Jean Cloutier

SRAP polymorphisms associated with superior freezing tolerance in alfalfa (Medicago sativa spp. sativa)

Sequence-related amplified polymorphism (SRAP) analysis was used to uncover genetic polymorphisms among alfalfa populations recurrently selected for superior tolerance to freezing (TF populations). Bulk DNA samples (45 plants/bulk) from each of the cultivar Apica (ATF0), and populations ATF2, ATF4, ATF5, and ATF6 were evaluated with 42 different SRAP primer pairs. Several polymorphisms that progressively intensified or decreased with the number of recurrent cycles were identified. Four positive polymorphisms (F10-R14, Me4-R8, F10-R8 and F11-R9) that, respectively, yielded 540-, 359-, 213-, and 180-bp fragments were selected for further analysis. SRAP amplifications with genotypes within ATF populations confirmed that the polymorphisms identified with bulk DNA samples were reflecting changes in the frequency of their occurrence in response to selection. In addition, the number of genotypes cumulating multiple polymorphisms markedly increased in response to recurrent selection. Independent segregation of the four SRAP polymorphisms suggests location at unlinked loci. Homology search gave matches with BAC clones from syntenic Medicago truncatula for the four SRAP fragments. Analysis of the relationship with low temperature tolerance showed that multiple SRAP polymorphisms are more frequent in genotypes that maintain superior regrowth after freezing. These results show that SRAP analysis of bulk DNA samples from recurrent selections is an effective approach for the identification of genetic polymorphisms associated with quantitative traits in allogamous species. These polymorphisms could be useful tools for indirect selection of freezing tolerance in alfalfa.

Unusual Methyl-Branched α,β-Unsaturated Acyl Chain Substitutions in the Nod Factors of an Arctic Rhizobium,Mesorhizobium sp. Strain N33 (Oxytropis arctobia)

Rhizobia are soil bacteria, now classified in several genera (e.g., Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, Azorhizobium), which form a symbiotic association with legume plants. The interaction between bacteria and plants results in the formation of nodules on the host plant roots, in which rhizobia fix atmospheric nitrogen. The association between rhizobia and legumes is specific: each rhizobial strain has a defined host range. For example, Mesorhizobium sp. strain N33, isolated from Oxytropis arctobia, also nodulates Astragalus alpinus and Onobrychis viciifolia (19, 26). In contrast, Sinorhizobium meliloti efficiently nodulates alfalfa (Medicago sativa) as well as Melilotus and Trigonella species. Mesorhizobium sp. strain N33 was isolated in the Canadian high arctic and is able to grow and fix nitrogen at temperatures as low as 5°C. In addition, it was shown that arctic rhizobia promoted better growth of O. viciifolia at low temperatures than did temperate strains (27). Thus, it might be valuable to extend the host range of arctic rhizobia to agronomically important legumes, in order to improve nitrogen fixation by these plants at low temperatures. It is therefore important to understand the molecular mechanisms controlling the nodulation specificity of arctic rhizobia. Earlier work has shown that nodulation and host specificity in rhizobium-legume symbiosis are determined by signal exchanges between the bacteria and the host plant (21). Flavonoids excreted by the plant roots induce the expression of rhizobial nodulation (nod) genes. Most of these genes are involved in the biosynthesis and secretion of bacterial signals, the Nod factors, that can specifically induce symbiotic responses of the host plants. These responses include root hair deformation, division of root cortical cells and, in some instances, nodule formation (8, 32). The structures of Nod factors produced by several rhizobial species have been characterized. They are all lipochito-oligosaccharides (LCOs) consisting of β-1,4-linked oligomers of three to five N-acetylglucosamine residues with an amide-linked acyl chain on the nonreducing terminal residue (8, 10, 25). In addition, the glucosamine residues can carry various substitutions. Nod factor specificity is determined by the nature of these substitutions and of the N-acyl chain. A number of nodulation genes have been identified in Mesorhizobium sp. strain N33 (2, 3, 4). The nodABC genes, which are found in all rhizobial species, are responsible for the synthesis of the lipo-oligosaccharide core common to all Nod factors. In contrast to most rhizobial species, where nodABC belong to one operon, in Mesorhizobium sp. strain N33 nodA and nodBC belong to two different operons (2, 4). In addition to nodABC, several nod genes likely to be involved in Nod factor substitutions have been characterized (3, 4). The nodHPQ genes, which are also found in S. meliloti and Rhizobium tropici, have been shown to specify O sulfation of the Nod factor reducing end in these two species (11, 18, 29). The nodFE genes, which are also present in S. meliloti and R. leguminosarum, determine in these two species the synthesis of various polyunsaturated fatty acids with one or several double bounds conjugated to the carbonyl group (7, 34, 39). Therefore, one might expect the Mesorhizobium sp. strain N33 Nod factors to be sulfated and substituted by a fatty acid with conjugated double bonds. However, given the peculiar host range of this species, the Mesorhizobium sp. strain N33 Nod factors are likely to carry novel substitutions. In this paper, we describe the structure of the Mesorhizobium sp. strain N33 Nod factors. We show that they have original structures with substitutions never described before, and we study several features of their biosynthesis.

Molecular physiology and breeding at the crossroads of cold hardiness improvement.

Alfalfa (Medicago sativa L.) is a major forage legume grown extensively worldwide with important agronomic and environmental attributes. Insufficient cold hardiness is a major impediment to its reliable production in northern climates. Improvement of freezing tolerance using conventional breeding approaches is slowed by the quantitative nature of inheritance and strong interactions with the environment. The development of gene-based markers would facilitate the identification of genotypes with superior stress tolerance. Successive cycles of recurrent selection were applied using an indoor screening method to develop populations with significantly higher tolerance to freezing (TF). Bulk segregant analysis of heterogeneous TF populations identified DNA variations that are progressively enriched in frequency in response to selection. Polymorphisms resulting from intragenic variations within a dehydrin gene were identified and could potentially lead to the development of robust selection tools. Our results illustrate the benefits of feedback interactions between germplasm development programs and molecular physiology for a deeper understanding of the molecular and genetic bases of cold hardiness.

