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The objective of this study was to test the genetic response to 12 cycles of a recurrent selection program for grain yield and related traits. During two seasons the 39 S-derived families (three/C0–C12 populations) were evaluated under conventional (CT) and non-tillage (NT) systems. Grain and biomass yield were determined. The grain number per square meter and harvest index were also estimated. Flour protein content, sodium dodecyl sulphate sedimentation, and lactic acid solvent retention capacity were considered as end-use quality predictive tests. High molecular weight glutenin subunits (HMW-GS) variation was also analyzed. A significant increase in grain yield per selection cycle, estimated in 3.25 g m−2 (1.3%) per year, was found under NT, but there was a not significant genetic progress under CT. The decrease in the percentage of protein in the most advanced selection cycles (11.0% vs. 9.7%) did not negatively affect its quality. HMW-GS variation analysis showed the erosion of extreme subunits related to gluten strength. After 24 years of recurrent selection, the grain yield was improved without affecting its industrial quality.

Seed storage proteins (gliadins and glutenins) play a key role in the determination of dough and bread-making quality in bread wheat. This is due to the interaction between high and low molecular w...

Background: Plant miRNAs are a class of small non-coding RNAs that can repress gene expression at the post-transcriptional level by targeting RNA degradation or promoting translational repression. There is increasing evidence that some miRNAs can derive from a group of non-autonomous class II transposable elements called Miniature Inverted-repeat Transposable Elements (MITEs). Results: We used public small RNA and degradome libraries from Triticum aestivum to screen for microRNAs production and predict their cleavage target sites. In parallel, we also created a comprehensive wheat MITE database by identifying novel elements and compiling known ones. When comparing both data sets, we found high homology between MITEs and 14% of all the miRNAs production sites detected. Furthermore, we show that MITE-derived miRNAs have preference for targeting degradation sites with MITE insertions in the 3’ UTR regions of the transcripts. Conclusions: Our results revealed that MITE-derived miRNAs can underlay the origin of some miRNAs and potentially shape a regulatory gene network. Since MITEs are found in millions of insertions in the wheat genome and are closely linked to genic regions, this kind of regulatory network could have a significant impact on the post-transcriptional control of gene expression., Fil: Crescente, Juan M. CONICET; Argentina, Fil: Zavallo, Diego. CONICET. INTA. Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CICVyA; Argentina, Fil: del Vas, Mariana. CONICET. INTA. Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CICVyA; Argentina, Fil: Asurmendi, Sebastián. CONICET. INTA. Instituto de Agrobiotecnología y Biología Molecular (IABIMO), CICVyA; Argentina, Fil: Helguera, Marcelo. EEA INTA; Argentina, Fil: Fernandez, Elmer. Universidad Católica de Córdoba. Centro de Investigación y Desarrollo en Inmunología y Enfermedades Infecciosas (CIDIE-CONICET); Argentina, Fil: Fernandez, Elmer. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales; Argentina, Fil: Vanzetti, Leonardo S. CONICET; Argentina, Fil: Vanzetti, Leonardo S. EEA INTA; Argentina

Grain characteristics (hardness, protein content/quality, starch properties, enzymatic activity, etc.) play an important role in the definition of end use quality for wheat-based products. Among them, gluten strength and extensibility, mostly determined by glutenin and gliadin composition, are two of the main factors that determine gluten quality. The complex inheritance of most quality traits has led to the development of indirect tests used in breeding for early and advanced generation selection. The main focus of breeders is adding resistance to biotic stress (fungi, insects, nematodes, etc.) and increasing grain yield while selection for quality often occurs in later generations. This often results in the propagation of poor quality lines that must be later discarded. Evaluation of quality in early generations requires suitable tests, preferably non-destructive. Increasing knowledge of the genes involved in quality will facilitate more precise and effective selection. Recent advances in wheat genome sequencing and the extensive genotyping of mapping populations has led to a precise molecular characterization of high molecular weight (HMW) and low molecular weight (LMW) glutenins, as well as the discovery of genes associated with quality traits like grain hardness, starch composition (e.g., waxy genes), etc. Massive genomic data will impact in breeding programs allowing quality fine tuning by precise selection of glutenins, starch, hardness and other traits, for specific end uses through marker assisted selection, genomic selection, etc. This chapter will describe different methods used for quality selection in breeding programs and research, and some examples of integration of local breeding programs with the extremely diverse end-uses of wheat based on a series of case-studies. Current and potential approaches to quality evaluation in durum wheat, wild relatives and synthetic wheat breeding programs will be also presented.

Bread wheat (Triticum aestivum L.) is a staple food crop eaten in different ways like pan and other food products. High molecular weight glutenin subunits (HMW-GS) are major determinants of the different wheat end-use qualities. Ethyl-methanesulfonate (EMS) mutagenized populations in plants can be used for the discovery of valuable mutants for basic research and breeding purposes. In this study, we report the identification of 27 HMW-GS M3 mutants based on SDS-PAGE patterns from an EMS mutagenized population of the cultivar Baguette Premium 11. Nine mutations were detected in Ax2*, five in Bx7, four in By8, six in Dx5 and three in Dy10 subunit. Two Ax2* null mutants were characterized at molecular level finding in both cases premature stop codons associated. EMS would tend to generate more premature stop codons in glutenins genes than in others because these have a high frequency of glutamine codons. This type of mutation generates null alleles, therefore they are easily detectable by a low cost protocol like SDS-PAGE. The potential use of knock-out (null alleles) and SDS-PAGE size altered mutants for HMW-GS in wheat quality and nutrition is discussed.

Next generation sequencing (NGS) and assembly are key pieces in the success of cereal crops genomes sequencing. One interesting strategy for reducing the complexity in large genomes, the case of several cereal crops, is the sequencing of individual chromosomes. This has been done with success by flow cytometric chromosome sorting followed by sequencing using available next generation (high throughput) sequencing platforms. In this chapter, methodologies for sequencing and assembly of flow sorted chromosomes and whole genomes in cereal crops, with special emphasis on the case of the International Wheat Genome Sequencing Consortium (IWGSC), are reviewed.

