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The article presents an optimization of the key parameters for the identification of SNPs in sugarcane using a GBS protocol based on two Illumina NextSeq and NovaSeq platforms. Sugarcane (Saccharum sp.), a world-wide known feedstock for sugar production, bioethanol, and energy, has an extremely complex genome, being highly polyploid and aneuploid. A double-digestion restriction site-associated DNA sequencing protocol (ddRADseq) was tested in four commercial sugarcane hybrids and one high-fibre biotype for the detection of single nucleotide polymorphisms (SNPs). In this work we tested two Illumina sequencing platforms, read size (70 vs. 150 bp), different sequencing coverage per individual (medium and high coverage), and single-reads versus paired-end reads. We also explored different variant calling strategies (with and without reference genome) and filtering schemes [combining two minor allele frequencies (MAFs) with three depth of coverage thresholds]. For the discovery of a large number of novel SNPs in sugarcane, we recommend longer size and paired-end reads, medium sequencing coverage per individual and Illumina platform NovaSeq6000 for a cost-effective approach, and filter parameters of lower MAF and higher depth coverages thresholds. Although the de novo analysis retrieved more SNPs, the reference-based method allows downstream characterization of variants. For the two best performing matrices, the number of SNPs per chromosome correlated positively with chromosome length, demonstrating the presence of variants throughout the genome. Multivariate comparisons, with both matrices, showed closer relationships among commercial hybrids than with the high-fibre biotype. Functional analysis of the SNPs demonstrated that more than half of them landed within regulatory regions, whereas the other half affected coding, intergenic and intronic regions. Allelic distances values were lower than 0.07 when analysing two replicated genotypes, confirming the protocol robustness.

Cultivar pedigrees from two sugarcane origins, 9 Argentine (AR) and 7 American (AM) have been reconstructed, and their genetic similarities (based on coefficient of parentage, COP, estimates) show an average of 0.206 ± 0.054. CP clones that enter the pedigrees of AM cultivars are parents or grandparents of AR cultivars, demonstrating that these genotypes have a strong genetic lineage in common. On average, AR pedigrees are smaller and contain less number of founding species than AM pedigrees. However, the lower height of the former is not explained by the different participation of founding species in the pedigrees. The presence of founding species in AR and AM pedigrees increases with the year of cultivar selection, indicating that more founding species entered the pedigree in recently selected cultivars than in older ones. The ancestry of the 16 cultivars trace back to 3 founding species: Saccharum officinarum, S. barberi, and S. spontaneum, with S. officinarum showing the greatest percentage of participation. As S. sinensis participates in 13 pedigrees and S. robustum in 3, the 5 founding species are present in 2 out of 16 pedigrees. Interestingly, the genus Sorghum enters the pedigree of LCP 85-384 pedigree. Industrial parameters assayed indicate that AR and AM cultivars were preferentially selected for their relatively high sucrose content and sugar yield in accordance with two facts: (i) the genetic background and the sucrose genes of sugarcane hybrids provided by the 68% of participation S. officinarum clones, and (ii) no clones of high fiber S. spontaneum have entered the pedigrees in earlier generations.

The potato species exhibit gametophytic self-incompatibility system controlled by a single S-locus with multiple S-haplotypes. Each S-haplotype carries two genetically linked genes, S-RNase and SLF, controlling the female and male specificities, respectively. In the style, the S-RNase gene codes for an allele-specific ribonuclease that is involved in the rejection of pollen that carries the same S-haplotype. This gene has five conserved regions (C1–C5) and two hypervariable domains located between C2 and C3 that play a role in S-RNase allele specificity. Presently, eight nucleotide sequences of S-RNase alleles from Solanum tuberosum and S. chacoense have been reported in potato species. In an effort to discriminate these S-RNase alleles and eventually detect new allelic variants, these sequences were analyzed by in silico PCR, using primers designed on the basis of four conserved regions, and were further examined by restriction enzyme patterns. The development of the in silico approach led to the creation of a novel dichotomous key which was able to unequivocally identify the eight potato S-RNase alleles already known and eventually detect putative alleles when the size and/or the restriction pattern of the amplicons were different from those predicted. Experimental data validates the utility and efficiency of the dichotomous key.

