Jens Léon

Advanced backcross QTL analysis in barley (Hordeum vulgare L.)

Abstract. This paper reports on the first advanced backcross-QTL (quantitative trait locus) project which utilizes spring barley as a model. A BC2F2 population was derived from the initial cross Apex (Hordeum vulgare ssp. vulgare, hereafter abbreviated with Hv) × ISR101-23 (H. v. ssp. spontaneum, hereafter abbreviated with Hsp). Altogether 136 BC2F2 individuals were genotyped with 45 SSR (simple sequence repeat) markers. Subsequently, field data for 136 BC2F2 families were collected for 13 quantitative traits measured in a maximum of six environments. QTLs were detected by means of a two-factorial ANOVA with a significance level of P < 0.01 for a marker main effect and a marker × environment (M × E) interaction, respectively. Among 585 marker × trait combinations tested, 86 putative QTLs were identified. At 64 putative QTLs, the marker main effect and at 27 putative QTLs, the M × E interaction were significant. In five cases, both effects were significant. Among the putative QTLs, 29 (34%) favorable effects were identified from the exotic parent. At these marker loci the homozygous Hsp genotype was associated with an improvement of the trait compared to the homozygous Hv genotype. In one case, the Hsp allele was associated with a yield increase of 7.7% averaged across the six environments tested. A yield QTL in the same chromosomal region was already reported in earlier barley QTL studies.

Mapping new EMBL-derived barley microsatellites and their use in differentiating German barley cultivars

Abstract By searching the EMBL DNA sequence database, we were able to develop 39 new, database-derived barley microsatellites. Eighteen of these EMBL microsatellites were mapped either to the interspecific barley map Lerche×BGRC41936 (L×41), the Igri×Franka map (I×F, Graner et al. 1991), or to both maps simultaneously. In addition, all 39 EMBL microsatellites were assigned to individual barley chromosomes by PCR screening of wheat barley addition lines. Both studies verified a random distribution of the microsatellites within the barley genome. Subsequently, 22 EMBL microsatellites were used to assess the genetic similarity among a set of 28, mainly German, barley cultivars and two wild form accessions. Spring and winter cultivars could be easily differentiated using the first coordinate of a principal coordinate analysis. Whereas the group of spring barley cultivars appeared rather homogeneous, winter barley cultivars could be divided into three subgroups. Two H. v. ssp. spontaneum accessions were included in the assessment of genetic similarity. They were placed among the winter barley cultivars. Based on the assessment of the 30 barley cultivars and accessions, the polymorphism information content (PIC) of each EMBL microsatellite has been calculated. The average PIC value among the EMBL microsatellites was equal to 0.38, which ascertains the value of these microsatellites as a genetic tool in barley genome research projects.

Development of candidate introgression lines using an exotic barley accession (Hordeum vulgare ssp. spontaneum) as donor

In the present paper, we report on the selection of two sets of candidate introgression lines (pre-ILs) in spring barley. Two BC2DH populations, S42 and T42, were generated by introgressing an accession of Hordeum vulgare ssp. spontaneum (ISR42-8, from Israel) into two different spring barley cultivars, Scarlett (S) and Thuringia (T). From these BC2DH populations two sets with 49 (S42) and 43 (T42) pre-ILs were selected, and their genomic architecture as revealed by SSR marker analysis was characterised. The selected pre-ILs cover at least 98.1% (S42) and 93.0% (T42) of the exotic genome in overlapping introgressions and contain on average 2 (S42) and 1.5 (T42) additional non-target introgressions. In order to illustrate a potential application and validation of these pre-ILs, the phenotypic effect of the exotic introgression at the locus of the major photoperiod response gene Ppd-H1 was analysed. Pre-ILs carrying the introgression at the Ppd-H1 locus flowered significantly earlier than the elite parents, and the introgression maintained its effect across the two genetic backgrounds and across four tested environments. The selected pre-ILs represent a first promising step towards the assessment and utilization of genetic variation present in exotic barley. They may promote the breeding progress, serve for the verification of QTL effects and provide a valuable resource for the unravelling of gene function, e.g. by expression profiling or map-based cloning.

