Identification of quantitative trait loci influencing aerial morphogenesis in the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

In many legume crops, especially in forage legumes, aerial morphogenesis defined as growth and development of plant organs, is an essential trait as it determines plant and seed biomass as well as forage quality (protein concentration, dry matter digestibility). Medicago truncatula is a model species for legume crops. A set of 29 accessions of M. truncatula was evaluated for aerial morphogenetic traits. A recombinant inbred lines (RILs) mapping population was used for analysing quantitative variation in aerial morphogenetic traits and QTL detection. Genes described to be involved in aerial morphogenetic traits in other species were mapped to analyse co-location between QTLs and genes. A large variation was found for flowering date, morphology and dynamics of branch elongation among the 29 accessions and within the RILs population. Flowering date was negatively correlated to main stem and branch length. QTLs were detected for all traits, and each QTL explained from 5.2 to 59.2% of the phenotypic variation. A QTL explaining a large part of genetic variation for flowering date and branch growth was found on chromosome 7. The other chromosomes were also involved in the variation detected in several traits. Mapping of candidate genes indicates a co-location between a homologue of Constans gene or a flowering locus T (FT) gene and the QTL of flowering date on chromosome 7. Other candidate genes for several QTLs are described. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Polymorphism of the <Emphasis Type="Italic">CONSTANS</Emphasis> gene in <Emphasis Type="Italic">Brassica</Emphasis> plants

Using a direct amplification of genomic DNA from two Brassica rapa forms, we obtained two homologs of the CONSTANS gene, which controls the photoperiodic induction of flowering in Arabidopsis plants. The cloned fragments of B. rapa genome were identified as members of the CONSTANS-LIKE1 class. By aligning the nucleotide sequences of the CONSTANS gene and its homologs, three classes, CONSTANS, CONSTANS-LIKE1, and CONSTANS-LIKE2, were distinctly discerned by their primary structure. The pattern of restriction fragment length polymorphisms (RFLP) of the CONSTANS homologs in B. carinata, B. juncea, B. napus, B. nigra, B. oleracea, and B. rapa were genome-specific; in addition, the CONSTANS homologs were classified by plant geographic origin, and we assume that such classification is related to plant photoperiodic response.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 274–281.Original Russian Text Copyright © 2005 by Martynov, Khavkin.This revised version was published online in April 2005 with a corrected cover date.     

Identification of QTL for resistance and susceptibility to <Emphasis Type="Italic">Stagonospora meliloti</Emphasis> in autotetraploid lucerne

In eastern Australia and California, USA, one of the major lethal fungal diseases of lucerne (Medicago sativa) is Stagonospora root and crown rot, caused by Stagonospora meliloti. Quantitative trait loci (QTL) involved in resistance and susceptibility to S. meliloti were identified in an autotetraploid lucerne backcross population of 145 individuals. Using regression analysis and interval mapping, we detected one region each on linkage groups 2, 6 and 7 that were consistently associated with disease reaction to S. meliloti in two separate experiments. The largest QTL on linkage group 7, which is associated with resistance to S. meliloti, contributed up to 17% of the phenotypic variation. The QTL located on linkage group 2, which is potentially a resistance allele in repulsion to the markers for susceptibility to S. meliloti, contributed up to 8% of the phenotypic variation. The QTL located on linkage group 6, which is associated with susceptibility to S. meliloti, contributed up to 16% of the phenotypic variation. A further two unlinked markers contributed 5 and 8% of the phenotypic variation, and were detected in only one experiment. A total of 517 simple sequence repeat (SSR) markers from Medicago truncatula were screened on the parents of the mapping population. Only 27 (6%) SSR markers were polymorphic and could be incorporated into the autotetraploid map of M. sativa. This allowed alignment of our M. sativa linkage map with published M. truncatula maps. The markers linked to the QTL we have reported will be useful for marker assisted selection for partial resistance to S. meliloti in lucerne.     

Genetic determinism of reproductive fitness traits under drought stress in the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Fitness traits that determine the reproductive ability of individuals and the persistence of populations are affected by drought stress. Medicago truncatula that commonly encounters drought stress in its natural area, and for which large natural diversity and genetic tools are available, is a suitable species to investigate genetic determinism of fitness traits under stress. In a common garden, three successive cycles of short drought stress were applied after flowering, during the reproductive stage that is the most susceptible to drought for that species. Ten genotypes derived from natural populations and a mapping population were used to investigate the genetic determinism of vegetative and reproductive traits as components of fitness. A large genetic variation was observed and transgressive genotypes (more resistant or more susceptible than the parental genotypes) were found in the mapping population. Fitness traits were reduced by 5–74% in drought condition compared to well-watered condition. The most affected characters were total pod number per plant and total pod weight per plant. A total of 49 QTL, explaining between 6 and 38% of phenotypic variation for vegetative and reproductive fitness traits, were detected on all chromosomes except chromosome 6. A major QTL for flowering date (R ² of 19 and 38%) that co-located with QTL for reproductive fitness traits were found on chromosome 7. In this study, no major QTL specific to drought-stressed or well-watered conditions were detected. We, thus, showed that QTL explaining fitness traits were numerous with small effects, in accordance with the genetic determinism of a complex trait.     

