Towards carotenoid biofortification in wheat: identification of XAT-7A1, a multicopy tandem gene responsible for carotenoid esterification in durum wheat.

Yellow pigment content, mainly due to the accumulation of carotenoids, is a quality trait in durum wheat grain as it confers the bright yellow color to pasta preferred by consumers. Also, carotenoids are essential nutrients exerting important biological functions in human health. Consequently, biofortification strategies have been developed in many crops to increase carotenoid content. In this context, carotenoid esterification is emerging as a new breeding target for wheat biofortification, as carotenoid esters have been found to promote both carotenoid accumulation and stability. Until recently, no carotenoid esters have been identified in significant proportions in durum wheat grains, and interspecific breeding programs have been started to transfer esterification ability from common wheat and Hordeum chilense.In this work, XAT-7A1 is identified as the gene responsible for carotenoid esterification in durum wheat. Sequencing, copy number variation and mapping results show that XAT-7A1 is organized as tandem or proximal GDSL esterase/lipase copies in chromosome 7A. Three XAT-7A1 haplotypes are described: Type 1 copies, associated with high levels of carotenoid esters (diesters and monoesters) production and high expression in grain development; Type 2 copies, present in landraces with low levels of carotenoid esters (monoesters) or no esters; and Type 3 copies, without the signal peptide, resulting in zero-ester phenotypes.The identification of XAT-7A1 is a necessary step to make the carotenoid esterification ability available for durum and bread wheat breeding, which should be focused on the Type 1 XAT-7A1 haplotype, which may be assessed as a single gene since XAT-7A1 copies are inherited together.

Encefalopatías y encefalitis durante la infección aguda por SARS-CoV2. Registro de la Sociedad Española de Neurología SEN COVID-19 / Encephalopathy and encephalitis during acute SARS-CoV-2 infection. Spanish Society of Neurology COVID-19 Registry

OBJETIVOS: Desde el inicio de la pandemia por el virus SARS-CoV2 la Sociedad Española de Neurología (SEN) creó un registro de afectación neurológica para informar al neurólogo clínico. Las encefalopatías y encefalitis fueron una de las complicaciones más descritas. Analizamos las características de las mismas. PACIENTES Y MÉTODOS: Estudio descriptivo retrospectivo, observacional multicéntrico, de pacientes con sintomatología compatible con encefalitis o encefalopatía, introducidos en el Registro SEN COVID-19 desde el 17 de marzo hasta el 6 de junio de 2020. RESULTADOS: Se han registrado 232 casos con síntomas neurológicos, 51 casos de encefalopatía/encefalitis (21,9%). Ningún paciente era trabajador sanitario. Los síndromes más frecuentes fueron: cuadro confusional leve-moderado (33%) y encefalopatía grave o coma (9,8%). El tiempo medio entre el inicio de la infección y la clínica neurológica fue de 8,02 días. Punción lumbar en el 60,8% de pacientes; solo hubo un caso con PCR positiva. Resonancia craneal en el 47% de los pacientes (alterada en el 7,8% de ellos). Se realizó electroencefalograma en el 41,3% de los casos (alterado en el 61,9% de los mismos). CONCLUSIONES: Las encefalopatías y encefalitis son dos de las complicaciones más frecuentes descritas en el SEN COVID-19. Más de un tercio de los pacientes presentó un cuadro de síndrome confusional leve o moderado. El tiempo medio de aparición de la sintomatología neurológica desde el inicio de la infección fue de 8 días (hasta 24 h antes en mujeres que en hombres). El electroencefalograma fue la prueba más sensible en estos pacientes, encontrando muy pocos casos con alteraciones en las pruebas de neuroimagen. Todos los pacientes que recibieron tratamiento con bolos de corticoides o inmunoglobulinas tuvieron una evolución favorable

