Cristina Rodríguez-Suárez

Development of SCAR markers linked to a gene controlling absence of tannins in faba bean

Faba beans are inexpensive, nutrient-dense sources of plant protein, but anti-nutritional factors such as condensed tannins reduce the biological value of their protein. Two recessive genes, zt-1 and zt-2, control the absence of tannins in faba bean seeds and also determine a white flower character on the plant. However, crosses between them produce coloured F1 plants with tannins that contaminate the crop. Therefore, it is important to identify the gene present in all tannin-free cultivars and gene bank accessions to enable breeders to choose appropriate genitors for their crosses. The aim of this study was the identification of markers linked to zt-1, one of the genes governing free tannin content in faba bean. A segregating F2 population derived from the cross between the coloured flower and high tannin content genotype Vf6 and a zt-1 line was developed and characterized phenotypically. Bulked Segregant Analysis (BSA) was used to identify Random Amplified Polymorphic DNA (RAPD) markers linked to the zt-1 gene. Four RAPD loci (OPC5551, OPG15600, OPG111171 and OPAF20776) showed polymorphism between the contrasting bulks. The markers were sequenced to develop specific Sequence Characterised Amplified Regions (SCARs). Amplification of SCC5551 produced a single product which was only observed in the white flowered and zero tannin content genotypes, whereas SCAR SCG111171only produced a band in F2 plants with coloured flower and high tannin content. SCARs SCC5551 and SCG111171 were tested for their applicability for routine screening in 37 faba bean genotypes differing in flower colour and tannin content. SCC5551, allowed the prediction of the zt-1 genotypes with a 95% of accuracy, underscoring the potential of this SCAR marker as a cost-effective tool for MAS in large faba bean breeding populations.

Genetic analysis of the response to eleven Colletotrichum lindemuthianum races in a RIL population of common bean (Phaseolus vulgaris L.)

BackgroundBean anthracnose is caused by the fungus Colletotrichum lindemuthianum (Sacc. & Magnus) Lams.- Scrib. Resistance to C. lindemuthianum in common bean (Phaseolus vulgaris L.) generally follows a qualitative mode of inheritance. The pathogen shows extensive pathogenic variation and up to 20 anthracnose resistance loci (named Co-), conferring resistance to specific races, have been described. Anthracnose resistance has generally been investigated by analyzing a limited number of isolates or races in segregating populations. In this work, we analyzed the response against eleven C. lindemuthianum races in a recombinant inbred line (RIL) common bean population derived from the cross Xana × Cornell 49242 in which a saturated linkage map was previously developed.ResultsA systematic genetic analysis was carried out to dissect the complex resistance segregations observed, which included contingency analyses, subpopulations and genetic mapping. Twenty two resistance genes were identified, some with a complementary mode of action. The Cornell 49242 genotype carries a complex cluster of resistance genes at the end of linkage group (LG) Pv11 corresponding to the previously described anthracnose resistance cluster Co-2. In this position, specific resistance genes to races 3, 6, 7, 19, 38, 39, 65, 357, 449 and 453 were identified, with one of them showing a complementary mode of action. In addition, Cornell 49242 had an independent gene on LG Pv09 showing a complementary mode of action for resistance to race 453. Resistance genes in genotype Xana were located on three regions involving LGs Pv01, Pv02 and Pv04. All resistance genes identified in Xana showed a complementary mode of action, except for two controlling resistance to races 65 and 73 located on LG Pv01, in the position of the previously described anthracnose resistance cluster Co-1.ConclusionsResults shown herein reveal a complex and specific interaction between bean and fungus genotypes leading to anthracnose resistance. Organization of specific resistance genes in clusters including resistance genes with different modes of action (dominant and complementary genes) was also confirmed. Finally, new locations for anthracnose resistance genes were identified in LG Pv09.

Allelic variation, alternative splicing and expression analysis of Psy1 gene in Hordeum chilense Roem. et Schult.