Mapping of quantitative trait loci for resistance to <Emphasis Type="Italic">Mycosphaerella pinodes</Emphasis> in <Emphasis Type="Italic">Pisum sativum</Emphasis> subsp. <Emphasis Type="Italic">syriacum</Emphasis>

Aschochyta blight, caused by Mycosphaerella pinodes, is one of the most economically serious pea pathogens, particularly in winter sowings. The wild Pisum sativum subsp. syriacum accession P665 shows good levels of resistance to this pathogen. Knowledge of the genetic factors controlling resistance to M. pinodes in this wild accession would facilitate gene transfer to pea cultivars; however, previous studies mapping resistance to M. pinodes in pea have never included this wild species. The objective of this study was to identify quantitative trait loci (QTL) controlling resistance to M. pinodes in P. sativum subsp. syriacum and to compare these with QTLs previously described for the same trait in P. sativum. A population formed by 111 F_6:7 recombinant inbred lines derived from a cross between accession P665 and a susceptible pea cultivar (Messire) was analysed using morphological, isozyme, RAPD, STS and EST markers. The map developed covered 1214 cM and contained 246 markers distributed in nine linkage groups, of which seven could be assigned to pea chromosomes. Six QTLs associated with resistance to M. pinodes were detected in linkage groups II, III, IV and V, which collectively explained between 31 and 75% of the phenotypic variation depending of the trait. While QTLs MpIII.1 and MpIII.2 were detected both for seedlings and field resistance, MpV.1 and MpII.1 were specific for growth chamber conditions and MpIII.3 and MpIV.1 for field resistance. Quantitative trait loci MpIII.1, MpII.1, MpIII.2 and MpIII.3 may coincide with other QTLs associated with resistance to M. pinodes previously described in P. sativum. Four QTLs associated with earliness of flowering were also identified. While dfIII.2 and dfVI.1, may correspond with other genes and QTLs controlling earliness in P. sativum, dfIII.1 and dfII.1 may be specific to P. sativum subsp. syriacum. Flowering date and growth habit were strongly associated with resistance to M. pinodes in the field evaluations. The relation observed between earliness, growth habit and resistance to M. pinodes is discussed.     

Mapping of a thermo-sensitive earliness <Emphasis Type="Italic">per se</Emphasis> gene on <Emphasis Type="Italic">Triticum monococcum</Emphasis> chromosome 1A<Superscript>m</Superscript>

An earliness per se gene, designated Eps-A^m1, was mapped in diploid wheat in F₂ and single-seed descent mapping populations from the cross between cultivated (DV92) and wild (G3116) Triticum monococcum accessions. A QTL with a peak on RFLP loci Xcdo393 and Xwg241, the most distal markers on the long arm of chromosome 1A^m, explained 47% of the variation in heading date (LOD score 8.3). Progeny tests for the two F_2:3 families with critical recombination events between Xcdo393 and Xwg241 showed that the gene was distal to Xcdo393 and linked to Xwg241. Progeny tests and replicated experiments with line #3 suggested that Eps-A^m1 was distal to Xwg241. This gene showed a large effect on heading date in the controlled environment experiments, and a smaller, but significant, effect under natural conditions. Eps-A^m1 showed significant epistatic interactions with photoperiod and vernalization treatments, suggesting that the different classes of genes affecting heading date interact as part of a complex network that controls the timing of flowering induction. Besides its interactions with other genes affecting heading date, Eps-A^m1 showed a significant interaction with temperature. The effect of temperature was larger in plants carrying the DV92 allele for late flowering than in those carrying the G3116 allele for early flowering. Average differences in heading date between the experiments performed at 16 °C and 23 °C were approximately 11 days (P < 0.001) for the lines carrying the Eps-A^m1 allele for early flowering but approximately 50 days (P < 0.0001) for the lines carrying the allele for late flowering. The large differences in heading time (average 80 days) observed between plants carrying the G3116 and DV92 alleles when grown at 16 °C, suggest that it would be possible to produce very detailed maps for this gene to facilitate its future positional cloning.     