Next generation sequencing (NGS) and assembly are key pieces in the success of cereal crops genomes sequencing. One interesting strategy for reducing the complexity in large genomes, the case of several cereal crops, is the sequencing of individual chromosomes. This has been done with success by flow cytometric chromosome sorting followed by sequencing using available next generation (high throughput) sequencing platforms. In this chapter, methodologies for sequencing and assembly of flow sorted chromosomes and whole genomes in cereal crops, with special emphasis on the case of the International Wheat Genome Sequencing Consortium (IWGSC), are reviewed.

The occurrence of flowering time in wheat is affected not only by the vernalization and photoperiod response, but also by earliness per se (EPS). The effects of this trait facilitate the fine tunning of the life cycle for regional adaptation within mega environments. To contribute to the knowledge of the wheat germplasm grown in Argentina and to the understanding of the genetic basis underlying variation in EPS, this study aimed to characterize EPS in a set of modern local bread wheat varieties and to develop a biparental recombinant inbred line (RIL) population based on parental contrasting for EPS. The variation observed in flowering time, which was measured under satisfied vernalization and photoperiod demands and assignment of chromosomic regions by QTL analysis, constitutes the first genetic dissection of EPS control in Argentine wheat adapted materials, providing valuable information for regional adaptation in breeding programs. Based on EPS variation analysis, Baguette Premium 11 (late-heading variety) and BioINTA 2001 (early-heading variety) were selected to develop the mapping population. Six QTLs for EPS were detected in the biparental RIL population and QTL QEps.imj-5D1, in chromosome 5D, showed the greatest effect on EPS and is the first QTL to be reported in this chromosome.

Understanding genes regulating wheat plant height is important to optimize harvest index and maximize grain yield. In modern wheat varieties grown under high-input conditions, the gibberellin-insensitive semi-dwarfing alleles Rht-B1b and Rht-D1b have been used extensively to confer lodging tolerance and improve harvest index. However, negative pleiotropic effects of these alleles (e.g., poor seedling emergence and reduced biomass) can cause yield losses in hot and dry environments. As part of current efforts to diversify the dwarfing alleles used in wheat breeding, we identified a quantitative trait locus (QHt.ucw-6AS) affecting plant height in the proximal region of chromosome arm 6AS (< 0.4 cM from the centromere). Using a large segregating population (~ 2800 gametes) and extensive progeny tests (70–93 plants per recombinant family), we mapped QHt.ucw-6AS as a Mendelian locus to a 0.2 cM interval (144.0–148.3 Mb, IWGSC Ref Seq v1.0) and show that it is different from Rht18. QHt.ucw-6AS is officially designated as Rht25, with Rht25a representing the height-increasing allele and Rht25b the dwarfing allele. The average dwarfing effect of Rht25b was found to be approximately half of the effect observed for Rht-B1b and Rht-D1b, and the effect is greater in the presence of the height-increasing Rht-B1a and Rht-D1a alleles than in the presence of the dwarfing alleles. Rht25b is gibberellin-sensitive and shows significant pleiotropic effects on coleoptile length, heading date, spike length, spikelet number, spikelet density, and grain weight. Rht25 represents a new alternative dwarfing locus that should be evaluated for its potential to improve wheat yield in different environments. Fil: Mo, Youngjun. Rural Development Administration; Corea del Sur. University of California; Estados Unidos Fil: Vanzetti, Leonardo Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Hale, Iago. University Of new Hampshire; Estados Unidos Fil: Spagnolo, Emiliano J.. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Guidobaldi, Fabio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Al-Oboudi, Jassim. University of California; Estados Unidos Fil: Odle, Natalie. University of California; Estados Unidos Fil: Pearce, Stephen. Colorado State University; Estados Unidos Fil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria; Argentina Fil: Dubcovsky, Jorge. Howard Hughes Medical Institute; Estados Unidos. University of California; Estados Unidos

Background: Miniature inverted-repeat transposable elements (MITEs) are short, non-autonomous class II transposable elements present in a high number of conserved copies in eukaryote genomes. An accurate identification of these elements can help to shed light on the mechanisms controlling genome evolution and gene regulation. The structure and distribution of these elements are well-defined and therefore computational approaches can be used to identify MITEs sequences. Results: Here we describe MITE Tracker, a novel, open source software program that finds and classifies MITEs using an efficient alignment strategy to retrieve nearby inverted-repeat sequences from large genomes. This program groups them into high sequence homology families using a fast clustering algorithm and finally filters only those elements that were likely transposed from different genomic locations because of their low scoring flanking sequence alignment. Conclusions: Many programs have been proposed to find MITEs hidden in genomes. However, none of them are able to process large-scale genomes such as that of bread wheat. Furthermore, in many cases the existing methods perform high false-positive rates (or miss rates). The rice genome was used as reference to compare MITE Tracker against known tools. Our method turned out to be the most reliable in our tests. Indeed, it revealed more known elements, presented the lowest false-positive number and was the only program able to run with the bread wheat genome as input. In wheat, MITE Tracker discovered 6013 MITE families and allowed the first structural exploration of MITEs in the complete bread wheat genome. Fil: Crescente, Juan Manuel. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Zavallo, Diego. Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina Fil: Vanzetti, Leonardo Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigaciones Agropecuarias. Instituto de Fisiología y Recursos Genéticos Vegetales; Argentina