OBJECTIVES: Since the beginning of the COVID-19 pandemic, the Spanish Society of Neurology has run a registry of patients with neurological involvement for the purpose of informing clinical neurologists. Encephalopathy and encephalitis were among the most frequently reported complications. In this study, we analyse the characteristics of these complications. PATIENTS AND METHODS: We conducted a retrospective, descriptive, observational, multicentre study of patients with symptoms compatible with encephalitis or encephalopathy, entered in the Spanish Society of Neurology's COVID-19 Registry from 17 March to 6 June 2020. RESULTS: A total of 232 patients with neurological symptoms were registered, including 51 cases of encephalopathy or encephalitis (21.9%). None of these patients were healthcare professionals. The most frequent syndromes were mild or moderate confusion (33%) and severe encephalopathy or coma (9.8%). The mean time between onset of infection and onset of neurological symptoms was 8.02 days. Lumbar puncture was performed in 60.8% of patients, with positive PCR results for SARS-CoV-2 in only one case. Brain MRI studies were performed in 47% of patients, with alterations detected in 7.8% of these. EEG studies were performed in 41.3% of cases, detecting alterations in 61.9%. CONCLUSIONS: Encephalopathy and encephalitis are among the complications most frequently reported in the registry. More than one-third of patients presented mild or moderate confusional syndrome. The mean time from onset of infection to onset of neurological symptoms was 8 days (up to 24 hours earlier in women than in men). EEG was the most sensitive test in these patients, with very few cases presenting alterations in neuroimaging studies. All patients treated with boluses of corticosteroids or immunoglobulins progressed favourably

Encephalopathy and encephalitis during acute SARS-CoV-2 infection. Spanish Society of Neurology COVID-19 Registry. / Encefalopatías y encefalitis durante la infección aguda por SARS-CoV2. Registro de la Sociedad Española de Neurología SEN COVID-19.

OBJECTIVES: Since the beginning of the COVID-19 pandemic, the Spanish Society of Neurology has run a registry of patients with neurological involvement for the purpose of informing clinical neurologists. Encephalopathy and encephalitis were among the most frequently reported complications. In this study, we analyse the characteristics of these complications. PATIENTS AND METHODS: We conducted a retrospective, descriptive, observational, multicentre study of patients with symptoms compatible with encephalitis or encephalopathy, entered in the Spanish Society of Neurology's COVID-19 Registry from 17 March to 6 June 2020. RESULTS: A total of 232 patients with neurological symptoms were registered, including 51 cases of encephalopathy or encephalitis (21.9%). None of these patients were healthcare professionals. The most frequent syndromes were mild or moderate confusion (33%) and severe encephalopathy or coma (9.8%). The mean time between onset of infection and onset of neurological symptoms was 8.02 days. Lumbar puncture was performed in 60.8% of patients, with positive PCR results for SARS-CoV-2 in only one case. Brain MRI studies were performed in 47% of patients, with alterations detected in 7.8% of these. EEG studies were performed in 41.3% of cases, detecting alterations in 61.9%. CONCLUSIONS: Encephalopathy and encephalitis are among the complications most frequently reported in the registry. More than one-third of patients presented mild or moderate confusional syndrome. The mean time from onset of infection to onset of neurological symptoms was 8 days (up to 24hours earlier in women than in men). EEG was the most sensitive test in these patients, with very few cases presenting alterations in neuroimaging studies. All patients treated with boluses of corticosteroids or immunoglobulins progressed favourably.

Carotenoid content in tritordeum is not primarily associated with esterification during grain development.

Tritordeums show a significant proportion of lutein esters which increases carotenoid stability and retention throughout the food chain. Esterification is a common means of carotenoid sequestration. A putative association between lutein esters formation acting as a metabolic sink during early stages of grain development and the high carotenoid content of tritordeums is analyzed in this work. Compared to wheat, tritordeums accumulated significantly higher lutein contents from 20 days post anthesis (dpa) but lutein esters were not detected until 36 dpa. Thus esterification is not acting as a metabolific sink before 36 dpa. The presence of lutein esters at late stages of grain development may have a complementary role in carotenoid accumulation by reducing and/or counteracting their catabolism. The differences for lutein esterification among tritordeums suggest the existence of diversity for xanthophyll acyl transferases that could be exploited to increase lutein retention in this cereal and through the food chain.

Lutein ester profile in wheat and tritordeum can be modulated by temperature: Evidences for regioselectivity and fatty acid preferential of enzymes encoded by genes on chromosomes 7D and 7Hch.

The increase of lutein retention through the food chain is desirable for wheat breeding. Lutein esters are more stable than free lutein during post-harvest storage and two loci on chromosomes 7D and 7Hch are important for esterification. We investigated the effect of temperature during grain filling on carotenoid accumulation and lutein ester profile including fatty acid selectivity (palmitic vs. linoleic) and regioselectivity (esterification at positions 3 vs. 3'). Three different temperature regimes were assayed (controlled, semi-controlled and non-controlled). Lutein esters were more stable than free carotenoids in vivo and the enzymes encoded by chromosomes 7Hch and 7D are complementary. Indeed, they show differential preferences for the fatty acid (palmitic and linoleic, respectively) and regioselectivity (3 and 3', respectively). Besides, H. chilense has additional genes for esterification. Finally, the increase of temperature favoured the accumulation of lutein esters with linoleic acid and the synthesis of regioisomers at position 3'.