Overexpression of the Gm<Emphasis Type="Italic">GAL2</Emphasis> Gene Accelerates Flowering in <Emphasis Type="BoldItalic">Arabidopsis</Emphasis>

A soybean MADS box gene GmGAL2 (Glycine max AGAMOUS Like 2), a homolog of AGL11/STK, was investigated in transgenic Arabidopsis lines. Ectopic expression of GmGAL2 in Arabidopsis enhanced flowering, under both long-day and short-day conditions, by promoting expression of key flowering genes, CONSTANS (CO) and FLOWERING LOCUS T (FT), and lowering expression of floral inhibiter FLOWERING LOCUS C (FLC). Moreover, frequency of silique pod set was also lower in transgenic compared to control Arabidopsis plants. RT-PCR results revealed that GmGAL2 was primarily expressed in the flowers and pods of soybean plants, GmGAL2 expressed higher in SD than LD in soybean.     

Flower form alteration by genetic transformation with the class B MADS-box genes of <Emphasis Type="Italic">Agapanthus</Emphasis><Emphasis Type="Italic">praecox</Emphasis> spp. <Emphasis Type="Italic">orientalis</Emphasis> in transgenic dicot and monocot plants

The class B genes, which belong to the MADS-box gene family, play important roles in regulating petal and stamen development in flowering plants. These genes exist in two different types termed DEF- and GLO-like genes, and the B-function is provided by heterodimers of a DEF- and a GLO-like gene product. In the present study, dicot (tobacco and lettuce) and monocot (Tricyrtis hirta) plants were transformed with the GLO-like gene of Agapanthus praecox ssp. orientalis ApGLO alone or in combination with the DEF-like gene of the same plant ApDEF. In two out of 10 transgenic tobacco plants containing ApGLO, sepals partially converted into petaloid organs. For lettuce, ray florets of four out of nine transgenic plants containing ApGLO also developed additional petaloid organs. In two out of five transgenic T. hirta plants containing both ApGLO and ApDEF, organs developed in whorl 4 showed noticeable morphological alteration: they were much longer compared with carpels of non-transgenic plants, and had purple spots overall on the surface as filaments of non-transgenic plants. No morphological alterations were observed in vegetative organs between transgenic and non-transgenic plants for all the three species. The results obtained in the present study indicate a possibility of molecular breeding for flower form alteration by genetic transformation with the class B MADS-box gene(s) of heterologous plant species.     

<Emphasis Type="Italic">GmFtsH9</Emphasis> expression correlates with in vivo photosystem II function: chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence transient analysis and eQTL mapping in soybean

Filamentation temperature-sensitive H (FtsH) is an ATP-dependent zinc metalloprotease involved in diverse biological functions. There are 12 FtsH proteins in Arabidopsis, among which AtFtsH2 plays an important role in regulating the turnover of photosystem II (PSII) reaction center D1 protein and the development of the photosynthetic apparatus. Here, we have identified 11 FtsH genes in the soybean genome by a bioinformatics approach. These soybean FtsH genes corresponded to seven Arabidopsis FtsH genes, suggesting that the main characteristics of soybean FtsH genes were formed before the evolutionary split of soybean and Arabidopsis. Phylogenetic analyses allowed us to clone a soybean AtFtsH2-like gene designated as GmFtsH9. The predicted protein of GmFtsH9 consists of 690 amino acids and contains three typical FtsH proteins conserved domains. The expression level of GmFtsH9 was determined in a soybean recombinant inbred line population under a pot experiment conducted for measuring chlorophyll a fluorescence transient parameters, photosynthetic CO₂ fixation rate (P _N), and seed yield. Expression quantitative trait loci (eQTL) mapping revealed two trans-acting eQTLs for GmFtsH9. The significant correlation of gene expression level with chlorophyll a fluorescence transient parameters and the presence of overlapping eQTL (QTL) between gene expression level and chlorophyll a fluorescence transient parameters indicated that GmFtsH9 could be involved in regulating PSII function. These results further lead to the understanding of the mechanism underlying FtsH gene expression, and contribute to the development of marker-assisted selection breeding programs for modulating soybean FtsH gene expression.     

