Differential gene expression between viruliferous and non-viruliferous Schizaphis graminum (Rondani).

An experiment was performed to measure the effect of Cereal Yellow-Dwarf Virus (CYDV), strain CYDV-RPV, on gene expression in its insect vector, greenbug aphid (Schizaphis graminum (Rondani)). RNA was sampled in three replicates from four treatments (biotypes B and H with or without carried CYDV), at 0, 1, 2, 3, 5, 10, 15 and 20 days from the introduction of carrier and virus-free greenbugs to uninfected wheat cv. 'Newton'. Illumina paired-end sequencing produced 1,840,820,000,000 raw reads that yielded 1,089,950,000 clean reads, which were aligned to two greenbug, Trinity transcriptome assemblies with bowtie2. Read counts to contigs were analyzed with principal components and with DESeq2 after removing contaminating contigs of wheat or microbial origin. Likelihood ratio tests with one transcriptome showed that CYDV influenced gene expression about seven-fold less than time or biotype, which were approximately equal. With the other transcriptome, virus, time, and biotype were about equally important. Pairwise comparisons of virus to no virus for each timepoint yielded estimates of fold-change that comprised expression profiles for each contig when ordered by timepoint. Hierarchical clustering separated expression profiles into 20 groups of contigs that were significantly differentially expressed for at least one timepoint. Contigs were also sorted by timepoint of maximally differential expression between virus and no virus. All contigs that were significantly differentially expressed at FDR = 0.05 were annotated by blast searches against NCBI nr and nt databases. Interesting examples of up-regulation with virus included a lysosomal-trafficking regulator, peptidylprolylisomerase, RNA helicase, and two secreted effector proteins. However, carried virus did not consistently change aphid gene expression overall. Instead there was complex interaction of time, biotype, host response, and virus.

Molecular characterization of eliminated chromosomes in Hessian fly (Mayetiola destructor (Say)).

Like other cecidomyiid Diptera, Hessian fly has stable S chromosomes and dispensable E chromosomes that are retained only in the germ line. Amplified fragment length polymorphisms (AFLP), suppressive subtractive hybridization (SSH), fluorescent in-situ hybridization (FISH), and sequencing were used to investigate similarities and differences between S and E chromosomes. More than 99.9% of AFLP bands were identical between separated ovary and somatic tissue, but one band was unique to ovary and resembled Worf, a non-LTR retrotransposon. Arrayed clones, derived by SSH of somatic from ovarian DNA, showed no clones that were unique to ovary. FISH with BAC clones revealed a diagnostic banding pattern of BAC positions on both autosomes and both sex chromosomes, and each E chromosome shared a pattern with one of the S chromosomes. Sequencing analysis showed that E chromosomes are nearly identical to S chromosomes, since no sequence could be confirmed to belong only to E chromosomes. There were a few questionably E-specific sequences that are candidates for further investigation. Thus, the E chromosomes appear to be derived from S chromosomes by the acquisition or conversion of sequences that produce the negatively heteropycnotic region around the centromere.

slag: A program for seeded local assembly of genes in complex genomes.

Although finished genomes have become more common, there is still a need for assemblies of individual genes or chromosomal regions when only unassembled reads are available. slag (Seeded Local Assembly of Genes) fulfils this need by performing iterative local assembly based on cycles of matching-read retrieval with blast and assembly with cap3, phrap, spades, canu or unicycler. The target sequence can be nucleotide or protein. Read fragmentation allows slag to use phrap or cap3 to assemble long reads at lower coverage (e.g., 5×) than is possible with canu or unicycler. In simple, nonrepetitive genomes, a slag assembly can cover a whole chromosome, but in complex genomes the growth of target-matching contigs is limited as additional reads are consumed by consensus contigs consisting of repetitive elements. Apart from genomic complexity, contig length and correctness depend on read length and accuracy. With pyrosequencing or Illumina reads, slag-assembled contigs are accurate enough to allow design of PCR primers, while contigs assembled from Oxford Nanopore or pre-HiFi Pacific Biosciences long reads are generally only accurate enough to design baiting sequences for further targeted sequencing. In an application with real reads, slag successfully extended sequences for four wheat genes, which were verified by cloning and Sanger sequencing of overlapping amplicons. slag is a robust alternative to atram2 for local assemblies, especially for read sets with less than 20× coverage. slag is freely available at https://github.com/cfcrane/SLAG.