El constante incremento de las temperaturas –como consecuencia del cambio climático– en las regiones donde se cultiva trigo, exige ajustar el ciclo del cultivo a períodos más cortos para evitar o disminuir los estreses causados por las altas temperaturas. En este sentido, el descubrimiento de fuentes genéticas innovadoras de modulación de la espigazón, así como también el desarrollo de herramientas moleculares que permitan capitalizarlas, tienen un rol central en el mejoramiento de este cultivo. En el presente estudio se evalúa, bajo condiciones controladas, el efecto de los genes Ppd-1 sobre la espigazón en una población biparental de trigo pan. Para tal fin, se desarrollaron marcadores moleculares inéditos para los genes Ppd-A1 y Ppd-B1. Los resultados obtenidos muestran un efecto desigual de los loci Ppd-1 (Ppd-A1, Ppd-B1 y Ppd-D1) sobre la respuesta al fotoperíodo. Ppd-D1 fue el locus con mayor efecto sobre la respuesta al fotoperíodo, mientras que Ppd-B1 y Ppd-A1 presentaron un efecto menor y nulo, respectivamente. Se concluye de este estudio, que la estimulación en condiciones de días largos no es un proceso esencial para que se produzca la espigazón en el trigo. The constant increase in temperatures, as a consequence of climate change, affects the regions where wheat is grown and thus demands an adjustment of crop cycle to shorter periods with the purpose of decreasing the high temperature stress. Therefore, the discovery of new genetic sources of flowering modulation and the development of molecular tools that enable their use, take a main role in wheat breeding. In this study, the effect of Ppd-1 on flowering time was evaluated in a bread wheat biparental population, under controlled conditions. Novel molecular markers for Ppd-A1 and Ppd-B1 genes were developed. The results showed an unequal effect of Ppd-1 loci (Ppd-A1, Ppd-B1 and Ppd-D1) on photoperiod response. Ppd-D1 was the locus with the greatest effect on photoperiod response, while Ppd-B1 and Ppd-A1 presented minor and null effects, respectively. It could be concluded that long day stimulation is not essential to wheat flowering. EEA Marcos Juárez Fil: Lombardo, Lucio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Nisi, María Mercedes. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Ghione, Celina Elena. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Fisore, G.D. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina

MS INTA 416 is a hard red winter wheat selected for high yield potential and good bread-making quality, combined with moderate resistance to Fusarium-head-blight and high resistance to leaf-rust, due mainly to presence of resistance genes Fhb1 and Lr47. MS INTA 416 is adapted to main production areas of Central-Argentina Fil: Bainotti, Carlos Tomas. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Lewis, Silvina. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Recursos Biológicos; Argentina Fil: Campos, Pablo Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bordenave; Argentina Fil: Alberione, Enrique Javier. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Salines, Nicolas. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Gomez, Dionisio Tomas. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Fraschina, Jorge Alberto. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Salines, José Héctor. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Formica, Maria Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Donaire, Guillermo Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Vanzetti, Leonardo Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Lombardo, Lucio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Nisi, María Mercedes. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Cuniberti, Martha Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Mir, Leticia Raquel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Conde, María Belén. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina Fil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez; Argentina

Background: Research projects often involve observation, registration, and data processing starting from information obtained in field experiments. In many cases, these tasks are carried out by several persons in different places, times, and ways, adding different levels of complexity and error in data collecting. Furthermore, data processing can be time consuming, and input errors may produce unwanted results. Results: We have developed a novel, open source software called Phenobook, an easy, flexible, and intuitive tool to organize, collect, and save experimental data for further analyses. Phenobook was conceived to collect phenotypic observations in a user-friendly, cost-effective way. It consists of a web-based software for experiment design, data input and visualization, and exportation, combined with a mobile application for remote data collecting. We provide in this article a detailed description of the developed tool. Conclusion: Phenobook is a software tool that can be easily implemented in collaborative research and development projects involving data collecting and forward analyses. Adopting Phenobook is expected to improve the involved processes by minimizing input errors, resulting in higher quality and reliability of the research outcomes Fil: Crescente, Juan Manuel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Guidobaldi, Fabio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Demichelis, Melina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Formica, Maria Beatriz. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Vanzetti, Leonardo Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Predicting phenology, in particular heading time, is crucial to avoid and/or minimize risk of damage offrost, drought and high temperatures during grain filling. Although some of the major genes controllingdevelopment, associated with photoperiod and vernalization responses, were identified, the associationbetween the molecular characterization of those genes and photoperiod sensitivity, vernalizationresponses and earliness per se has been poorly studied. The present study was conducted to determinethe effects of photoperiod and vernalization genes (and their allelic combination characterized by molecularapproach) on heading time and its correlation with the phenological parameters determined by fieldexperiments in a wide range of Argentine bread wheat commercial cultivars. Additionally, the associationbetween photoperiod and vernalization responses with earliness per se was analyzed. Molecularcharacterization showed that most of the commercial Argentine wheat cultivars available in the marketcorrespond to spring growth habit with dominant insensitive photoperiod alleles (SI) followed by springhabit sensitive to photoperiod (SS), while winter insensitive (WI) habit represented a minority group. Allgenotypes included in the present study (even those classified as SI and WI) were photoperiod sensitivewhen that trait was quantified from a physiological analysis as the slope of the relationship betweenduration of a particular phase and mean photoperiod sensed during the period between emergence andheading. SI showed lower photoperiod sensitivity than SS and WI, without clear differences betweenboth later groups. In all cases, photoperiod sensitivity was the main attribute that determined the differencesin time to heading even when vernalization requirements were not completely fulfilled in the WI.The genotypes with different photoperiod and vernalization allele combinations showed a wide range ofduration of earliness per se. However, differences in earliness per se did not show any particular associationwith the groups classified by molecular markers for photoperiod and vernalization. The informationincluded in the present study can be used to build a gene-based model for predicting phenology. However,the variations in photoperiod and/or vernalization sensitivity within the same allelic combinationcould still determine mismatching in the prediction of the models based on Ppd-1 and Vrn-1 genes. Fil: Gomez, Dionisio Tomas. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuario Marcos Juarez; Argentina Fil: Vanzetti, Leonardo Sebastián. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuario Marcos Juarez; Argentina Fil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuario Marcos Juarez; Argentina Fil: Lombardo, Lucio Andrés. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuario Marcos Juarez; Argentina Fil: Fraschina, Jimena. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuario Marcos Juarez; Argentina Fil: Miralles, Daniel Julio. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Fisiológicas y Ecológicas Vinculadas a la Agricultura; Argentina. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Vegetal. Cátedra de Cerealicultura; Argentina