Characterization of two novel cold-inducible K3 dehydrin genes from alfalfa (Medicago sativa spp. sativa L.).

Dehydrin defines a complex family of intrinsically disordered proteins with potential adaptive value with regard to freeze-induced cell dehydration. Search within an expressed sequence tags library from cDNAs of cold-acclimated crowns of alfalfa (Medicago sativa spp. sativa L.) identified transcripts putatively encoding K3-type dehydrins. Analysis of full-length coding sequences unveiled two highly homologous sequence variants, K3-A and K3-B. An increase in the frequency of genotypes yielding positive genomic amplification of the K3-dehydrin variants in response to selection for superior tolerance to freezing and the induction of their expression at low temperature strongly support a link with cold adaptation. The presence of multiple allelic forms within single genotypes and independent segregation indicate that the two K3 dehydrin variants are encoded by distinct genes located at unlinked loci. The co-inheritance of the K3-A dehydrin with a Y2K4 dehydrin restriction fragment length polymorphism with a demonstrated impact on freezing tolerance suggests the presence of a genome domain where these functionally related genes are located. These results provide additional evidence that dehydrin play important roles with regard to tolerance to subfreezing temperatures. They also underscore the value of recurrent selection to help identify variants within a large multigene family in allopolyploid species like alfalfa.

Characterization and mutational analysis of nodHPQ genes of Rhizobium sp. strain N33.

We have shown, by sequencing the nodulation gene region of Rhizobium sp. strain N33 previously isolated from the Canadian high arctic, that the nodHPQ genes are located in a 4.8-kb region downstream of nodBCIJ. The open reading frames of nodHPQ are 747, 906, and 1941 nucleotides long, respectively. The strain N33 genome contains one copy of nodH and two copies of nodPQ that are homologous to those genes in Rhizobium meliloti. Tn5 insertions in the nodHPQ genes of strain N33 did not affect the formation of nodules on the two homologous hosts, Astragalus cicer and Onobrychis viciifolia. Since strain N33 contains the nodBCIJHPQ genes and the recently sequenced nodAFEG genes, we looked for similar host range with R. meliloti. Strain N33 and R. meliloti strains A2 and RCR2011 were shown to induce the formation of root nodules on plants of O. viciifolia. However, strain N33, compared with R. meliloti strains, was able to elicit a few, white, empty, root nodules on Medicago sativa. R. meliloti strains, compared with strain N33, were shown to induce only few nodules containing bacteria on A. cicer. Induction of nod genes transcription in strain N33 was shown to be induced by a variety of flavonoid compounds that are different from those inducing nod genes from R. meliloti.

Sequence and Mutational Analysis of the 6.7-kb Region Containing nodAFEG Genes of Rhizobium sp. Strain N33: Evidence of DNA Rearrangements

A 6.7-kb region upstream of nodBC genes in Rhizobium sp. strain N33 was shown to contain the nodAFEG genes and an open reading frame designated orfZ. The open reading frames for these genes contain 591, 282, 1209, 738, and 1,338 nucleotides respectively. Homologues of these genes were found in other rhizobia with the exception of orfZ, for which there was no counterpart found in the Genbank/EMBL database. Tn5 mutagenesis in nodEG and in the intergenic nodG-B region has shown a Nod+ phenotype on their temperate hosts Onobrychis viciifolia and Astragalus cicer. The nodules formed on O. viciifolia plants by these mutants were altered in shape and size. However, on A. cicer there was only a reduction in the number of nodules formed, compared with the wild-type strain. Sequence analysis of the orfZ-nodA and nodG-B intergenic regions indicates the presence of truncated nodD genes.

Characterization of Dehydrin Variants Linked to Freezing Tolerance in Alfalfa at the DNA and Post-transcriptional Levels

Dehydrins are highly hydrophilic proteins that are thought to play key adaptive roles with regard to tolerance to freezing-induced cell desiccation. We recently identified a DNA polymorphism between alfalfa populations recurrently selected for superior freezing tolerance that was associated with size variations of the C-terminal coding region of a dehydrin homolog (msaCIG). These size variants were caused by the presence of small and large indels.We identified a fragment that was preferentially amplified using pooled DNA from genotypes with the dehydrin polymorphism initially uncovered on Southern blots. In the current study, the amplification of the C-terminal region using cDNA templates from cold-acclimated plants confirmed the cold-induced accumulation of transcripts of the expected molecular sizes. Western blot hybridization with antibodies raised against dehydrins revealed variations in polypeptide profiles that closely matched the allelic pattern uncovered with DNA amplifications.

Development of Marker-Assisted Selection for the Improvement of Freezing Tolerance in Alfalfa

Marker-assisted selection (MAS) accelerates conventional breeding approaches in the improvement of multigenic traits. We used a bulk segregant analysis (BSA) approach to identify genetic polymorphisms closely associated to cold adaptation among populations of alfalfa (Medicago sativa L.) recurrently selected for increased tolerance to freezing (TF). Using bulk DNA samples from cultivar Apica (A-TF0) and populations (A-TF2 and A-TF5) derived from that initial background, we observed both the intensification and the disappearance of several DNA fragments in response to selection pressure. Subsequent assessment of freezing tolerance of individual genotypes confirmed a close relationship between some of these polymorphisms and freezing tolerance. Our results illustrate that the combination of BSA and populations recurrently selected for the improvement of polygenic traits are effective tools to develop MAS applications in alfalfa.