Development of wild barley (Hordeum chilense)-derived DArT markers and their use into genetic and physical mapping.

Diversity arrays technology (DArT) genomic libraries were developed from H. chilense accessions to support robust genotyping of this species and a novel crop comprising H. chilense genome (e.g., tritordeums). Over 11,000 DArT clones were obtained using two complexity reduction methods. A subset of 2,209 DArT markers was identified on the arrays containing these clones as polymorphic between parents and segregating in a population of 92 recombinant inbred lines (RIL) developed from the cross between H. chilense accessions H1 and H7. Using the segregation data a high-density map of 1,503 cM was constructed with average inter-bin density of 2.33 cM. A subset of DArT markers was also mapped physically using a set of wheat-H. chilense chromosome addition lines. It allowed the unambiguous assignment of linkage groups to chromosomes. Four segregation distortion regions (SDRs) were found on the chromosomes 2H(ch), 3H(ch) and 5H(ch) in agreement with previous findings in barley. The new map improves the genome coverage of previous H. chilense maps. H. chilense-derived DArT markers will enable further genetic studies in ongoing projects on hybrid wheat, seed carotenoid content improvement or tritordeum breeding program. Besides, the genetic map reported here will be very useful as the basis to develop comparative genomics studies with barley and model species.

Molecular and cytological characterization of an extra acrocentric chromosome that restores male fertility of wheat in the msH1 CMS system.

A new CMS system designated as 'msH1' has been reported in bread wheat using the cytoplasm of H. chilense. While testing this system in different wheat backgrounds, a highly fertile line with chromosome number 42 plus an extra acrocentric chromosome was obtained. The extra chromosome did not pair with any wheat chromosome at meiosis, and progeny from this line which lack the acrocentric chromosome showed pollen abortion and male sterility. In order to establish the origin of this chromosome, FISH using H. chilense genomic DNA as probe was used and showed that it had originated from H. chilense chromosome(s). The novel chromosome did not possess sequences similar to wheat rDNA; however, the probe pSc119.2 from S. cereale containing the 120 bp family was found to occur at the end of its long arm. Data obtained from FISH and EST molecular markers confirm that the long arm of the acrocentric chromosome is indeed, the short arm of chromosome 1H(ch) from H. chilense. We suggest that the novel chromosome originated from a deletion of the distal part of the long arm of chromosome 1H(ch). Neither the 1H(ch)S short arm, nor the whole chromosome 1H(ch) restores pollen fertility of the alloplasmic wheat. Therefore, the restorer gene on the acrocentric chromosome must be located on the retained segment from the hypothetical 1H(ch)L, while some pollen fertility inhibitor could be present on the deleted 1H(ch)L distal segment. Disomic addition of the acrocentric chromosome was obtained and this line resulted fully stable and fertile.

Introgression of wheat chromosome 2D or 5D into tritordeum leads to free-threshing habit.

Hexaploid tritordeum is the amphiploid derived from the cross between the diploid wild barley Hordeum chilense and durum wheat. The non-free-threshing habit is a constraint to this species becoming a new crop. Three tritordeum lines (HT374, HT376, and HT382) showing the free-threshing habit were selected from crosses between tritordeum and bread wheat. All three lines were euploids, as revealed by mitotic chromosome counting. Genomic in situ hybridization analysis made it possible to distinguish differences among these lines. While the line HT382 carries only 10 chromosomes from H. chilense, the lines HT374 and HT376 have 12. These results suggest that HT382 is a double chromosome substitution line between H. chilense and the wheat D genome, while HT374 and HT376 each have one pair of H. chilense (Hch) chromosomes substituted by wheat D chromosomes. Molecular characterization revealed that HT382 is a 1D/(1Hch), 2D/(2Hch) chromosome substitution line, whereas HT374 and HT376 have 5D/(5Hch) substitutions. On the basis of previous knowledge, it seems that the absence of chromosome 2Hch or 5Hch is more important for producing the free-threshing habit than the presence of chromosome 2D or 5D, while chromosome 1Hch seems to be unrelated to the trait. These free-threshing tritordeum lines constitute an important advance in the tritordeum breeding program.

Effects of reciprocal crosses on agronomic performance of tritordeum.