In wheat, stem elongation phase (SEP) duration is critical for grain number (GN) per unit of area determination, as it is the phase in which the spikes grow. Lengthening SEP, for instance by photoperiodic sensitivity, without altering the cycle to anthesis (AT) has been proposed as an alternative way to increase spike dry weight, and in turn GN. As most works supporting this idea have modified only SEP by artificial manipulation (e.g. photoperiod extensions), it is relevant to evaluate this hypothesis in populations segregating for this attribute in natural conditions. The aim of this work was to analyse the variability in SEP duration relative to AT in two F4 populations; in order to select contrasting phenotypes to evaluate the impact of this attribute on grain yield components and to analyse the selection response of this attribute. These segregating populations (Las Rosas INTA × Triguero 230 (A) and Klein Estrella × ProINTA B. Alazan (B)) were derived from parental lines with similar flowering time but different relative duration of their pre-anthesis phases. Two field experiments with previous vernalization treatment in cool chamber were carried out. In 2006, F4 populations were characterized and from one of them (population B, which presented higher variability) four groups were selected, which presented contrasting phenotypes in the attribute under study. Progenies of these groups (F5), together with remnant F4 full-sib of each one, were studied during 2007. Grain yield per plant was higher, due to GN increases, when duration of the SEP was lengthened. However, selection response to longer SEP with similar cycle to AT could not be found, possibly as the result of a high environmental influence on this attribute. The phenotypic variability evidenced in this attribute was not clearly associated with major adaptation genes evaluated (i.e. Ppd and/or Vrn), suggesting that other minor genes could be associated.

Background: The number and complexity of repetitive elements varies between species, being in general most represented in those with larger genomes. Combining the flow-sorted chromosome arms approach to genome analysis with second generation DNA sequencing technologies provides a unique opportunity to study the repetitive portion of each chromosome, enabling comparisons among them. Additionally, different sequencing approaches may produce different depth of insight to repeatome content and structure. In this work we analyze and characterize the repetitive sequences of Triticum aestivum cv. Chinese Spring homeologous group 4 chromosome arms, obtained through Roche 454 and Illumina sequencing technologies, hereinafter marked by subscripts 454 and I, respectively. Repetitive sequences were identified with the RepeatMasker software using the interspersed repeat database mips-REdat_v9.0p. The input sequences consisted of our 4DS454 and 4DL454 scaffolds and 4ASI, 4ALI, 4BSI, 4BLI, 4DSI and 4DLI contigs, downloaded from the International Wheat Genome Sequencing Consortium (IWGSC). Results: Repetitive sequences content varied from 55% to 63% for all chromosome arm assemblies except for 4DLI, in which the repeat content was 38%. Transposable elements, small RNA, satellites, simple repeats and low complexity sequences were analyzed. SSR frequency was found one per 24 to 27 kb for all chromosome assemblies except 4DLI, where it was three times higher. Dinucleotides and trinucleotides were the most abundant SSR repeat units. (GA)n/(TC)n was the most abundant SSR except for 4DLI where the most frequently identified SSR was (CCG/CGG)n. Retrotransposons followed by DNA transposons were the most highly represented sequence repeats, mainly composed of CACTA/En-Spm and Gypsy superfamilies, respectively. This whole chromosome sequence analysis allowed identification of three new LTR retrotransposon families belonging to the Copia superfamily, one belonging to the Gypsy superfamily and two TRIM retrotransposon families. Their physical distribution in wheat genome was analyzed by fluorescent in situ hybridization (FISH) and one of them, the Carmen retrotransposon, was found specific for centromeric regions of all wheat chromosomes. Conclusion: The presented work is the first deep report of wheat repetitive sequences analyzed at the chromosome arm level, revealing the first insight into the repeatome of T. aestivum chromosomes of homeologous group 4. Fil: Garbus, Ingrid. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida(i); Argentina. Universidad Nacional del Sur; Argentina Fil: Romero, José Rodolfo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida(i); Argentina. Universidad Nacional del Sur; Argentina Fil: Valarik, Miroslav . Centre of the Region Haná for Biotechnological and Agricultural Research. Institute of Experimental Botany; República Checa Fil: Vanžurová, Hana . Centre of the Region Haná for Biotechnological and Agricultural Research. Institute of Experimental Botany; República Checa Fil: Karafiátová, Miroslava . Centre of the Region Haná for Biotechnological and Agricultural Research. Institute of Experimental Botany; República Checa Fil: Cáccamo, Mario . Norwich Research Park. The Genome Analysis Centre; Reino Unido Fil: Doležel, Jeroslav . Centre of the Region Haná for Biotechnological and Agricultural Research. Institute of Experimental Botany; República Checa Fil: Tranquilli, Gabriela . Instituto Nacional de Tecnología Agropecuaria. Centro Nacional de Investigaciones Agropecuarias. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; Argentina Fil: Helguera, Marcelo . Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuario Marcos Juarez; Argentina Fil: Echenique, Carmen Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Bahía Blanca. Centro de Recursos Naturales Renovables de la Zona Semiárida(i); Argentina. Universidad Nacional del Sur; Argentina