The Russian wheat aphid (RWA) is one of the most aggressive pests of barley and wheat. The outbreak of RWA occurred in Argentina in 2008 caused serious damage to barley cultivars. The most effective and sustainable method of RWA control is to identify new resistance genes. The purpose of the current research was to map RWA resistance genes in a set of double haploid (DH) lines of the Oregon-Wolfe Barley (OWB) mapping population derived from the cross between OWBDOM and OWBREC. The DH and both parental lines were screened for antixenosis, tolerance and antibiosis to RWA. There was significant variation among the DH lines in most of the traits studied. However, only tolerance resulted in significant quantitative trait loci (QTLs) associated with the molecular markers. Two main QTLs were identified. These explained 90 and 79 % of the variability of foliar area and chlorophyll content, respectively, of infested and control plants. The initial and final foliar area and the variation in foliar area were associated with the same molecular markers on chromosome 2H (BmAc0125, Vrs1, BmAc0144f and BmAg0113e). The positive alleles were provided by OWBDOM. The content of chlorophyll was associated with the marker loci WMC1E8, MWG912, ABC261, MWG2028 and Blp on chromosome 1H, with the positive alleles provided by OWBREC. Both parents contributed to different tolerance traits, with foliar area and chlorophyll content remaining as the plant traits most affected by aphid feeding. The QTLs found in this population are new RWA resistance loci. A sequence homology search was performed to derive the putative function of the genes linked to the QTLs.

Sexual self-incompatibility in wild diploid potato species is controlled by a single multiallelic S-locus encoding a polymorphic stylar ribonuclease (S-RNase) that is responsible for the female function in pollen–pistil recognition. In this study, an approach using PCR-based markers were originally developed to amplify the S-RNase alleles in Solanum chacoense. Subsequently, to investigate their general applicability in Solanum, this molecular approach was successfully tested on S. spegazzinii and S. kurtzianum. Application of PCR-SSCP approach revealed potentially new S-RNase alleles in the three species, demonstrating for the first time the existence of S-RNase genetic variability within and between populations of wild diploid potato species. Species-specific SSCP markers that may be successfully used in gene flow studies was also detected in this investigation.

In several plant species, lesionmimic mutants simulate the diseaseresistance response in the absence of pathogens. Interestingly, some of these mutants confer broadspectrum resistance to diverse pathogens. We previously demonstrated that the HLP (hypersensitivelike phenotype) mutant of bread wheat (Triticum aestivum L.) exhibited spontaneous hypersensitive response (HR) in the absence of any pathogen input. However, when HLP plants showing HR phenotype were challenged with leafrust (Puccinia triticina) they were more resistant than plants of the motherline of comparable developmental stage that did not show spontaneous HR, suggesting that the HLP mutation may confer enhanced resistance to the fungus. In this paper we validate the aforementioned finding in several wheat genetic backgrounds. Two way crosses were performed among the HLP mutant and eight wheat commercial stocks, and third backcross progenies with and without spontaneous HR were challenged with leafrust to investigate the response to the fungus. Backcrosses to cv. Sinvalocho M.A., the mother line, and cv. Purplestraw, highly susceptible to leafrust attack, were used as controls. Third backcross progenies of cvs. Sinvalocho M.A., Purplestraw, Buck Guarani and Pro INTA Imperial bearing spontaneous HR phenotype were more resistant to the fungal pathogen than third backcross progenies that did not carry the HLP mutation. Other four wheat stocks were as healthy as the HLP mutant. As expected, backcross to the motherline demonstrated that the HLP mutation conferred an additional resistance to the already healthy performance displayed by the motherline at adult plant stage. The introgression of the HLP mutation conferred heigh tened leafrust resistance and caused no kernel weight reduction on the backcrossed progenies. Taken together, these data validate the direct use of this type of mutations in diseaseresis tance breeding.

We have investigated the antioxidant status in RPr79/4, a CAT-deficient mutant of barley, and in its motherline, cv. Maris Mink. Seedlings of the CAT-deficient mutant that were grown in a growth chamber under a 14-h photoperiod (200 mol quanta m −2 s −1 ), exhibited higher concentrations of glutathione and ascorbate peroxidase as compared to wild-type plants. An additional mitochondrial MnSOD isoenzyme, was also detected in RPr79/4. When seedlings of the CAT-deficient mutant were grown at higher light intensities (370 mol quanta m − 2 s − 1 ), a Cu/ZnSOD isoform and the cytosolic glutamine synthetase isoenzyme were concomitantly induced. Taken together, these results suggest that several defense mechanisms operating in different subcellular compartments respond in concert to compensate for CAT deficiency in barley seedlings exposed to oxidative stress. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Wild potato species have a gametophytic self-incompatibility system controlled by a single multiallelic S locus. In the style, the S-RNase gene codes for an allele-specific ribonuclease that is involved in the rejection of pollen that carries the same S haplotype. This gene has 5 conserved regions (C1–C5) and highly variable regions outside of these areas that play a role in S-RNase allele specificity. In this work, PCR-mediated amplification of genomic DNA from 2 Solanum chacoense accessions was performed using primers designed on the basis of the C1 and C4 conserved regions. By sequencing the PCR products, a new S-RNase allele (S16) was identified in 1 plant of the QBCM argentinian accession. Comparison of the partial sequence (from C2 to C3) of S16 RNase with those of 11 S-RNase genes of other Solanaceae species showed the highest and the lowest similarity scores within the same plant species (respectively, 71% with the S11 and S13 RNase and 35% with the S2 RNase). Differences at the nucleotide level between S16 and S11 RNase alleles are discussed.Key words: gametophytic self-incompatibility, Solanum chacoense, S16 RNase allele, nucleotide and amino acid sequence variability.

