Use of synteny to identify candidate genes underlying QTL controlling stomatal traits in faba bean (Vicia faba L.)

Key message

We have identified QTLs for stomatal characteristics on chromosome II of faba bean by applying SNPs derived from M. truncatula , and have identified candidate genes within these QTLs using synteny between the two species.

Abstract

Faba bean (Vicia faba L.) is a valuable food and feed crop worldwide, but drought often limits its production, and its genome is large and poorly mapped. No information is available on the effects of genomic regions and genes on drought adaptation characters such as stomatal characteristics in this species, but the synteny between the sequenced model legume, Medicago truncatula, and faba bean can be used to identify candidate genes. A mapping population of 211 F₅ recombinant inbred lines (Mélodie/2 × ILB 938/2) were phenotyped to identify quantitative trait loci (QTL) affecting stomatal morphology and function, along with seed weight, under well-watered conditions in a climate-controlled glasshouse in 2013 and 2014. Canopy temperature (CT) was evaluated in 2013 under water-deficit (CTd). In total, 188 polymorphic single nucleotide polymorphisms (SNPs), developed from M. truncatula genome data, were assigned to nine linkage groups that covered ~928 cM of the faba bean genome with an average inter-marker distance of 5.8 cM. 15 putative QTLs were detected, of which eight (affecting stomatal density, length and conductance and CT) co-located on chromosome II, in the vicinity of a possible candidate gene—a receptor-like protein kinase found in the syntenic interval of M. truncatula chromosome IV. A ribose-phosphate pyrophosphokinase from M. truncatula chromosome V, postulated as a possible candidate gene for the QTL for CTd, was found some distance away in the same chromosome. These results demonstrate that genomic information from M. truncatula can successfully be translated to the faba bean genome.     

New evidence of ancestral polyploidy in the Genistoid legume Lupinus angustifolius L. (narrow-leafed lupin)

Key message

This is the first clear evidence of duplication and/or triplication of large chromosomal regions in a genome of a Genistoid legume, the most basal clade of Papilionoid legumes.

Abstract

Lupinus angustifolius L. (narrow-leafed lupin) is the most widely cultivated species of Genistoid legume, grown for its high-protein grain. As a member of this most basal clade of Papilionoid legumes, L. angustifolius serves as a useful model for exploring legume genome evolution. Here, we report an improved reference genetic map of L. angustifolius comprising 1207 loci, including 299 newly developed Diversity Arrays Technology markers and 54 new gene-based PCR markers. A comparison between the L. angustifolius and Medicago truncatula genomes was performed using 394 sequence-tagged site markers acting as bridging points between the two genomes. The improved L. angustifolius genetic map, the updated M. truncatula genome assembly and the increased number of bridging points between the genomes together substantially enhanced the resolution of synteny and chromosomal colinearity between these genomes compared to previous reports. While a high degree of syntenic fragmentation was observed that was consistent with the large evolutionary distance between the L. angustifolius and M. truncatula genomes, there were striking examples of conserved colinearity of loci between these genomes. Compelling evidence was found of large-scale duplication and/or triplication in the L. angustifolius genome, consistent with one or more ancestral polyploidy events.     

Six <Emphasis Type="Italic">Medicago truncatula</Emphasis> Dicer-like protein genes are expressed in plant cells and upregulated in nodules

Key message

Here we report the existence of six putative Dicer-like genes in the Medicago truncatula genome. They are ubiquitously expressed throughout the plant and significantly induced in root nodules.