Highlights • Survey sequence of T. aestivum chromosome 4D was obtained by pyrosequencing. • Near 5700 genes were predicted on 4D chromosome, ∼2200 on 4DS and ∼3500 on 4DL. • A 4D virtual gene order based on synteny with orthologous gene loci is proposed. • Among group 4, higher collinearity exists between 4D and 4B as compared to 4A. • Complementary data to that provided by IWGSC is presented, available at NCBI., Survey sequencing of the bread wheat (Triticum aestivum L.) genome (AABBDD) has been approached through different strategies delivering important information. However, the current wheat sequence knowledge is not complete. The aim of our study is to provide different and complementary set of data for chromosome 4D. A survey sequence was obtained by pyrosequencing of flow-sorted 4DS (7.2×) and 4DL (4.1×) arms. Single ends (SE) and long mate pairs (LMP) reads were assembled into contigs (223 Mb) and scaffolds (65 Mb) that were aligned to Aegilops tauschii draft genome (DD), anchoring 34 Mb to chromosome 4. Scaffolds annotation rendered 822 gene models. A virtual gene order comprising 1973 wheat orthologous gene loci and 381 wheat gene models was built. This order was largely consistent with the scaffold order determined based on a published high density map from the Ae. tauschii chromosome 4, using bin-mapped 4D ESTs as a common reference. The virtual order showed a higher collinearity with homeologous 4B compared to 4A. Additionally, a virtual map was constructed and ∼5700 genes (∼2200 on 4DS and ∼3500 on 4DL) predicted. The sequence and virtual order obtained here using the 454 platform were compared with the Illumina one used by the IWGSC, giving complementary information.

New leaf rust resistance genes are needed in wheat (Triticum aestivum L.) to provide additional sources of resistance to the highly variable and dynamic leaf rust pathogen Puccinia triticina Eriks. Leaf rust resistance gene Lr51, located within a segment of Triticum speltoides Taush chromosome IS translocated to the long arm of chromosome 1B of bread wheat, is resistant to the current predominant races of leaf rust in the USA. The objectives of this study were to determine the genetic length of the translocated IS segment, develop a PCR marker for Lr51, and use this marker to generate isogenic lines for this gene. Characterization of two translocation lines (F-7-3 and F-7-12) with 10 molecular markers indicated that F-7-3 has an interstitial T. speltoides chromosome segment of 14 to 32 cM long including loci XAga7 and Xmwg710, whereas line F-7-12 has a complex series of translocations among chromosomes of homeologous group 1. On the basis of the DNA sequence of the A, B, D, and S alleles of the XAga7 locus, we designed a cleavage amplified polymorphic sequence (CAPS) marker for the S genome allele. Primers S30-13L and AGA7-759R preferentially amplified the XAga7 1S (819 bp) and 1B alleles (783 bp). These amplification products can be separated in agarose gels after digestion with PstI or BamHI restriction enzymes. This CAPS marker was validated in a collection of 32 common wheat cultivars and was used to develop three pairs of hard red spring isogenic lines from the donor parent F-7-3. These isogenic lines will be valuable for future assessment of the effect of this chromosome introgression on agronomic performance and end-use quality.

Rust resistance genes Lr37, Sr38, and Yr17 are located within a segment of Triticum ventricosum (Tausch) Cess. chromosome 2NS translocated to the short arm of bread wheat chromosome 2AS. Characterization of this chromosome segment by 13 restriction fragment length polymorphism (RFLP) markers indicated that the 2NS translocation replaced approximately half of the short arm of chromosome 2A (distal 25-38 centimorgans, cM). The objective of this study was to develop polymerase chain reaction (PCR) assays based on RFLP marker cMWG682 to facilitate the transfer of this cluster of rust resistance genes into commercial wheat (Triticum aestivum L.) cultivats. DNA sequence was obtained from the A-, B-, D-, and N-alleles of cMWG682 and was used to design N-allele specific primers. The 2NS fragment amplified by PCR primers cosegregated with the presence of the RFLP-2NS band in all backcross populations. A cleaved amplified polymorphic sequence (CAPS) was used to develop a marker for the 2A-allele. This marker can be used to differentiate homozygous and heterozygous plants carrying the 2NS translocation in the final cycle of backcross introgression or in screenings for homozygous plants in segregating populations. Finally, a third PCR assay was developed by means of TaqMan technology as a high-throughput alternative for selection of the 2NS/2AS translocation in large segregating populations in breeding programs that have access to real time PCR equipment. These molecular markers were used to develop four hard red spring isogenic lines homozygous for the 2NS chromosome segment. One of the isogenic lines, derived from 'Anza,' did not show the expected resistance in spite of the presence of all the RFLP markers for the 2NS chromosome segment. Analysis of F1 hybrids suggested that a suppressor of the Lr37 gene is present in Anza. These isogenic lines will provide a valuable tool to test the effects of the large 2NS translocation on quality and agronomic performance.

With 3 figures and 1 table Abstract This work describes a novel null Wx-A1 allele detected in one cultivar and two breeding lines from a collection of 48 durum wheats from Argentina. The electrophoretic patterns of waxy proteins showed 45 accessions displaying Wx-A1 and Wx-B1 proteins and three accessions that lacked the Wx-A1 protein. Partial sequence of the Wx-A1 gene obtained from the three lines lacking the Wx-A1 protein showed a 1-bp deletion in exon 6 that produces a frameshift and a premature stop codon in the gene sequence. In addition, a PCR-RFLP marker suitable to detect the null Wx-A1 allele in segregating populations is described.