Background The wild barley Hordeum chilense Roem. et Schult. is a valuable source of genes for increasing carotenoid content in wheat. Tritordeums, the amphiploids derived from durum or common wheat and H. chilense, systematically show higher values of yellow pigment colour and carotenoid content than durum wheat. Phytoene synthase 1 gene (Psy1) is considered a key step limiting the carotenoid biosynthesis, and the correlation of Psy1 transcripts accumulation and endosperm carotenoid content has been demonstrated in the main grass species. Methodology/Principal findings We analyze the variability of Psy1 alleles in three lines of H. chilense (H1, H7 and H16) representing the three ecotypes described in this species. Moreover, we analyze Psy1 expression in leaves and in two seed developing stages of H1 and H7, showing mRNA accumulation patterns similar to those of wheat. Finally, we identify thirty-six different transcripts forms originated by alternative splicing of the 5′ UTR and/or exons 1 to 5 of Psy1 gene. Transcripts function is tested in a heterologous complementation assay, revealing that from the sixteen different predicted proteins only four types (those of 432, 370, 364 and 271 amino acids), are functional in the bacterial system. Conclusions/Significance The large number of transcripts originated by alternative splicing of Psy1, and the coexistence of functional and non functional forms, suggest a fine regulation of PSY activity in H. chilense. This work is the first analysis of H. chilense Psy1 gene and the results reported here are the bases for its potential use in carotenoid enhancement in durum wheat.

Increase in transcript accumulation of Psy1 and e-Lcy genes in grain development is associated with differences in seed carotenoid content between durum wheat and tritordeum

Carotenoid rich diets have been associated with lower risk of certain diseases. The great importance of cereals in human diet has directed breeding programs towards carotenoid enhancement to alleviate these deficiencies in developing countries and to offer new functional foods in the developed ones. The new cereal tritordeum (×Tritordeum Ascherson et Graebener) derived from durum wheat (Triticum turgidum ssp. durum) and the wild barley Hordeum chilense, naturally presents carotenoid levels 5–8 times higher than those of durum wheat. The improvement of tritordeum properties as a new functional food requires the elucidation of biosynthetic steps for carotenoid accumulation in seeds that differ from durum wheat. In this work expression patterns of nine genes from the isoprenoid and carotenoid biosynthetic pathways were monitored during grain development in durum wheat and tritordeum. Additionally, a fine identification and quantification of pigments (chlorophylls and carotenoids) during grain development and in mature seeds has been addressed. Transcript levels of Psy1, Psy2, Zds, e-Lcy and b-Lcy were found to correlate to carotenoid content in mature grains. The specific activation of the homeologous genes Psy1, e-Lcy from H. chilense and the high lutein esterification found in tritordeum may serve to explain the differences with durum wheat in carotenoid accumulation.

Progress and perspectives for carotenoid accumulation in selected Triticeae species

Plant carotenoids are C40 isoprenoids with multiple biological roles. Breeding for carotenoid content in rice, maize and wheat is a relevant issue, both for their importance in human health and nutrition and for their influence in food colouration in products such as pasta from durum wheat. Regarding human health, vitamin A deficiency (VAD) is one of the major causes of malnutrition in the world. As many as 500 000 children become blind due to VAD each year with many of them dying from VAD-related illness within 1 year. This review presents the main results in the improvement of endosperm carotenoid levels in rice, maize and wheat considering the methodology used, either transgenic or non-transgenic; the breeding target, such as provitamin A or total carotenoid content; the identification of new carotenogenic genes/alleles related to the available variation for this trait; and the development of new functional markers for marker-assisted selection. A comparative overview among these species and key areas for further improvement are also identified. Carotenoid enhancement in grasses would benefit from comparative studies among Triticeae species since they allow the understanding of the diversity basis. Therefore, the comparative overview given in this work will be relevant not only to rice, maize and wheat but also to other Triticeae species.

