Defining the mutation sites in chickpea nodulation mutants PM233 and PM405.

BACKGROUND: Like most legumes, chickpeas form specialized organs called root nodules. These nodules allow for a symbiotic relationship with rhizobium bacteria. The rhizobia provide fixed atmospheric nitrogen to the plant in a usable form. It is of both basic and practical interest to understand the host plant genetics of legume root nodulation. Chickpea lines PM233 and PM405, which harbor the mutationally identified nodulation genes rn1 and rn4, respectively, both display nodulation-deficient phenotypes. Previous investigators identified the rn1 mutation with the chickpea homolog of Medicago truncatula nodulation gene NSP2, but were unable to define the mutant rn1 allele. We used Illumina and Nanopore sequencing reads to attempt to identify and characterize candidate mutation sites responsible for the PM233 and PM405 phenotypes. RESULTS: We aligned Illumina reads to the available desi chickpea reference genome, and did a de novo contig assembly of Nanopore reads. In mutant PM233, the Nanopore contigs allowed us to identify the breakpoints of a ~ 35 kb deleted region containing the CaNSP2 gene, the Medicago truncatula homolog of which is involved in nodulation. In mutant PM405, we performed variant calling in read alignments and identified 10 candidate mutations. Genotyping of a segregating progeny population narrowed that pool down to a single candidate gene which displayed homology to M. truncatula nodulation gene NIN. CONCLUSIONS: We have characterized the nodulation mutation sites in chickpea mutants PM233 and PM405. In mutant PM233, the rn1 mutation was shown to be due to deletion of the entire CaNSP2 nodulation gene, while in mutant PM405 the rn4 mutation was due to a single base deletion resulting in a frameshift mutation between the predicted RWP-RK and PB1 domains of the NIN nodulation gene. Critical to characterization of the rn1 allele was the generation of Nanopore contigs for mutant PM233 and its wild type parent ICC 640, without which the deletional boundaries could not be defined. Our results suggest that efforts of prior investigators were hampered by genomic misassemblies in the CaNSP2 region of both the desi and kabuli reference genomes.

Gene loss and genome rearrangement in the plastids of five Hemiparasites in the family Orobanchaceae.

BACKGROUND: The chloroplast genomes (plastome) of most plants are highly conserved in structure, gene content, and gene order. Parasitic plants, including those that are fully photosynthetic, often contain plastome rearrangements. These most notably include gene deletions that result in a smaller plastome size. The nature of gene loss and genome structural rearrangement has been investigated in several parasitic plants, but their timing and contributions to the adaptation of these parasites requires further investigation, especially among the under-studied hemi-parasites. RESULTS: De novo sequencing, assembly and annotation of the chloroplast genomes of five photosynthetic parasites from the family Orobanchaceae were employed to investigate plastome dynamics. Four had major structural rearrangements, including gene duplications and gene losses, that differentiated the taxa. The facultative parasite Aureolaria virginica had the most similar genome content to its close non-parasitic relative, Lindenbergia philippensis, with similar genome size and organization, and no differences in gene content. In contrast, the facultative parasite Buchnera americana and three obligate parasites in the genus Striga all had enlargements of their plastomes, primarily caused by expansion within the large inverted repeats (IRs) that are a standard plastome feature. Some of these IR increases were shared by multiple investigated species, but others were unique to particular lineages. Gene deletions and pseudogenization were also both shared and lineage-specific, with particularly frequent and independent loss of the ndh genes involved in electron recycling. CONCLUSIONS: Five new plastid genomes were fully assembled and compared. The results indicate that plastome instability is common in parasitic plants, even those that retain the need to perform essential plastid functions like photosynthesis. Gene losses were slow and not identical across taxa, suggesting that different lineages had different uses or needs for some of their plastome gene content, including genes involved in some aspects of photosynthesis. Recent repeat region extensions, some unique to terminal species branches, were observed after the divergence of the Buchnera/Striga clade, suggesting that this otherwise rare event has some special value in this lineage.

Roles of three Fusarium graminearum membrane Ca2+ channels in the formation of Ca2+ signatures, growth, development, pathogenicity and mycotoxin production.