The influence of high molecular weight glutenin subunits (HMW-GS) on wheat breadmaking quality has been extensively studied but the effect of different Glu-1 alleles on cookie quality is still poorly understood. This study was conducted to analyze the effect of HMW-GS composition and wheat-rye translocations on physicochemical flour properties and cookie quality of soft wheat flours. Alleles encoded at Glu-A1, Glu-B1 and Glu-D1 locus had a significant effect over physicochemical flour properties and solvent retention capacity (SRC) profile. The null allele for Glu-A1 locus presented the highest cookie factor observed (CF = 7.10), whereas 1BL/1RS and 1AL/1RS rye translocations had a negative influence on CF. The three cultivars that showed the highest CF (19, 44 and 47) had the following combination: Glu-A1 = null, Glu-B1 = 7 + 8, Glu-D1 = 2 + 12 and no secalins. Two prediction equations were developed to estimate soft wheat CF: one using the HMW-GS composition and the other using physicochemical flour parameters, where SRCsuc, SRC carb, water-soluble pentosans, damaged starch and protein turned out to be better CF predictors. This data suggests that grain protein allelic composition and physicochemical flour properties can be useful tools in breeding programs to select soft wheat of good cookie making quality. Fil: Moiraghi, Malena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentina Fil: Vanzetti, Leonardo Sebastián. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Marcos Juarez; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Pflüger, Laura. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación de Recursos Naturales. Instituto de Recursos Biológicos; Argentina Fil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Córdoba. Estación Experimental Agropecuaria Marcos Juarez; Argentina Fil: Perez, Gabriela Teresa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Ciencia y Tecnología de Alimentos Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentina

The identification of genetically homogeneous groups of individuals is an ancient issue in population genetics and in the case of crops like wheat, it can be valuable information for breeding programs, genetic mapping and germplasm resources. In this work we determined the genetic structure of a set of 102 Argentinean bread wheat (Triticum aestivum L.) elite cultivars using 38 biochemical and molecular markers (functional, closely linked to genes and neutral ones) distributed throughout 18 wheat chromosomes. Genetic relationships among these lines were examined using model-based clustering methods. In the analysis three subpopulations were identified which correspond largely to the origin of the germplasm used by the main breeding programs in Argentina. Fil: Vanzetti, Leonardo Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Yerkovich, Nadia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Chialvo, Eugenia. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Lombardo, Lucio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Vaschetto, Luis. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina

Fusarium Head Blight (FHB) caused by Fusarium graminearum is a common and devastating disease of hexaploid wheat (Triticum aestivum L.) in all temperate growing regions of the world, including Argentina. Host resistance has been considered a highly economical and efficient way to manage the disease, however, progress of breeding for FHB resistance has been limited because of the lack of effective resistance to FHB and the complex inheritance of the partial resistance currently available in wheat. First part of this chapter deals with the impact of FHB in bread and durum wheat from Argentina, as well as the early efforts to improve for FHB resistance in bread wheat. The second part addresses some examples of successful introgression of the locusFhb1 (the best validated gene for FHB resistance from Sumai 3) into local backgrounds by marker assisted selection, and more briefly, the recent progress in the identification of alternative sources of FHB, future projects for QTL mapping, and the development of molecular tools for marker assisted selection.

International audience; Partial genomic sequences from an unknown garlic potyvirus and from an onion isolate of the onion yellow dwarf potyvirus (OYDV) were obtained. Comparison of the deduced amino acid sequences showed a similarity of 88% between the respective viral coat proteins. The garlic potyvirus coat protein was expressed inE. coli cells, purified, and subjected to Western blot analysis using antibodies raised against different garlic-infecting viruses. The expression protein was consistently recognised by anti-OYDV antibodies and did not react with antibodies specific for leek yellow stripe potyvirus (LYSV), garlic common latent carlavirus (GCLV) and shallot latent carlavirus (SLV). Besides, the garlic potyvirus coat protein was obtained as a fusion protein and used as antigen to produce polyclonal antibodies. These antibodies reacted with purified OYDV virions, but failed to recognise LYSV particles. In the light of this evidence the garlic potyvirus was identified as the garlic strain of OYDV.

Leaf rust, caused by Puccinia triticina Eriks. is a common and widespread disease of bread wheat (Triticum aestivum L.), in Argentina. Host resistance is the most economical, effective and ecologically sustainable method of controlling the disease. Gene postulation helps to determine leaf rust resistance genes (Lr genes) that may be present in a large group of wheat germplasm. Additionally presence of Lr genes can be determined using associated molecular markers. The objective of this study was to identify Lr genes that condition leaf rust resistance in 66 wheat cultivars from Argentina. Twenty four differential lines with individual known leaf rust resistance genes were tested with 17 different pathotypes of leaf rust collected from Argentina. Leaf rust infection types produced on seedling plants of the 66 local cultivars were compared with the infection types produced by the same pathotypes on Lr differentials to postulate which seedling leaf rust genes were present. Presence of Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, Lr25, Lr26, Lr29, Lr34, Lr35, Lr37, Lr47 and Lr51 was also determined using molecular markers. Eleven different Lr genes were postulated in the material: Lr1, Lr3a, Lr3ka, Lr9, Lr10, Lr16, Lr17, Lr19, Lr24, Lr26, Lr41. Presence of Lr21, Lr25, Lr29, and Lr47 could not be determined with the seventeen pathotypes used in the study because all were avirulent to these genes. Eleven cultivars (16.7%) were resistant to all pathotypes used in the study and the remaining 55 (83.3%) showed virulent reaction against one or more local pathotypes. Cultivars with seedling resistance gene combinations including Lr16 or single genes Lr47 (detected with molecular marker), Lr19 and Lr41, showed high levels of resistance against all pathotypes or most of them. On the opposite side, cultivars with seedling resistance genes Lr1, Lr3a, Lr3a + Lr24, Lr10, Lr3a + Lr10, Lr3a + Lr10 + Lr24 showed the highest number of virulent reactions against local pathotypes. Occurrence of adult plant resistance genes Lr34, Lr35 and Lr37 in local germplasm was evaluated using specific molecular markers confirming presence of Lr34 and Lr37. Our data suggest that combinations including seedling resistance genes like Lr16, Lr47, Lr19, Lr41, Lr21, Lr25 and Lr29, with adult plant resistance genes like Lr34, SV2, Lr46 will probably provide durable and effective resistance to leaf rust in the region. EEA Marcos Juárez Fil: Vanzetti, Leonardo Sebastian. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Campos, Pablo Eduardo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Bordenave. Area Fitopatología; Argentina Fil: Demichelis, Melina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Lombardo, Lucio. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Aurelia, Paola Romina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Vaschetto, Luis María. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina Fil: Bainotti, Carlos Tomas. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Mejoramiento de Trigo; Argentina Fil: Helguera, Marcelo. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Marcos Juárez. Grupo Biotecnología y Recursos Genéticos; Argentina

'BIOINTA 2004' (Reg. No. CV-1030, PI 655312) is a hard red winter wheat (Triticum aestivum L.) developed and released by the Marcos Juárez Experimental Station from the National Wheat Breeding Program of the National Institute of Agricultural Technology, Argentina. BIOINTA 2004, previously designated R4001, was selected for its excellent grain yield potential, resistance to leaf rust (caused by Puccinia triticina Eriks.) conferred mainly by the Lr47 gene selected by marker assisted selection (MAS), and its good bread-making quality. © Crop Science Society of America.