Tritordeums (Tritordeum Ascherson et Graebner) are the amphiploids derived from the crosses between Hordeum chilense and durum or bread wheats. Primary tritordeums are obtained using H. chilense as female parent and therefore they exhibit H. chilense cytoplasm. The effect of wheat cytoplasm on agronomic performance of tritordeums was investigated. We developed four pairs of reciprocal F1 lines only differing in their cytoplasm, donated from wheat or H. chilense alternatively. The agronomic performance of reciprocal F1 lines contrasting for their cytoplasm was evaluated. The following traits were assessed: leave and tillers number one month after sowing, plant height, anthesis date, total number of ears, number of spikelets per spike, fertility of the main spike, length and wide of the flag leaf in the main stem and thousand kernel weight. Reciprocal F1 lines did not differ for any of the agronomic traits evaluated with the exception of anthesis date in the pair THC1726/HTC1727. Therefore, both wheat and H. chilense cytoplasms can be used in tritordeum breeding.

Development of a new diagnostic marker for growth habit selection in faba bean (Vicia faba L.) breeding.

Faba bean varieties with determinacy of the apical meristem are relevant to green production. A diagnostic CAPS (cleavage amplification polymorphic sequence) marker for determinate growth habit (ti) in faba bean was previously developed by Avila et al. (Mol Breed 17:185-190, 2006) but was effective only on a limited range of cultivars or genotypes. In this study, we studied the reasons for this limited application and developed a new marker useful for most faba bean-breeding programs. By designing a new set of primers, the complete genomic Vf_TFL1 sequences from different genotypes contrasting for the character were obtained and additional base changes associated with the ti phenotype were identified. The comparison among faba bean sequences showed that the previous CAPS marker was based on a SNP (single nucleotide polymorphism) at position 469 in the intron 2-3, a silent mutation. On the contrary, a SNP at position 26 that distinguishes determinate and indeterminate growth habit genotypes lead to an amino acid change (Leu-9 to Arg) in the determinate growth habit genotypes that could account for the ti phenotype. A dCAPS marker based on this SNP that creates a TaqI site in the ti allele was developed. The marker was 100% successful in predicting ti phenotypes in a broad range of faba bean germplasm representing all major cultivars historically grown in Europe. The outcome confirms the utility of the new dCAPS in worldwide marker-assisted selection programs.

Identification of QTLs influencing combustion quality in Miscanthus sinensis Anderss. II. Chlorine and potassium content.

Chlorine and potassium content are important traits related to combustion quality of Miscanthus species. These traits were analysed in a cross between F(1.1) and F(1.7) entries of Miscanthus sinensis Anderss, both lines offspring of the cross between MS-90-2 and MS-88-110. Quantitative trait locus (QTL) analyses were performed on a previous linkage map constructed with the offspring cross mapping strategy. The mapqtl 4.0 package was used to perform QTL analyses. Six potential QTLs were detected with data collected over a 2-year period. Of these, four were associated with chlorine and two with potassium. These results could be used as an initial step to develop a marker-aided selection programme for biomass with low mineral content.

Identification of QTLs influencing agronomic traits in Miscanthus sinensis Anderss. I. Total height, flag-leaf height and stem diameter.

We have developed the first quantitative trait locus (QTL) analyses for agronomic traits in a cross between F(1.1) (P1) and F(1.7) (P7) entries of Miscanthus sinensis Anderss. Both lines are offspring of the cross between MS-90-2 and MS-88-110. A map based on random amplified polymorphic DNA markers previously constructed was used to perform the QTL analyses. This map was developed using a new mapping strategy that has been designated offspring cross. Eleven QTLs were detected for height, panicle height and diameter using the programme mapqtl 4.0 and the multiple QTL method. QTL significance was determined using several analyses, including Kruskal-Wallis analyses, empirical determination of LOD critical values using permutation tests, QTLs validation with field data over 2 years and co-localization of QTLs for correlated traits. The results obtained could be the first step in developing a marker-assisted selection programming in this species for biomass production.

Effects of bromodeoxyuridine, cytosine arabinoside and Colcemid upon in vitro development of mouse blastocysts.

Mouse blastocysts in culture have been treated with increasing concentrations of cytosine arabinoside, bromodeoxyuridine or Colcemid. Concentrations of all three antimetabolites have been found which interfere with neither hatching of the blastocysts from their zona pellucidae nor subsequent attachment of the blastocysts to the culture dish, but which eventually result in death of the inner cell mass (ICM) and its derivatives. The effect upon the ICM is selective at these antimetabolite concentrations since many or, in some cases, all trophoblast cells continue to survive, and by a number of criteria, undergo normal patterns of differentiation and development.