Study of malting quality and frost tolerance in the winter x spring 'Nure x Tremois’ doubled haploid population and development of a Marker-Assisted Selection programme

Abstract

Recently, it has been reported the development of a new barley map derived from a two-rowed cross ‘winter’ x ‘spring’ (Francia et al. 2004). The winter parent ‘Nure’ is a frost tolerant feed cultivar, whereas the spring parent ‘Tremois’ is a frost susceptible malting variety. The genetic map built on the ‘Nure’ x ‘Tremois’ (NxT) doubled-haploid (DH) population is currently the only one where malting quality traits were expected to segregate together with winterhardiness and vernalization requirement, and this represented a valuable tool to investigate in a unique genetic system with multiple agronomical traits, such as malting quality and tolerance to abiotic stresses. By using the DH population NxT, the objectives were to study the genetic basis of the quantitative variation observed for malting quality, to validate the genetic determinants of frost tolerance (Fr-H1 and Fr-H2 loci) and to identify a molecular marker set to select winter malting barley. QTL analysis of malting quality traits allowed us to identify QTLs on six chromosomes, with a main cluster on chromosome 1H. For wort viscosity and malt extract, the favourable alleles at the two QTLs on chromosome 5H were carried by the winter feeding parent ‘Nure’. Doubled-haploid lines with malting quality higher than the spring cultivar ‘Tremois’ were individuated in the NxT population. These higher quality lines all showed either a facultative or a winter growth habit, and a level of frost tolerance comparable to that of the winter, tolerant parent ‘Nure’. This showed that, at least in the present cross, positive contributions to malting quality can be found in winter feed barleys. In the present study, marker-assisted selection was conducted among progenies from the same cross in which the frost tolerance and malting quality QTLs have been mapped. Our results indicate that using the molecular marker set identified in this work the selection for winter, frost tolerance and high malting lines is possible, at least in material genetically derived from the ‘Nure’ x ‘Tremois’ cross. At last, two low temperature QTLs were found to segregate in the NxT population on the long arm of chromosome 5H (Fr-H1, distal; Fr-H2, proximal). With the final aim of positional cloning of the genetic determinants of Fr-H1 and Fr-H2, a large segregating population of 1,849 F2 plants between parents ‘Nure’ and ‘Tremois’ was prepared. These two QT loci were first validated by using a set of F3 families, marker-selected to harbor pairs of reciprocal haplotypes, with one QTL fixed at homozygosity and the alternate one in heterozygous phase. The study was then focused towards the isolation of the determinant of Fr-H2. Subsequent recombinant screens and phenotypic evaluation of F4 segregants allowed us to estimate a refined genomic interval of Fr-H2 (4.6 cM). Several barley genes with the CBF transcription factor signature had been already roughly mapped in cluster at Fr-H2, and they represent likely candidate genes underlying this QTL. Using the large segregating population (3,698 gametes) a high-resolution genetic map of the HvCBF gene cluster was then constructed, and after fine mapping, six recombinations between the HvCBFs were observed. It was therefore possible to genetically divide seven HvCBF subclusters in barley, in a region spanning 0.81 cM, with distances among them varying from 0.03 to 0.32 cM. The few recombinants between the different HvCBF sub-clusters are being marker-selected and taken to homozygosity, to phenotypically separate the effects of the single HvCBF genes.