The concentration of yellow carotenoid pigments in durum wheat grain is an important quality criterion and is determined both by their accumulation and by their degradation by lipoxygenase enzymes (Lpx loci). The existence of a duplication at the Lpx-B1 locus and the allelic variation for a deletion of the Lpx-B1.1 copy is reported. This deletion was associated with a 4.5-fold reduction in lipoxygenase activity and improved pasta color (P

Leaf rust resistance gene Lr47 is located within a interstitial segment of Triticum speltoides Taush. 7S chromosome translocated to the short arm of chromosome 7A of bread wheat. This gene is resistant against currently predominant races of leaf rust from Argentina. The objectives of this study were to identify microsatellites linked to this source of resistance as a potential tool to introgress this source of resistance. Isogenic lines with and without Lr47 developed from 10 cultivars/breeding lines as well as 10 microsatellites previously mapped in 7AS chromosome were used in this study. Microsatellite gwm 60 was the only marker that co-segregated completely linked to Lr47. These data indicate that gwm 60 could be a valuable marker to introgress Lr47 in wheat germplasm. © 2006 by Pontificia Universidad Católica de Valparaíso.

Mol Gen Genomics (2005) 273: 54–65 DOI 10.1007/s00438-004-1095-4 O R I GI N A L P A P E R Daolin Fu AE Pe´ter Szu } cs AE Liuling Yan Marcelo Helguera AE Jeffrey S. Skinner Jarislav von Zitzewitz AE Patrick M. Hayes Jorge Dubcovsky Large deletions within the first intron in VRN-1 are associated with spring growth habit in barley and wheat Received: 15 September 2004 / Accepted: 22 November 2004 / Published online: 3 February 2005 Springer-Verlag 2005 Abstract The broad adaptability of wheat and barley is in part attributable to their flexible growth habit, in that spring forms have recurrently evolved from the ancestral winter growth habit. In diploid wheat and barley growth habit is determined by allelic variation at the VRN-1 and/or VRN-2 loci, whereas in the polyploid wheat species it is determined primarily by allelic variation at VRN-1. Dominant Vrn-A1 alleles for spring growth habit are frequently associated with mutations in the promoter region in diploid wheat and in the A genome of common wheat. However, several dominant Vrn-A1, Vrn-B1, Vrn-D1 (common wheat) and Vrn-H1 (barley) alleles show no polymorphisms in the promoter region relative to their respective recessive alleles. In this study, we sequenced the complete VRN-1 gene from these accessions and found that all of them have large dele- tions within the first intron, which overlap in a 4-kb region. Furthermore, a 2.8-kb segment within the 4-kb region showed high sequence conservation among the different recessive alleles. PCR markers for these dele- tions showed that similar deletions were present in all the accessions with known Vrn-B1 and Vrn-D1 alleles, and in 51 hexaploid spring wheat accessions previously shown to have no polymorphisms in the VRN-A1 pro- moter region. Twenty-four tetraploid wheat accessions Communicated by W.R. McCombie D. Fu AE L. Yan AE J. Dubcovsky (&) Department of Plant Sciences, University of California, One Shields Av, Davis, CA 95616, USA P. Szu } cs Agricultural Research Institute of the Hungarian Academy of Sciences, 2462 Martonva´sa´r, Hungary M. Helguera EEA INTA Marcos Jua´rez, CC 21, 2580 Marcos Jua´rez, Argentina J. S. Skinner AE J. von Zitzewitz AE P. M. Hayes Department of Crop and Soil Science and Horticulture, Oregon State University, Corvallis, OR 97331, USA had a similar deletion in VRN-A1 intron 1. We hypothesize that the 2.8-kb conserved region includes regulatory elements important for the vernalization requirement. Epistatic interactions between VRN-H2 and the VRN-H1 allele with the intron 1 deletion suggest that the deleted region may include a recognition site for the flowering repression mediated by the product of the VRN-H2 gene of barley. Keywords Wheat AE Barley AE Vernalization AE VRN-1 AE Allelic variation Introduction Many cereal crops, such as wheat, barley and oat, are divided into spring and winter types based on their growth habit. Winter varieties require an extended per- iod of exposure to cold in order to flower, a process known as vernalization. Vernalization is defined as ‘‘the acquisition or acceleration of the ability to flower by a chilling treatment’’ (Chouard 1960). The physiology of vernalization has been studied extensively in cereals, but only a few genes from this molecular pathway are cur- rently known (Yan et al. 2003, 2004b). In wheat and barley, the determination of the ver- nalization requirement involves an epistatic interaction between the genetic loci VRN-1 and VRN-2 (Takahashi and Yasuda 1971; Tranquilli and Dubcovsky 2000). Positional cloning of these two genes from diploid wheat (Triticum monococcum L., 2n=14, genome A m A m ) re- vealed that VRN-1 encodes a MADS-box transcription factor that is orthologous to the Arabidopsis meristem identity gene APETALA1 (Yan et al. 2003), while the VRN-2 gene codes for a zinc finger-CCT domain tran- scription factor with no clear orthologues in Arabidopsis or rice (Yan et al. 2004b). The VRN-1 gene is dominant for the spring growth habit and it is up-regulated by vernalization in winter lines (Danyluk et al. 2003; Tre- vaskis et al. 2003; Yan et al. 2003), whereas the VRN-2