AB-QTL analysis in spring barley. I. Detection of resistance genes against powdery mildew, leaf rust and scald introgressed from wild barley

The objective of this study was to map new resistance genes against powdery mildew (Blumeria graminis f. sp. hordei L.), leaf rust (Puccinia hordei L.) and scald [Rhynchosporium secalis (Oud.) J. Davis] in the advanced backcross doubled haploid (BC2DH) population S42 derived from a cross between the spring barley cultivar ‘Scarlett’ and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). Using field data of disease severity recorded in eight environments under natural infestation and genotype data of 98 SSR loci, we detected nine QTL for powdery mildew, six QTL for leaf rust resistance and three QTL for scald resistance. The presence of the exotic QTL alleles reduced disease symptoms by a maximum of 51.5, 37.6 and 16.5% for powdery mildew, leaf rust and scald, respectively. Some of the detected QTL may correspond to previously identified qualitative (i.e. Mla) and to quantitative resistance genes. Others may be newly identified resistance genes. For the majority of resistance QTL (61.0%) the wild barley contributed the favourable allele demonstrating the usefulness of wild barley in the quest for resistant cultivars.

Association of barley photoperiod and vernalization genes with QTLs for flowering time and agronomic traits in a BC2DH population and a set of wild barley introgression lines

The control of flowering time has important impacts on crop yield. The variation in response to day length (photoperiod) and low temperature (vernalization) has been selected in barley to provide adaptation to different environments and farming practices. As a further step towards unraveling the genetic mechanisms underlying flowering time control in barley, we investigated the allelic variation of ten known or putative photoperiod and vernalization pathway genes between two genotypes, the spring barley elite cultivar ‘Scarlett’ (Hordeum vulgare ssp. vulgare) and the wild barley accession ‘ISR42-8’ (Hordeum vulgare ssp. spontaneum). The genes studied are Ppd-H1, VRN-H1, VRN-H2, VRN-H3, HvCO1, HvCO2, HvGI, HvFT2, HvFT3 and HvFT4. ‘Scarlett’ and ‘ISR42-8’ are the parents of the BC2DH advanced backcross population S42 and a set of wild barley introgression lines (S42ILs). The latter are derived from S42 after backcrossing and marker-assisted selection. The genotypes and phenotypes in S42 and S42ILs were utilized to determine the genetic map location of the candidate genes and to test if these genes may exert quantitative trait locus (QTL) effects on flowering time, yield and yield-related traits in the two populations studied. By sequencing the characteristic regions of the genes and genotyping with diagnostic markers, the contrasting allelic constitutions of four known flowering regulation genes were identified as ppd-H1, Vrn-H1, vrn-H2 and vrn-H3 in ‘Scarlett’ and as Ppd-H1, vrn-H1, Vrn-H2 and a novel allele of VRN-H3 in ‘ISR42-8’. All candidate genes could be placed on a barley simple sequence repeat (SSR) map. Seven candidate genes (Ppd-H1, VRN-H2, VRN-H3, HvGI, HvFT2, HvFT3 and HvFT4) were associated with flowering time QTLs in population S42. Four exotic alleles (Ppd-H1, Vrn-H2, vrn-H3 and HvCO1) possibly exhibited significant effects on flowering time in S42ILs. In both populations, the QTL showing the strongest effect corresponded to Ppd-H1. Here, the exotic allele was associated with a reduction of number of days until flowering by 8.0 and 12.7%, respectively. Our data suggest that Ppd-H1, Vrn-H2 and Vrn-H3 may also exert pleiotropic effects on yield and yield-related traits.

