Sergio G. Atienza

Developing a core collection of olive (Olea europaea L.) based on molecular markers (DArTs, SSRs, SNPs) and agronomic traits

Molecular markers (SSR, SNP and DArT) and agronomical traits have been used in the world’s largest olive (Olea europaea L.) germplasm collection (IFAPA, Centre Alameda del Obispo, Cordoba, Spain) to study the patterns of genetic diversity and underlying genetic structure among 361 olive accessions. In addition the marker data were used to construct a set of core collections by means of two different algorithms (MSTRAT and PowerCore) based on M (maximization) strategy. Our results confirm that the germplasm collection is a useful source of genetically diverse material. We also found that geographical origin is an important factor structuring genetic diversity in olive. Subsets of 18, 27, 36, 45 and 68 olive accessions, representing respectively 5%, 7.5%, 10%, 12.5% and 19% of the whole germplasm collection, were selected based on the information obtained by all the data set as well as each marker type considered individually. According to our results, the core collections that represent between 19% and 10% of the total collection size could be considered as optimal to retain the bulk of the genetic diversity found in this collection. Due to its high efficiency at capturing all the alleles/traits states found in the whole collection, the core size of 68 accessions could be of special interest for genetic conservation applications in olive. The high average genetic distance and diversity and the almost equal representation of accessions from different geographical regions indicate that the core size of 36 accessions, could be the working collection for olive breeders.

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

Abstract Chlorine and potassium content are important traits related to combustion quality of Miscanthus species. These traits were analysed in a cross between F1.1 and F1.7 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 F1.1 (P1) and F1.7 (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.

Male fertility restoration of wheat in Hordeum chilense cytoplasm is associated with 6HchS chromosome addition

We report a new cytoplasmic male sterility (CMS) source in bread wheat (Triticum aestivum L.) designated as msH1. CMS has been identified during the process of obtaining alloplasmic bread wheat in different Hordeum chilense Roem. Schultz. cytoplasms. It was observed that when using the H. chilense H1 accession, the corresponding alloplasmic line was male sterile. This alloplasmic wheat is stable under different environmental conditions and it does not exhibit developmental or floral abnormalities, showing only slightly reduced height and some delay in heading. On examining microsporogenesis in the alloplasmic line, it was found that different stages of meiosis were completed normally, but abnormal development occurred at the uninucleate-pollen stage at the first mitosis, resulting in failure of anther exertion and pollen abortion. Fertility restoration of the CMS phenotype caused by the H. chilense cytoplasm was associated with the addition of chromosome 6HchS from H. chilense accession H1. Thus, some fertility restoration genes appear to be located in this chromosome arm. Considering the features displayed by the msH1 system, we consider that it has a great potential for the development of viable technology for hybrid wheat production.

The development of a PCR-based marker for PSY1 from Hordeum chilense, a candidate gene for carotenoid content accumulation in tritordeum seeds

Hexaploid tritordeums are the amphiploids derived from the cross between the wild barley Hordeum chilense and durum wheat. Tritordeums are characterised by higher yellow pigment content in their seeds than their durum wheat progenitors due to certain H. chilense genes located on the α arm of chromosome 7Hch. In this work a candidate gene approach based on the phytoene synthase gene (PSY) was followed to investigate whether PSY1 may be responsible for the high carotenoid content in tritordeum and to develop a diagnostic marker for H. chilense PSY. This gene codes for the first step in the carotenoid biosynthetic pathway. It was first demonstrated that PSY is duplicated in H. chilense, Triticum urartu, and durum wheat (PSY1 and PSY2), and subsequently a diagnostic cleaved amplified polymorphism (CAP) marker able to differentiate between H. chilense and durum wheat PSY1 was developed. Using this CAP marker and a set of H. chilense-common wheat addition lines it was found that PSY1 is located on the α arm of chromosome 7Hch, where the gene(s) for yellow pigment content are located. PSY1 is located on chromosomes 7A and 7B of durum wheat as demonstrated using Langdon substitution lines. Furthermore, synteny between rice and wheat indicates that PSY1 should be located on the long arms of chromosomes 7A and 7B, in agreement with QTL data for yellow pigment content. Together, these results suggest that PSY1 may be a good candidate gene for further work with yellow pigment content in both durum wheat and tritordeum. In addition, the diagnostic CAP marker developed will be used in our breeding program to transfer H. chilense genes to durum wheat, to evaluate their potential for durum wheat improvement.

Identification of QTLs associated with yield and its components in Miscanthus sinensis Anderss

Yield and its components (stem-, leaf- and top-yield) were analyzed in a population derived from the cross between F1.1 andF1.7 entries of Miscanthus sinensisAnders. Both lines are offspring of the cross between MS-90-2 and MS-88-110. The aim of this work was to identify QTLs for yield and its components suitable to develop a Marker Aided Selection (MAS) program in M. sinensis.QTL analyses were performed using a previous linkage map based on RAPD markers which was constructed using a new mapping strategy named ‘offspring cross’ that is useful for mapping in forest and fruit trees. The MapQTL 4.0 package was used to perform QTL analyses. Twenty potential QTLs were detected over two years of analyses. Out of these, 6 were associated with yield,8 with stem-yield, 2 with leaf-yield and 4with top-yield. These results constitute an initial step to develop a MAS program for biomass production.

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.

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.

Chromosome engineering in wheat to restore male fertility in the msH1 CMS system

Pollen fertility restoration of the CMS phenotype caused by H. chilense cytoplasm in wheat was associated with the addition of chromosome 6HchS from H. chilense accession H1. In order to develop an euploid restored line, different genomic combinations substituting the 6HchS arm for another homoeologous chromosome in wheat were evaluated, with the conclusion that the optimal combination was the translocation T6HchS·6DL. The double translocation T6HchS·6DL in H. chilense cytoplasm was obtained. This line is fertile and stable under different environmental conditions. However, a single dose of the T6HchS·6DL translocation is insufficient for fertility restoration when chromosome 6D is also present. Restoration in the msH1 system is promoted by interaction between two or more genes, and in addition to the restorer of fertility (Rf) located on chromosome 6HchS, one or more inhibitor of fertility (Fi) genes may be present in chromosome 6DL.

Application of near infrared reflectance spectroscopy to the determination of carotenoid content in tritordeum for breeding purposes

For pasta production, the yellow colour, mainly caused by carotenoids, is a worldwide requirement. Hexaploid tritodeums are the amphiploids derived from the cross between Hordeum chilense and Triticum turgidum. They show a higher carotenoid content than their wheat parents. This work aimed to develop a non-destructive method for carotenoid content determination to assist the tritordeum breeding program. We assessed the ability of near infrared reflectance spectroscopy (NIRS) to predict carotenoid content in whole grains of tritordeum. In total, 285 samples were scanned by NIRS. After non-destructive NIRS scanning, the seeds were analysed for carotenoid content and a calibration equation was developed. It is characterised by a coefficient of multiple determination (R2) of 0.85. This equation was initially evaluated by cross validation showing an r2 of 0.81 and a standard error of cross validation (SECV) of 1.49. It was further evaluated using external validation with a different set of samples not included in the calibration. This analysis showed an r2 of 0.81 and a standard error of performance (SEP) of 1.51. This equation allows discrimination between low and high carotenoid content lines in a non-destructive way. These results constitute a substantial advance for tritordeum breeding programs whose final aim is to develop high carotenoid content tritordeums useful for durum wheat breeding.