Vernalization, the requirement of a long exposure to low temperatures to induce flowering, is an essential adaptation of plants to cold winters. We have shown recently that the vernalization gene VRN-1 from diploid wheat Triticum monococcum is the meristem identity gene APETALA1, and that deletions in its promoter were associated with spring growth habit. In this study, we characterized the allelic variation at the VRN-1 promoter region in polyploid wheat. The Vrn-A1a allele has a duplication including the promoter region. Each copy has similar foldback elements inserted at the same location and is flanked by identical host direct duplications (HDD). This allele was found in more than half of the hexaploid varieties but not among the tetraploid lines analyzed here. The Vrn-A1b allele has two mutations in the HDD region and a 20-bp deletion in the 5′ UTR compared with the winter allele. The Vrn-A1b allele was found in both tetraploid and hexaploid accessions but at a relatively low frequency. Among the tetraploid wheat accessions, we found two additional alleles with 32 bp and 54 bp deletions that included the HDD region. We found no size polymorphisms in the promoter region among the winter wheat varieties. The dominant Vrn-A1 allele from two spring varieties from Afghanistan and Egypt (Vrn-A1c allele) and all the dominant Vrn-B1 and Vrn-D1 alleles included in this study showed no differences from their respective recessive alleles in promoter sequences. Based on these results, we concluded that the VRN-1 genes should have additional regulatory sites outside the promoter region studied here. © Springer-Verlag 2004.

Winter wheats require several weeks at low temperature to flower. This process, vernalization, is controlled mainly by the VRN1 gene. Using 6,190 gametes, we found VRN1 to be completely linked to MADS-box genes AP1 and AGLG1 in a 0.03-centimorgan interval flanked by genes Cysteine and Cytochrome B5 . No additional genes were found between the last two genes in the 324-kb Triticum monococcum sequence or in the colinear regions in rice and sorghum. Wheat AP1 and AGLG1 genes were similar to Arabidopsis meristem identity genes AP1 and AGL2 , respectively. AP1 transcription was regulated by vernalization in both apices and leaves, and the progressive increase of AP1 transcription was consistent with the progressive effect of vernalization on flowering time. Vernalization was required for AP1 transcription in apices and leaves in winter wheat but not in spring wheat. AGLG1 transcripts were detected during spike differentiation but not in vernalized apices or leaves, suggesting that AP1 acts upstream of AGLG1 . No differences were detected between genotypes with different VRN1 alleles in the AP1 and AGLG1 coding regions, but three independent deletions were found in the promoter region of AP1 . These results suggest that AP1 is a better candidate for VRN1 than AGLG1 . The epistatic interactions between vernalization genes VRN1 and VRN2 suggested a model in which VRN2 would repress directly or indirectly the expression of AP1 . A mutation in the promoter region of AP1 would result in the lack of recognition of the repressor and in a dominant spring growth habit.

Rust resistance genes Lr37, Sr38, and Yr17 are located within a segment of Triticum ventricosum (Tausch) Cess. chromosome 2NS translocated to the short arm of bread wheat chromosome 2AS. Characterization of this chromosome segment by 13 restriction fragment length polymorphism (RFLP) markers indicated that the 2NS translocation replaced approximately half of the short arm of chromosome 2A (distal 25-38 centimorgans, cM). The objective of this study was to develop polymerase chain reaction (PCR) assays based on RFLP marker cMWG682 to facilitate the transfer of this cluster of rust resistance genes into commercial wheat (Triticum aestivum L.) cultivats. DNA sequence was obtained from the A-, B-, D-, and N-alleles of cMWG682 and was used to design N-allele specific primers. The 2NS fragment amplified by PCR primers cosegregated with the presence of the RFLP-2NS band in all backcross populations. A cleaved amplified polymorphic sequence (CAPS) was used to develop a marker for the 2A-allele. This marker can be used to differentiate homozygous and heterozygous plants carrying the 2NS translocation in the final cycle of backcross introgression or in screenings for homozygous plants in segregating populations. Finally, a third PCR assay was developed by means of TaqMan technology as a high-throughput alternative for selection of the 2NS/2AS translocation in large segregating populations in breeding programs that have access to real time PCR equipment. These molecular markers were used to develop four hard red spring isogenic lines homozygous for the 2NS chromosome segment. One of the isogenic lines, derived from 'Anza,' did not show the expected resistance in spite of the presence of all the RFLP markers for the 2NS chromosome segment. Analysis of F1 hybrids suggested that a suppressor of the Lr37 gene is present in Anza. These isogenic lines will provide a valuable tool to test the effects of the large 2NS translocation on quality and agronomic performance.

The leaf rust resistance gene Lr47 confers resistance to a wide spectrum of leaf rust strains. This gene was recently transferred from chromosome 7S of Triticum speltoides to chromosome 7A of hexaploid wheat Triticum aestivum. To facilitate the transfer of Lr47 to commercial varieties, the completely linked restriction fragment length polymorphism (RFLP) locus Xabc465 was converted into a PCR-based marker. Barley clone ABC465 is orthologous to the type-I wheat sucrose synthase gene and primers were designed for the conserved regions between the two sequences. These conserved primers were used to amplify, clone and sequence different alleles from T. speltoides and T. aestivum. This sequence information was used to identify the T. speltoides sequence, detect allele-specific mutations, and design specific primers. Cosegregation of the PCR product of these primers and the T. speltoides chromosome segment was confirmed in four backcross-populations. To complement this dominant marker, a cleavage amplified polymorphic sequence (CAPS) was developed for the 7A allele of Xabc465. This CAPS marker is useful to select homozygous Lr47 plants from F2 or backcross-F2 segregating populations, and in combination with the T- speltoides specific primers is expected to facilitate the deployment of Lr47 in new bread wheat varieties.