Early drought stress detection in cereals: simplex volume maximisation for hyperspectral image analysis

Early water stress recognition is of great relevance in precision plant breeding and production. Hyperspectral imaging sensors can be a valuable tool for early stress detection with high spatio-temporal resolution. They gather large, high dimensional data cubes posing a significant challenge to data analysis. Classical supervised learning algorithms often fail in applied plant sciences due to their need of labelled datasets, which are difficult to obtain. Therefore, new approaches for unsupervised learning of relevant patterns are needed. We apply for the first time a recent matrix factorisation technique, simplex volume maximisation (SiVM), to hyperspectral data. It is an unsupervised classification approach, optimised for fast computation of massive datasets. It allows calculation of how similar each spectrum is to observed typical spectra. This provides the means to express how likely it is that one plant is suffering from stress. The method was tested for drought stress, applied to potted barley plants in a controlled rain-out shelter experiment and to agricultural corn plots subjected to a two factorial field setup altering water and nutrient availability. Both experiments were conducted on the canopy level. SiVM was significantly better than using a combination of established vegetation indices. In the corn plots, SiVM clearly separated the different treatments, even though the effects on leaf and canopy traits were subtle.

AB-QTL analysis in spring barley: III. Identification of exotic alleles for the improvement of malting quality in spring barley (H. vulgare ssp. spontaneum)

Malting quality is genetically determined by the complex interaction of numerous traits which are expressed prior to and, in particular, during the malting process. Here, we applied the advanced backcross quantitative trait locus (AB-QTL) strategy (Tanksley and Nelson, Theor Appl Genet 92:191–203, 1996), to detect QTLs for malting quality traits and, in addition, to identify favourable exotic alleles for the improvement of malting quality. For this, the BC2DH population S42 was generated from a cross between the spring barley cultivar Scarlett and the wild barley accession ISR42-8 (Hordeum vulgare ssp. spontaneum). A QTL analysis in S42 for seven malting parameters measured in two different environments yielded 48 QTLs. The exotic genotype improved the trait performance at 18 (37.5%) of 48 QTLs. These favourable exotic alleles were detected, in particular, on the chromosome arms 3HL, 4HS, 4HL and 6HL. The exotic allele on 4HL, for example, improved α-amylase activity by 16.3%, fermentability by 0.8% and reduced raw protein by 2.4%. On chromosome 6HL, the exotic allele increased α-amylase by 16.0%, fermentability by 1.3%, friability by 7.3% and reduced viscosity by 2.9%. Favourable transgressive segregation, i.e. S42 lines exhibiting significantly better performance than the recurrent parent Scarlett, was recorded for four traits. For α-amylase, fermentability, fine-grind extract and VZ45 20, 16, 2 and 26 S42 lines, respectively, surpassed the recurrent parent Scarlett. The present study hence demonstrates that wild barley does harbour valuable alleles, which can enrich the genetic basis of cultivated barley and improve malting quality traits.

Multi-parent advanced generation inter-cross in barley: high-resolution quantitative trait locus mapping for flowering time as a proof of concept

The choice of mapping population is one of the key factors in understanding the genetic effects of complex traits and determines the power and precision of quantitative trait locus (QTL) mapping. We present the results of the first eight-way multi-parent advanced generation inter-cross (MAGIC) doubled haploid (DH) population in barley (Hordeum vulgare ssp. vulgare) applied to mapping complex traits. The results of the genetic architecture within the barley MAGIC population allowed QTL mapping in 533 DH lines with 4,550 single nucleotide polymorphisms (SNPs) with a newly developed mixed linear model in SAS v9.2, incorporating multi-locus analysis and cross validation for flowering time. Two QTL mapping approaches, the binary approach (BA), which is widely used in QTL and association mapping, and a novel haplotype approach (HA) were compared based on their efficiency, precision for QTL detection and estimation of genetic effects. The analysis detected 17 QTLs, five of which were shared between the two approaches; five and two were specifically found with the BA and HA approaches, respectively. The combination of the two mapping approaches enabled high-precision QTL mapping for flowering time. The QTLs corresponded to the genomic regions of major flowering-time genes Vrn-H1, Vrn-H3, HvGI, Ppd-H1, HvFT2, HvFT4, Co1 and linked genes for plant height (sdw1). These results confirm the proof of concept of QTL mapping in a multi-parent population, highlight the advantages and demonstrate that the barley MAGIC DH lines in combination with an advanced QTL mapping approach are valuable resources for mapping complex traits.

