mass spectrometry detection.

Calafate (Berberis microphylla G. Forst) is a Patagonian barberry very rich in anthocyanins and one of the fruits with the highest levels of these polyphenols. Other phenolic compounds have also been described in calafate berries. However, to the best of our knowledge there is no available information on hydroxycinnamic acid derivatives. The complexity of hydroxycinnamic acids determination in calafate berries, due to their structure similarities and the interference of high anthocyanin concentration is addressed by means of solid liquid extraction, followed by solid phase extraction clean-up on MCX columns and HPLC-DAD-ESI-MS/MS. The optimized extraction, clean-up and HPLC separation method allowed the assignation of identity and quantification of 20 hydroxycinnamic acids from calafate fruits. 5-Caffeoylquinic acid was the main compound found in all the studied samples. Other 13 hydroxycinnamoyl quinic acids and 6 caffeic acid esters with aldaric acid derivatives assigned as glucaric acid were also identified. Moreover, the glucaric-based hydroxycinnamic acid derivatives accounted for almost the half of total content of this kind of phenolic compounds. The total concentration of hydroxycinnamic acids derivatives ranged between 0.32±0.00 µmol/g and 8.28±0.01 µmol/g. Effect of ripening and geographical location on hydroxycinnamic acid profiles and concentrations are also evaluated. The methodology allows the determination of hydroxycinnamic acids from calafate despite of the high anthocyanin concentrations, showing a much higher concentration of these acids than other widely consumed berries. Thus suggesting that calafate could be considered a very interesting fruit from the point of view of their nutraceutical composition. However, geographical location and ripening have incidence in levels of studied compounds.

Identification and characterization of microsatellites from calafate (Berberis microphylla, Berberidaceae).

PREMISE OF THE STUDY: Southern barberry or calafate (Berberis microphylla) is a shrub species endemic to the Patagonian region of South America that is used for human consumption. The fruit is very rich in vitamin C and anthocyanins and has a very high antioxidant capacity. There have been only a few genetic studies of this and other closely related species. • METHODS AND RESULTS: Here we present the first 18 microsatellite markers of B. microphylla that were characterized using 66 accessions of calafate from Patagonia. On average, they had 7.6 alleles per marker, with an expected heterozygosity of 0.688. The informativeness of these markers was also evaluated in another 15 Berberis species, including most of the native and endemic Chilean species. • CONCLUSIONS: The results confirm that these new simple sequence repeat markers are very polymorphic and potentially useful in genetic studies in any species of the genus Berberis.

Molecular, genetic and transcriptional evidence for a role of VvAGL11 in stenospermocarpic seedlessness in grapevine.

BACKGROUND: Stenospermocarpy is a mechanism through which certain genotypes of Vitis vinifera L. such as Sultanina produce berries with seeds reduced in size. Stenospermocarpy has not yet been characterized at the molecular level. RESULTS: Genetic and physical maps were integrated with the public genomic sequence of Vitis vinifera L. to improve QTL analysis for seedlessness and berry size in experimental progeny derived from a cross of two seedless genotypes. Major QTLs co-positioning for both traits on chromosome 18 defined a 92-kb confidence interval. Functional information from model species including Vitis suggested that VvAGL11, included in this confidence interval, might be the main positional candidate gene responsible for seed and berry development.Characterization of VvAGL11 at the sequence level in the experimental progeny identified several SNPs and INDELs in both regulatory and coding regions. In association analyses performed over three seasons, these SNPs and INDELs explained up to 78% and 44% of the phenotypic variation in seed and berry weight, respectively. Moreover, genetic experiments indicated that the regulatory region has a larger effect on the phenotype than the coding region. Transcriptional analysis lent additional support to the putative role of VvAGL11's regulatory region, as its expression is abolished in seedless genotypes at key stages of seed development. These results transform VvAGL11 into a functional candidate gene for further analyses based on genetic transformation.For breeding purposes, intragenic markers were tested individually for marker assisted selection, and the best markers were those closest to the transcription start site. CONCLUSION: We propose that VvAGL11 is the major functional candidate gene for seedlessness, and we provide experimental evidence suggesting that the seedless phenotype might be caused by variations in its promoter region. Current knowledge of the function of its orthologous genes, its expression profile in Vitis varieties and the strong association between its sequence variation and the degree of seedlessness together indicate that the D-lineage MADS-box gene VvAGL11 corresponds to the Seed Development Inhibitor locus described earlier as a major locus for seedlessness. These results provide new hypotheses for further investigations of the molecular mechanisms involved in seed and berry development.