Proteomic Profiling Unravels Insights into the Molecular Background Underlying Increased <Emphasis Type="Italic">Aphanomyces euteiches</Emphasis>-Tolerance of <Emphasis Type="Italic">Medicago truncatula</Emphasis>

To investigate the molecular mechanisms underlying susceptibility of legumes to the root pathogen Aphanomyces euteiches (oomycota), comparative proteomic studies have been carried out. In a first approach, we have analysed two Medicago truncatula lines of the French CORE collection (F83.005-5 (R2002) and F83.005-9 (R2002)), which showed either increased or decreased susceptibility to A. euteiches as compared to the widely adopted line A17. Several proteins were identified to be differentially induced after pathogen challenge in the two M. truncatula accessions with altered disease susceptibility, whereof proteins with increased abundances in the more resistant line F83.005-9 could be involved in mechanisms that lead to an improved disease resistance. Among these proteins, we identified two proteasome alpha subunits, which might be involved in defense response. To broaden our studies on A. euteiches-tolerance of M. truncatula, we investigated two other phenomena that lead to an either increased A. euteiches-resistance or to an enhanced susceptibility. The topic of an enhanced plant resistance to A. euteiches was studied in plants showing a bioprotective effect of a pre-established arbuscular mycorrhiza (AM) symbiosis. Evaluation of root fresh weights and pathogen spreading in the root system clearly indicate that mycorrhizal plants show increased A. euteiches-resistance as compared to non-mycorrhizal plants. Proteome analyses revealed the induction of similar protein patterns as in the M. truncatula accessions with comparatively high resistance level to A. euteiches. In a third approach, increased A. euteiches susceptibility was effected by exogenous abscisic acid (ABA) application prior to root infection. Evaluation of the abundance levels of a group of pathogenesis related class 10 (PR10)-like proteins, which were previously identified to be regulated after A. euteiches infection, revealed a correlation between the abundance levels of these proteins and the A. euteiches infection level or severity. Requests concerning seeds from the Medicago truncatula lines F83.005-5 and F83.005-9 should be addressed to Jean-Marie Prospéri, INRA-SGAP Laboratory, Laboratoire de Ressources Génétiques et d’Amélioration des Luzernes méditerranéennes, Mauguio, France, jean-marie.prosperi@ensam.inra.fr.     

Genetic and physical localization of an anthracnose resistance gene in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

Anthracnose of alfalfa, caused by the fungal pathogen Colletotrichum trifolii, is one of the most destructive diseases of alfalfa worldwide. An improved understanding of the genetic and molecular mechanisms underlying host resistance will facilitate the development of resistant alfalfa cultivars, thus providing the most efficient and environmentally sound strategy to control alfalfa diseases. Unfortunately, cultivated alfalfa has an intractable genetic system because of its tetrasomic inheritance and out-crossing nature. Nevertheless, the model legume Medicago truncatula, a close relative of alfalfa, has the potential to serve as a surrogate to map and clone the counterparts of agronomically important genes in alfalfa—particularly, disease resistance genes against economically important pathogens. Here we describe the high-resolution genetic and physical mapping of RCT1, a host resistance gene against C. trifolii race 1 in M. truncatula. We have delimited the RCT1 locus within a physical interval spanning ∼200 kb located on the top of M. truncatula linkage group 4. RCT1 is part of a complex locus containing numerous genes homologous to previously characterized TIR-NBS-LRR type resistance genes. The result presented in this paper will facilitate the positional cloning of RCT1 in Medicago.     

Genetic analysis of<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis> susceptibility in<Emphasis Type="Italic"> Brassica oleracea</Emphasis>

The genetic control and heritability of Agrobacterium tumefaciens susceptibility was investigated using a doubled haploid (DH) mapping population of Brassica oleracea and the associated RFLP map. Preliminary studies were carried out by analysis of an 8×8 diallel, for which the parental lines were selected to include a range of susceptibilities to A. tumefaciens. The variation observed within the diallel was attributed to both additive and dominant gene effects, with additive gene effects being more important. A broad sense heritability value of 0.95 suggested that 95% of the observed variation was due to genetic effects, with just 5% attributed to non-genetic or environmental effects. A high narrow-sense heritibility value of 0.79 suggested that 79% of this trait was controlled by additive gene effects and, therefore, the potential to introduce this trait into breeding material is high. Fifty-nine DH lines from the mapping population were screened for susceptibility towards A. tumefaciens. Variation in susceptibility was observed across the population. The results of the DH screen were entered into the mapping programme MAPQTL and a highly significant quantitative trait loci (QTL) associated with susceptibility to A. tumefaciens was identified on linkage group 09. The use of substitution lines covering this region confirmed the location of this QTL. This work shows that susceptibility to A. tumefaciens is a heritable trait, and the transfer of susceptibility into resistant lines is demonstrated. These findings may help to overcome genotype restrictions to genetic transformation.Communicated by G. Wenzel