Pasqualina Colasuonno

Comparison of genomic and EST-derived SSR markers in phylogenetic analysis of wheat

Microsatellite markers (simple sequence repeats, SSRs) are used for a wide range of crop genetic and breeding applications, including genetic diversity assessment, phylogenetic analysis, genotypic profiling and marker-assisted selection. Genomic SSR (gSSR) have attracted more attention because of abundance in plant genome, reproducibility, high level of polymorphism and codominant inheritance. Recently, the availability of data for expressed sequence tags (EST), has given more emphasis to EST-derived SSRs, which belong to the transcribed regions of DNA, and are expected to be more conserved and have a higher transferability rate across species than gSSR markers. In the present study, several gSSR and EST-SSR markers were investigated for their transferability and level of DNA polymorphism in different ancestral tetraploid and diploid Triticum and Aegilops species. The same gSSR and EST-SSR markers were also evaluated for their applicability in the phylogenetic analysis of wheat. Both gSSR and EST-SSR markers showed differences for the average transferability rate and the number of alleles/ locus. Phylogenetic trees based on gSSR and EST-SSR markers were in accordance with phylogenetic relations based on cytogenetic and molecular analyses.

The carotenoid biosynthetic and catabolic genes in wheat and their association with yellow pigments

BackgroundIn plants carotenoids play an important role in the photosynthetic process and photo-oxidative protection, and are the substrate for the synthesis of abscisic acid and strigolactones. In addition to their protective role as antioxidants and precursors of vitamin A, in wheat carotenoids are important as they influence the colour (whiteness vs. yellowness) of the grain. Understanding the genetic basis of grain yellow pigments, and identifying associated markers provide the basis for improving wheat quality by molecular breeding.ResultsTwenty-four candidate genes involved in the biosynthesis and catabolism of carotenoid compounds have been identified in wheat by comparative genomics. Single nucleotide polymorphisms (SNPs) found in the coding sequences of 19 candidate genes allowed their chromosomal location and accurate map position on two reference consensus maps to be determined. The genome-wide association study based on genotyping a tetraploid wheat collection with 81,587 gene-associated SNPs validated quantitative trait loci (QTLs) previously detected in biparental populations and discovered new QTLs for grain colour-related traits. Ten carotenoid genes mapped in chromosome regions underlying pigment content QTLs indicating possible functional relationships between candidate genes and the trait.ConclusionsThe availability of linked, candidate gene-based markers can facilitate breeding wheat cultivars with desirable levels of carotenoids. Identifying QTLs linked to carotenoid pigmentation can contribute to understanding genes underlying carotenoid accumulation in the wheat kernels. Together these outputs can be combined to exploit the genetic variability of colour-related traits for the nutritional and commercial improvement of wheat products.

Development of a new wheat microarray from a durum wheat totipotent cDNA library used for a powdery mildew resistance study

Totipotent cDNA libraries representative of all the potentially expressed sequences in a genome would be of great benefit to gene expression studies. Here, we report on an innovative method for creating such a library for durum wheat (Triticum turgidum L. var. durum) and its application for gene discovery. The use of suitable quantities of 5-azacytidine during the germination phase induced the demethylation of total DNA, and the resulting seedlings potentially express all of the genes present in the genome. A new wheat microarray consisting of 4925 unigenes was developed from the totipotent cDNA library and used to screen for genes that may contribute to differences in the disease resistance of two near-isogenic lines, the durum wheat cultivar Latino and the line 5BIL-42, which are respectively susceptible and resistant to powdery mildew. Fluorescently labeled cDNA was prepared from the RNA of seedlings of the two near-isogenic wheat lines after infection with a single powdery mildew isolate under controlled conditions in the greenhouse. Hybridization to the microarray identified six genes that were differently expressed in the two lines. Four of the sequences could be assigned putative functions based on their similarity to known genes in public databases. Physical mapping of the six genes localized them to two regions of the genome: the centromeric region of chromosome 5B, where the Pm36 resistance gene was previously localized, and chromosome 6B.

Genetic dissection of the relationships between grain yield components by genome-wide association mapping in a collection of tetraploid wheats.