AB-QTL analysis reveals new alleles associated to proline accumulation and leaf wilting under drought stress conditions in barley (Hordeum vulgare L.).

BackgroundLand plants have evolved several measures to maintain their life against abiotic stresses. The accumulation of proline is the most generalized response of plants under drought, heat or salt stress conditions. It is known as an osmoprotectant which also acts as an instant source of energy during drought recovery process. But, both its role and genetic inheritance are poorly understood in agriculture crops. In the present work, advanced backcross quantitative trait locus (AB-QTL) analysis was performed to elucidate genetic mechanisms controlling proline accumulation and leaf wilting in barley under drought stress conditions.ResultsThe analysis revealed eight QTL associated to proline content (PC) and leaf wilting (WS). QTL for PC were localized on chromosome 3H, 4H, 5H and 6H. The strongest QTL effect QPC.S42.5H was detected on chromosome 5H where drought inducible exotic allele was associated to increase PC by 54%. QTL effects QPC.S42.3H, QPC.S42.4H and QPC.S42.6H were responsible to heighten PC due to the preeminence of elite alleles over the exotic alleles which ranged from 26% to 43%. For WS, QTL have been localized on chromosome 1H, 2H, 3H and 4H. Among these, QWS.S42.1H and QWS.S42.4H were associated to decrease in WS due to the introgression of exotic alleles. In addition, two digenic epistatic interaction effects were detected for WS where the additive effect of exotic alleles imparted a favorable increase in the trait value.ConclusionsThe present data represents a first report on whole-genome mapping of proline accumulation and leaf wilting in barley. The detected QTL are linked to new alleles from both cultivated and wild accessions which bring out an initial insight on the genetic inheritance of PC and WS. These QTL alleles are fixed in the isogenic background of Scarlett, which will allow for positional cloning of underlying genes and to develop drought resilient barley cultivars.

Wild barley introgression lines revealed novel QTL alleles for root and related shoot traits in the cultivated barley (Hordeum vulgare L.)

BackgroundRoot is the prime organ that sucks water and nutrients from deep layer of soil. Wild barley diversity exhibits remarkable variation in root system architecture that seems crucial in its adaptation to abiotic stresses like drought. In the present study, we performed quantitative trait locus (QTL) mapping of root and related shoot traits under control and drought conditions using a population of wild barley introgression lines (ILs). This population (S42IL) comprising of genome-wide introgressions of wild barley accession ISR42-8 in the cultivar Scarlett background. Here, we aimed to detect novel QTL alleles for improved root and related shoot features and to introduce them in modern cultivars.ResultsThe cultivar Scarlett and wild barley accession ISR42-8 revealed significant variation of root and related shoot traits. ISR42-8 showed a higher performance in root system attributes like root dry weight (RDW), root volume (RV), root length (RL) and tiller number per plant (TIL) than Scarlett. Whereas, Scarlett exhibited erect type growth habit (GH) as compared to spreading growth habit in ISR42-8. The S42IL population revealed significant and wide range of variation for the investigated traits. Strong positive correlations were found among the root related traits whereas GH revealed negative correlation with root and shoot traits. The trait-wise comparison of phenotypic data with the ILs genetic map revealed six, eight, five, five and four QTL for RL, RDW, RV, TIL and GH, respectively. These QTL were linked to one or several traits simultaneously and localized to 15 regions across all chromosomes. Among these, beneficial QTL alleles of wild origin for RL, RDW, RV, TIL and GH, have been fixed in the cultivar Scarlett background.ConclusionsThe present study revealed 15 chromosomal regions where the exotic QTL alleles showed improvement for root and related shoot traits. These data suggest that wild barley accession ISR42-8 bears alleles different from those of Scarlett. Hence, the utility of genome-wide wild barley introgression lines is desirable to test the performance of individual exotic alleles in the elite gene pool as well as to transfer them in the cultivated germplasm.