Abstract

Over the past decade, small noncoding RNAs (sncRNA) have emerged as widespread and important regulatory molecules influencing both the structure and expression of plant genomes. One of the key factors involved in sncRNA biogenesis in plants is a group of RNase III-type nucleases known as Dicer-like (DCL) proteins. Based on functional analysis of DCL proteins identified in Arabidopsis thaliana, four types of DCLs were distinguished (DCL1-4). DCL1 mainly produces 21 nt miRNAs. The products generated by DCL2, DCL3, and DCL4 belong to various classes of siRNAs that are 22, 24 and 21 nt in length, respectively. M. truncatula is a model legume plant closely related to many economically important cultivable species. By screening the recent M. truncatula genome assembly, we were able to identify three new DCL genes in addition to the MtDCL1-3 genes that had been earlier characterized. The newly found genes include MtDCL4 and two MtDCL2 homologs. We showed that all six M. truncatula DCL genes are expressed in plant cells. The first of the identified MtDCL2 paralogs encodes a truncated version of the DCL2 protein, while the second undergoes substantial and specific upregulation in the root nodules. Additionally, we identified an alternative splicing variant of MtDCL1 mRNA, similar to the one found in Arabidopsis. Our results indicate that DCL genes are differently activated during Medicago symbiosis with nitrogen fixing bacteria and upon pathogen infection. In addition, we hypothesize that the alternative splicing variant of MtDCL1 mRNA may be involved in tissue-specific regulation of the DCL1 level.

     

Acetic acid acts as an elicitor exerting a chitosan-like effect on xanthone biosynthesis in <Emphasis Type="Italic">Hypericum perforatum</Emphasis> L. root cultures

Key message

Acetic acid acts as a signal molecule, strongly enhancing xanthone biosynthesis in Hypericum perforatum root cultures. This activity is specific, as demonstrated by the comparison with other short-chain monocarboxylic acids.

Abstract

We have recently demonstrated that Hypericum perforatum root cultures constitutively produce xanthones at higher levels than the root of the plant and that they respond to chitosan (CHIT) elicitation with a noteworthy increase in xanthone production. In the present study, CHIT was administered to H. perforatum root cultures using three different elicitation protocols, and the increase in xanthone production was evaluated. The best results (550 % xanthone increase) were obtained by subjecting the roots to a single elicitation with 200 mg l^?1 CHIT and maintaining the elicitor in the culture medium for 7 days. To discriminate the effect of CHIT from that of the solvent, control experiments were performed by administering AcOH alone at the same concentration used for CHIT solubilization. Unexpectedly, AcOH caused an increase in xanthone production comparable to that observed in response to CHIT. Feeding experiments with ¹³C-labeled AcOH demonstrated that this compound was not incorporated into the xanthone skeleton. Other short-chain monocarboxylic acids (i.e., propionic and butyric acid) have little or no effect on the production of xanthones. These results indicate that AcOH acts as a specific signal molecule, able to greatly enhance xanthone biosynthesis in H. perforatum root cultures.

     

Mapping and identification of a Cicer arietinum NSP2 gene involved in nodulation pathway

Key message

For the first time the putative NSP2 gene in chickpea has been identified using pairs of NILs differing for the Rn1 / rn1 nodulation gene that was located in LG5 of chickpea genetic map.

Abstract

An intraspecific cross between the mutant non-nodulating genotype PM233, carrying the recessive gene rn1, and the wild-type CA2139 was used to develop two pairs of near-isogenic lines (NILs) for nodulation in chickpea. These pairs of NILs were characterized using sequence tagged microsatellite site (STMS) markers distributed across different linkage groups (LGs) of the chickpea genetic map leading to the detection of polymorphic markers located in LG5. Using this information, together with the genome annotation in Medicago truncatula, a candidate gene (NSP2) known to be involved in nodulation pathway was selected for mapping in chickpea. The full length sequence obtained in chickpea wild-type (CaNSP2) was 1,503 bp. Linkage analysis in an F₃ population of 118 plants derived from the cross between the pair of NILS NIL7-2A (nod) × NIL7-2B (non-nod) revealed a co-localization between CaNSP2 and Rn1 gene. These data implicate the CaNSP2 gene as a candidate for identity to Rn1, and suggest that it could act in the nodulation signaling transduction pathway similarly to that in other legumes species.     

Different non-host resistance responses of two rice subspecies,japonica and indica,to Puccinia striiformis f. sp. tritici

Key message

Japonica and indica have different non-host resistance (NHR) abilities to Puccinia striiformis f. sp. tritici ( Pst ), and hydrogen peroxide (H ₂ O ₂ ) has a positive function in NHR to japonica against Pst.