Increasing grain yield potential in wheat has been a major target of most breeding programs. Genetic advance has been frequently hindered by negative correlations among yield components that have been often observed in segregant populations and germplasm collections. A tetraploid wheat collection was evaluated in seven environments and genotyped with a 90K SNP assay to identify major and stable quantitative trait loci (QTL) for grain yield per spike (GYS), kernel number per spike (KNS) and thousand-kernel weight (TKW), and to analyse the genetic relationships between the yield components at QTL level. The genome-wide association analysis detected eight, eleven and ten QTL for KNS, TKW and GYS, respectively, significant in at least three environments or two environments and the mean across environments. Most of the QTL for TKW and KNS were found located in different marker intervals, indicating that they are genetically controlled independently by each other. Out of eight KNS QTL, three were associated to significant increases of GYS, while the increased grain number of five additional QTL was completely or partially compensated by decreases in grain weight, thus producing no or reduced effects on GYS. Similarly, four consistent and five suggestive TKW QTL resulted in visible increase of GYS, while seven additional QTL were associated to reduced effects in grain number and no effects on GYS. Our results showed that QTL analysis for detecting TKW or KNS alleles useful for improving grain yield potential should consider the pleiotropic effects of the QTL or the association to other QTLs.

Characterization of Aldehyde Oxidase (AO) Genes Involved in the Accumulation of Carotenoid Pigments in Wheat Grain

Aldehyde Oxidase (AO) enzyme (EC 1.2.3.1) catalyzes the final steps of carotenoid catabolism and it is a key enzyme in the abscisic acid (ABA) biosynthesis. AO isoforms are located in the cytosolic compartment of tissues in many plants, where induce the oxidation of aldehydes into carboxylic acid, and in addition, catalyze the hydroxylation of some heterocycles. The goal of the present study was to characterize the AO genes involved in the accumulation of carotenoid pigments in wheat grain, an important quantitative trait controlled by multiple genes. The cDNAs corresponding to the four AO isoforms from Arabidopsis thaliana and five AO isoforms from Brachypodium distachyon were used as query in 454 sequence assemblies data for Triticum aestivum cv. Chinese Spring (https://urgi.versailles.inra.fr/blast/blast.php) to obtain the partial or whole orthologous wheat AO sequences. Three wheat isoforms, designated AO1, AO2, and AO3 were located on the chromosome groups 2, 5, and 7, respectively, and mapped on two consensus wheat maps by SNP markers located within the AO gene sequences. To validate the possible relationships between AO3 genes and carotenoid accumulation in wheat, the expression levels of AO-A3 and AO-B3 gene were determined during the kernel maturation stage of two durum wheat cultivars, Ciccio and Svevo, characterized by a low and high carotenoid content, respectively. Different AO-A3 gene expression values were observed between the two cultivars indicating that the AO-A3 allele present in Ciccio was more active in carotenoid degradation. A gene marker was developed and can be used for marker-assisted selection in wheat breeding programs.

Development of a High-Density SNP-Based Linkage Map and Detection of QTL for β-Glucans, Protein Content, Grain Yield per Spike and Heading Time in Durum Wheat

High-density genetic linkage maps of crop species are particularly useful in detecting qualitative and quantitative trait loci for important agronomic traits and in improving the power of classical approaches to identify candidate genes. The aim of this study was to develop a high-density genetic linkage map in a durum wheat recombinant inbred lines population (RIL) derived from two elite wheat cultivars and to identify, characterize and correlate Quantitative Trait Loci (QTL) for β-glucan, protein content, grain yield per spike and heading time. A dense map constructed by genotyping the RIL population with the wheat 90K iSelect array included 5444 single nucleotide polymorphism (SNP) markers distributed in 36 linkage groups. Data for β-glucan and protein content, grain yield per spike and heading time were obtained from replicated trials conducted at two locations in southern Italy. A total of 19 QTL were detected in different chromosome regions. In particular, three QTL for β-glucan content were detected on chromosomes 2A and 2B (two loci); eight QTL controlling grain protein content were detected on chromosomes 1B, 2B, 3B (two loci), 4A, 5A, 7A and 7B; seven QTL for grain yield per spike were identified on chromosomes 1A, 2B, 3A (two loci), 3B (two loci) and 6B; and one marker-trait association was detected on chromosome 2A for heading time. The last was co-located with a β-glucan QTL, and the two QTL appeared to be negatively correlated. A genome scan for genomic regions controlling the traits and SNP annotated sequences identified five putative candidate genes involved in different biosynthesis pathways (β-glucosidase, GLU1a; APETALA2, TaAP2; gigantea 3, TaGI3; 14-3-3 protein, Ta14A; and photoperiod sensitivity, Ppd-A1). This study provides additional information on QTL for important agronomic traits that could be useful for marker-assisted breeding to obtain new genotypes with commercial and nutritional relevance.