Cloned catalase probes from barley (Hordeum vulgare L.) and maize (Zea mays L.) were used to examine catalase gene expression in greened and etiolated leaves of several barley lines. Etiolated leaves had greater levels of an mRNA detected by barley Cat1, compared with greened leaves, in all lines. In contrast, a Cat2-like mRNA (homologous to Cat2 of maize) was induced by light and accumulated to high levels in greened leaves, compared to the negligible levels detected in etiolated leaves. This suggests that barley contains light-inducible and light-repressible catalase genes. In the catalase-deficient barley mutant RPr 79/4, no hybridization signal was detected when RNA from greened or etiolated leaves was probed with maize Cat2, indicating that this mutant is deficient for the light-induced Cat mRNA. In etiolated seedlings of both RPr 79/4 and its motherline, the level of the Cat1 mRNA increased coordinately with a steady increase in catalase activity. Even though the mutant RPr 79/4 was able to grow to maturity in normal air, it sustained chlorosis and significant head sterility, probably due to the lack of a light-inducible catalase. Although the mutant RPr 79/4 is not completely lacking catalase (EC 1.11.1.6), the loss of the CAT-1 isozyme is evidently harmful. This observation underscores the protective role of catalases in plants.

We have investigated the accumulation of Cat2 and Cat3 catalase transcripts in 6-7-day postimbibition leaves of normally pigmented and pigment-deficient maize seedlings under different light regimes. In seedlings of normal inbred maize lines Cat2 mRNA accumulates to significantly higher levels in either continuous light or a diurnal light/dark cycle than in continuous dark. In contrast to the high levels of the Cat2 message observed in their wild-type siblings, carotenoid-deficient mutants accumulate Cat2 mRNA at barely detectable levels. Mutants deficient in chlorophylls, but having normal carotenoid levels, accumulate normal levels of Cat2 mRNA. This suggests that both light and carotenoids are required for the normal accumulation of the Cat2 message. The steady-state level of Cat3 RNA exhibits a dramatic diurnal variation when seedlings are grown under a 24-hr light/dark cycle. We have previously shown that this variation is at the level of Cat3 gene transcription and is under the control of a novel circadian rhythm. In this study we show that both pigment-deficient mutants and their wild-type siblings exhibit the normal diurnal pattern of Cat3 RNA accumulation. This indicates that photosynthetic pigments, allelic variation, and genetic background do not directly affect the temporal pattern of Cat3 accumulation in leaves. We observed, however, that when normal plants are grown in either continuous light or continuous dark, the Cat3 transcript in leaves is present at uniformly high levels throughout the 24-hr sampling period. Because the Cat3 gene is continually transcribed in leaves in the absence of a cyclic light regime, the normally observed diurnal variation of Cat3 gene expression is apparently the result of a circadian-regulated transcriptional repressor.

The temporal and spatial patterns of expression of the Cat and Sod genes encoding the multiple catalases and superoxide dismutases in maize have been examined throughout stem development. Three stages of stem development have been defined based upon catalase activity profiles and stem internode elongation. At stage 1, catalase activity is low and internodes remain short; at stage 2, catalase activity dramatically increases and internodes rapidly elongate; and at stage 3, catalase activity decreases to levels intermediate to stage 1 and 2, and internode elongation ceases. Zymogram analysis and immunoassays show that only the CAT-3 catalase isozyme is present in the stem, even though both Cat1 and Cat3 mRNAs accumulate throughout stem development. Cat2 mRNA is not detectable in the developing stem. In full-grown stems catalase is localized primarily in the sclerenchyma beneath the epidermis and around the vascular bundles and may possibly play a role in lignification. Unlike catalase, all the superoxide dismutase (SOD) isozymes and transcripts are present in the developing stem. Thus, these two major antioxidant gene-enzyme systems show differential patterns of expression during stem development in maize. © 1992 Wiley-Liss, Inc.