Hordeum chilense genome, a useful tool to investigate the endosperm yellow pigment content in the Triticeae

BackgroundThe wild barley Hordeum chilense fulfills some requirements for being a useful tool to investigate the endosperm yellow pigment content (YPC) in the Triticeae including its diploid constitution, the availability of genetic resources (addition and deletion stocks and a high density genetic map) and, especially, its high seed YPC not silenced in tritordeums (amphiploids derived from H. chilense and wheat). Thus, the aim of this work was to test the utility of the H. chilense genome for investigating the YPC in the Triticeae.ResultsTwelve genes related to endosperm carotenoid content and/or YPC in grasses (Dxr, Hdr [synonym ispH], Ggpps1, Psy2, Psy3, Pds, Zds, e-Lcy, b-Lcy, Hyd3, Ccd1 and Ppo1) were identified, and mapped in H. chilense using rice genes to identify orthologs from barley, wheat, sorghum and maize. Macrocolinearity studies revealed that gene positions were in agreement in H. vulgare and H. chilense. Additionally, three main regions associated with YPC were identified in chromosomes 2Hch, 3Hch and 7Hch in H. chilense, the former being the most significant one.ConclusionsThe results obtained are consistent with previous findings in wheat and suggest that Ggpps1, Zds and Hyd3 on chromosome 2Hch may be considered candidate genes in wheat for further studies in YPC improvement. Considering the syntenic location of carotenoid genes in H. chilense, we have concluded that the Hch genome may constitute a valuable tool for YPC studies in the Triticeae.

Exploitation of nuclear and cytoplasm variability in Hordeum chilense for wheat breeding.

Our work in this area is supported by grants (to S. G. A.) AGL2008-03720, and P09-AGR-4817 from Spanish Ministry of Science and Innovation, Junta de Andalucia and FEDER. C. R.-S. acknowledges financial support from CSIC (JAE-Doc program).

Selection and molecular characterization of imidazolinone resistant mutation-derived lines of Tritordeum HT621

Imidazolinone herbicides resistant varieties, induced by mutations at the AHAS gene (acetohydroxyacid synthase), have been developed in many crops. Hexaploid tritordeum (Tritordeum Asch. & Graebn.) is the amphiploid derived from the cross between Hordeum chilense (HchHch) and durum wheat Triticum turgidum L. (Thell) (AABB). Tritordeums have the potential to become a new crop with high added-value for food or feed. Mutagenesis with EMS was conducted to obtain imidazolinone resistant lines derived of the tritordeum HT621. Eleven M3 plants were selected after imidazolinone treatment and five descendants of two of these lines (HT621-M3R1-3 and HT621-M3R10-1) were analyzed at the molecular level. Partial sequences of the three homologous AHAS loci in genomes A, B, and Hch were obtained as well as those of HT621. A partial sequence of the AHAS gene in Hordeum chilense is first described in this work, and the designation ahasL-Hch1 is proposed. A single Ser-Asn627 substitution at the AHAS locus in the B genome is responsible of resistance in both lines. We propose the name AhasL-B2 for this resistance allele. This is the first report of the selection of imidazolinone resistant lines of tritordeum and the molecular characterization of the mutation conferring this resistance.

Applicability of chromosome-specific SSR wheat markers for the introgression of Triticum urartu in durum wheat breeding programmes

This work has been performed with the financial support of the Spanish Ministry of Science and Innovation (AGL2009-11359) and the Consejeria Economia, Innovacion y Ciencia of the Junta de Andalucia (P09-AGR-4817) and FEDER. C. R.-S. acknowledges financial support from CSIC (JAE-Doc program).

Contribution of Chromosomes 1HchS and 6HchS to Fertility Restoration in the Wheat msH1 CMS System under Different Environmental Conditions.

Exploiting hybrid wheat heterosis has been long pursued to increase crop yield, stability and uniformity. Cytoplasmic male sterility (CMS) systems based in the nuclear-cytoplasmic incompatible interactions are a classic way for hybrid seed production, but to date, no definitive system is available in wheat. The msH1 CMS system results from the incompatibility between the nuclear genome of wheat and the cytoplasmic genome of the wild barley Hordeum chilense. Fertility restoration of the CMS phenotype was first associated with the disomic addition of the short arm of chromosome 6H from H. chilense. In further studies it was observed that chromosome arm 1HchS was also implicated, and the combination of genes in both chromosome arms restored fertility more efficiently. In this work we aim to dissect the effect of each chromosome in fertility restoration when combined in different genomic backgrounds and under different environmental conditions. We propose a model to explain how restoration behaves in the msH1 system and generate valuable information necessary to develop an efficient system for hybrid wheat production.