Abstract

Non-host interactions between Puccinia striiformis f. sp. tritici (Pst) and two rice subspecies were characterized using 23 rice varieties, including 11 japonica and 12 indica. Results showed that the infected fungal structures were easily produced in the leaves of indica, whereas only several substomatal vesicles and primary infection hyphae were observed in the leaves of japonica. This result indicated that indica is less resistant or more susceptible to Pst than japonica. Hydrogen peroxide accumulated in the initial phase of japonica–Pst interaction but not in indica–Pst interaction. A set of reactive oxygen species (ROS)-related genes was also induced in response to Pst infection, suggesting that ROS activation is one of the major mechanisms of non-host resistance of rice to Pst.     

Enhanced nodulation and symbiotic effectiveness of Medicago truncatula when co-inoculated with Pseudomonas fluorescens WSM3457 and Ensifer (Sinorhizobium) medicae WSM419

Aims

Low numbers of rhizobia in soil or inoculants delay nodulation and decrease symbiotic legume productivity. This study investigated the effect of co-inoculation with a helper bacterium, Pseudomonas fluorescens WSM3457 on the Medicago truncatula - Ensifer (Sinorhizobium) medicae WSM419 symbiosis challenged by a low inoculum dose.

Methods

In a glasshouse experiment the effect of co-inoculation with WSM3457 on the kinetics of nodule initiation and development was assessed 5, 7, 10, 14, 17, 21, and 42 days after inoculation of M. truncatula cv. Caliph with 10³ cells/plant of E. medicae WSM419.

Results

Co-inoculated plants had enhanced rate of nodule initiation and development, greater numbers of larger crown nodules, and by day 42 accumulated more N than plants inoculated with E. medicae WSM419 alone. Nodule development was altered by co-inoculation. Approximately 25% of nodule initials on co-inoculated plants formed in closely associated pairs, young nodules were larger with multiple meristems and developed into cluster-like multi-lobed nodules compared to those on WSM419 inoculated plants. Molecular typing showed WSM3457 occupied a significant proportion of root nodules on co-inoculated plants.

Conclusion

Co-inoculation with P. fluorescens WSM3457 enhanced symbiotic effectiveness of M. truncatula when inoculated with a low inoculum dose of E. medicae WSM419.     

Estimating the absorptive root area in Norway spruce by using the common direct and indirect earth impedance methods

Aims

Estimates of root absorption magnitude are needed for the balanced management of forest ecosystems, but no methods able to work on the whole tree and stand level were available. Modified earth impedance method was developed recently and here it was tested, by comparing the results with those obtained by combination of several classical methods.

Methods

We used direct (soil cores, scanning and microscopy) and indirect (sap flow patterns and modified earth impedance) methods in an attempt to estimate the absorptive root area indexes (RAI) at two sites of about 25 and 40-years-old Norway spruce. We considered the geometric surfaces of all scanned fine roots to be equal to the fine root absorptive area (RAI _scan ). To estimate the potentially physically permeable area of fine roots, we microscopically evaluated the point of secondary xylem appearance and calculated the geometric area of root portions with primary structure (RAI _micro ). We termed the area of electrically conductive root surface as the active (ion) absorptive area (RAI _mei ) and measured its extent by the modified earth impedance (MEI) method.

Results

The highest values for absorptive root areas at the two experimental sites we obtained with the scanning method (RAI _scan was considered to be 100%), followed by the RAI _micro (51%) and RAI _mei (32%). RAImei reached about 2/3 of RAImicro. The surface area of the ectomycorrhizal hyphae was an order of magnitude larger than that of all fine roots, but the MEI did not measure such increase.

Conclusions

We showed that the absorptive root area, indirectly estimated by the MEI, provides consistent results that approach the values obtained for fine roots with a primary structure estimated by traditional direct methods. The similar range of the values for the absorptive root surface area obtained by microscopy and by the MEI method indicates that this method is feasible and that it could be used to determine the extent of active absorptive root surface areas in forests.