The expression of the Cat and Sod genes encoding the multiple catalases and superoxide dismutases in maize has been studied in maize pollen at anther dehiscence. Pollen from three catalase activity variant inbred lines was examined. Zymorgram analysis, immunoassays, and RNA blots show that, in the lines used, only the CAT-1 catalase isozyme is expressed in mature pollen; the CAT-2 and CAT-3 isozymes are not. The data presented further demonstrate that the Cat1 gene is transcribed and translated after tetrad formation. The relative protein levels of the various superoxide dismutase (SOD) isozymes appear to be similar in pollen and scutella, and correspond to the relatively low levels observed for the different Sod transcripts in these tissues. The presence of "double" transcripts for Sod3 and Sod4A is discussed.

A lesion-mimic mutant was obtained from a mutagenic treatment performed with ethyl methanesulfonate on the Argentine bread wheat (Triticum aestivum) cultivar Sinvalocho M.A. The HLP (hypersensitive-like phenotype) mutant exhibited tiny, discrete, white lesions in the absence of any pathogen, resembling the typical hypersensitive response (HR). The lesions only became evident once the fifth or sixth leaf emerged, and spread at random along the leaf blades and leaf sheaths of the developing plant, including tissues of the spike. Because the lesion-mimic mutant showed no lesions at the seedling stage, the phenotypes of both the mutant and its mother line were identical at this point. Histochemical studies showed that spontaneous hypersensitive-like lesions in the HLP mutant corresponded to cell death. In leaf-rust (Puccinia triticina) infection experiments performed at seedling and adult-plant stages, adult HLP plants showed enhanced resistance to leaf-rust attack compared with plants of Sinvalocho M.A. of comparable developmental stage, suggesting that the HLP mutation may confer increased resistance to the fungus. Because enhanced resistance coincided with the presence of spontaneous HR lesions, activation of HLP plant defence responses appeared to be tightly linked to this phenomenon. Final plant height and yield components in the lesion-mimic mutant did not differ from those of the mother line, indicating that the HLP mutation caused no detrimental pleiotropic effects that significantly affected agronomic performance. These data support the direct use of mutations in disease-resistance breeding.

Field-grown plants of the catalase-deficient mutant RPr79/4 show necrotic lesions in leaves and preferentially die. Initially, necrotic lesions exhibited by RPr79/4 were used to indirectly assess the role of distinct levels of catalase on the survival and agronomic performance of field-grown barley progeny. The segregation of three control traits was also analyzed to eliminate the influence of any obvious meiotic disturbance in case a reduction of plant survival was observed. The RPr79/4 necrotic phenotype had recessive expression in field-grown F1 plants. F2 progeny studies performed in the greenhouse revealed that the inheritance of necrotic lesions was monofactorial, and that the control traits segregated as expected. Progeny test analyses of field-grown F2 plants demonstrated that necrotic homozygous plants died preferentially. While the few surviving necrotic homozygous families were catalase-deficient, healthy homozygous families had normal levels of catalase. Progeny test analyses of the control traits confirmed the inheritance calculated in F2. Taken together, these findings indicate that abnormal segregation of necrotic lesions cannot be attributed to any obvious abnormal meiotic behavior but to the incapacity of catalase-deficient plants to overcome field stress conditions. Thus, catalase deficiency in barley reduced survival and pleiotropically affected the agronomic performance by diminishing seed weight and yield.

Catalase gene expression was characterized in the scutellum of maize seedlings grown at normal (25 degrees C) and elevated temperatures (35 and 40 degrees C). Chronic elevated temperatures reduce scutellar catalase activity most noticeably in the inbred lines W64A and R6-67, which express all three CAT isozymes (CAT-1, CAT-2, and CAT-3). The observed decline in catalase activity is primarily attributed to a decrease in the amount of CAT-2 isozyme, due to diminished levels of the Cat2 transcript. As CAT-2 activity levels are regulated by the trans-acting gene locus Car1, it is possible that the Car1 gene product is inhibited at the elevated temperatures. In maize lines null for CAT-2 or both CAT-2 and CAT-3, the relative levels of Cat1 transcript, although steady throughout the 10 days post-imbibition scutellar profile, are slightly higher with increasing temperatures. This might indicate that, in thermally stressed seedlings, the accumulation and/or stability of Cat1 mRNA might compensate for the lack of Cat2 transcript in a tissue where Cat2 mRNA normally accumulates during the developmental period examined. These observations, along with the drastic reduction in seed germination and seedling height at chronically elevated growth temperatures, suggest that developmental arrest, rather than oxidative stress, might be the cause for the observed results relative to Cat gene expression under such conditions.