Similar to animals and plants, external stimuli cause dynamic spatial and temporal changes of cytoplasmic Ca2+ in fungi. Such changes are referred as the Ca2+ signature and control cellular responses by modulating the activity or location of diverse Ca2+-binding proteins (CBPs) and also indirectly affecting proteins that interact with CBPs. To understand the mechanism underpinning Ca2+ signaling, therefore, characterization of how Ca2+ moves to and from the cytoplasm to create Ca2+ signatures under different conditions is fundamental. Three genes encoding plasma membrane Ca2+ channels in a Fusarium graminearum strain that expresses a fluorescent protein-based Ca2+ indicator in the cytoplasm were mutagenized to investigate their roles in the generation of Ca2+ signatures under different growth conditions and genetic backgrounds. The genes disrupted include CCH1 and MID1, which encode a high affinity Ca2+ uptake system, and FIG1, encoding a low affinity Ca2+ channel. Resulting mutants were also analyzed for growth, development, pathogenicity and mycotoxin production to determine how loss of each of the genes alters these traits. To investigate whether individual genes influence the function and expression of other genes, phenotypes and Ca2+ signatures of their double and triple mutants, as well as their expression patterns, were analyzed.

Roles of three Fusarium oxysporum calcium ion (Ca(2+)) channels in generating Ca(2+) signatures and controlling growth.

Spatial and temporal changes of cytoplasmic calcium ions ([Ca(2+)]c), caused by external stimuli, are known as the Ca(2+) signature and presumably control cellular and developmental responses. Multiple types of ion channels, pumps, and transporters on plasma and organellar membranes modulate influx and efflux of Ca(2+) to and from the extracellular environment and internal Ca(2+) stores to form Ca(2+) signatures. Expression of a fluorescent protein-based Ca(2+) probe, Cameleon YC3.60, in Fusarium oxysporum enabled us to study how disruption of three Ca(2+) channel genes, including FoCCH1, FoMID1 and FoYVC1, affects Ca(2+) signature formation at polarized hyphal tips and whether specific changes in the Ca(2+) signature caused by these mutations are related to growth-related phenotypes. Resulting mutants displayed altered amplitude, interval, and duration of Ca(2+) pulses under various external Ca(2+) concentrations as well as changes in sporulation and growth. Loss of FoMID1 and FoCCH1, genes encoding putative plasma membrane channel proteins, had a major impact on Ca(2+) signatures and growth, while disruption of FoYVC1, which encodes a vacuolar channel, only subtly affected both traits. Results from our study provide new insights into the underpinning of Ca(2+) signaling in fungi and its role in controlling growth and also raise several new questions.

A pentaploid-based linkage map of the ancestral octoploid strawberry Fragaria virginiana reveals instances of sporadic hyper-recombination.

The first high-resolution genetic linkage map of the ancestral octoploid (2n = 8x = 56) strawberry species, Fragaria virginiana, was constructed using segregation data obtained from a pentaploid progeny population. This novel mapping population of size 178 was generated by crossing highly heterozygous F. virginiana hybrid "LB48" as a paternal parent with diploid (2n = 2x = 14) Fragaria vesca "Hawaii 4". The LB48 linkage map comprises 6055 markers genotyped on the Axiom® IStraw90 strawberry SNP array. The map consists of 28 linkage groups (LGs) organized into seven homoeology groups of four LGs each, and excludes a small 29th LG of undefined homoeology. One member of each homoeology group was assignable to an "A" subgenome associated with ancestral diploid Fragaria vesca, while no other subgenomes were defined. Despite an intriguing discrepancy within homoeology group VI, synteny comparisons with the previously published Fragaria ×ananassa DA × MO linkage map revealed substantial agreement. Following initial map construction, examination of crossover distributions revealed that six of the total 5162 (=29 chromosomes/individual × 178 individuals) chromosomes making up the data set exhibited abnormally high crossover counts, ranging from 15 to 48 crossovers per chromosome, as compared with the overall mean of 0.66 crossovers per chromosome. Each of these six hyper-recombinant (HypR) chromosomes occurred in a different LG and in a different individual. When calculated upon exclusion of the six HypR chromosomes, the canonical (i.e., broadly representative) LB48 map had 1851 loci distributed over a total map length of 1873 cM, while their inclusion increased the number of loci by 130, and the overall map length by 91 cM. Discovery of these hyper-recombinant chromosomes points to the existence of a sporadically acting mechanism that, if identified and manipulable, could be usefully harnessed for multiple purposes by geneticists and breeders.