Combating a Global Threat to a Clonal Crop: Banana Black Sigatoka Pathogen Pseudocercospora fijiensis (Synonym Mycosphaerella fijiensis) Genomes Reveal Clues for Disease Control.

Black Sigatoka or black leaf streak disease, caused by the Dothideomycete fungus Pseudocercospora fijiensis (previously: Mycosphaerella fijiensis), is the most significant foliar disease of banana worldwide. Due to the lack of effective host resistance, management of this disease requires frequent fungicide applications, which greatly increase the economic and environmental costs to produce banana. Weekly applications in most banana plantations lead to rapid evolution of fungicide-resistant strains within populations causing disease-control failures throughout the world. Given its extremely high economic importance, two strains of P. fijiensis were sequenced and assembled with the aid of a new genetic linkage map. The 74-Mb genome of P. fijiensis is massively expanded by LTR retrotransposons, making it the largest genome within the Dothideomycetes. Melting-curve assays suggest that the genomes of two closely related members of the Sigatoka disease complex, P. eumusae and P. musae, also are expanded. Electrophoretic karyotyping and analyses of molecular markers in P. fijiensis field populations showed chromosome-length polymorphisms and high genetic diversity. Genetic differentiation was also detected using neutral markers, suggesting strong selection with limited gene flow at the studied geographic scale. Frequencies of fungicide resistance in fungicide-treated plantations were much higher than those in untreated wild-type P. fijiensis populations. A homologue of the Cladosporium fulvum Avr4 effector, PfAvr4, was identified in the P. fijiensis genome. Infiltration of the purified PfAVR4 protein into leaves of the resistant banana variety Calcutta 4 resulted in a hypersensitive-like response. This result suggests that Calcutta 4 could carry an unknown resistance gene recognizing PfAVR4. Besides adding to our understanding of the overall Dothideomycete genome structures, the P. fijiensis genome will aid in developing fungicide treatment schedules to combat this pathogen and in improving the efficiency of banana breeding programs.

Use of microsatellite and SNP markers for biotype characterization in Hessian fly.

Exploration of the biotype structure of Hessian fly, Mayetiola destructor (Say) (Diptera: Cecidomyiidae), would improve our knowledge regarding variation in virulence phenotypes and difference in genetic background. Microsatellites (simple sequence repeats) and single-nucleotide polymorphisms (SNPs) are highly variable genetic markers that are widely used in population genetic studies. This study developed and tested a panel of 18 microsatellite and 22 SNP markers to investigate the genetic structure of nine Hessian fly biotypes: B, C, D, E, GP, L, O, vH9, and vH13. The simple sequence repeats were more polymorphic than the SNP markers, and their neighbor-joining trees differed in consequence. Microsatellites suggested a simple geographic association of related biotypes that did not progressively gain virulence with increasing genetic distance from a founder type. Use of the k-means clustering algorithm in the STRUCTURE program shows that the nine biotypes comprise six to eight populations that are related to geography or history within laboratory cultures.

The evolution of Hessian fly from the Old World to the New World: Evidence from molecular markers.

Eighteen polymorphic microsatellite loci and 11 single-nucleotide polymorphisms were genotyped in 1,095 individual Hessian fly specimens representing 23 populations from North America, southern Europe, and southwest Asia. The genotypes were used to assess genetic diversity and interrelationship of Hessian fly populations. While phylogenetic analysis indicates that the American populations most similar to Eurasian populations come from the east coast of the United States, genetic distance is least between (Alabama and California) and (Kazakhstan and Spain). Allelic diversity and frequency vary across North America, but they are not correlated with distance from the historically documented point of introduction in New York City or with temperature or precipitation. Instead, the greatest allelic diversity mostly occurs in areas with Mediterranean climates. The microsatellite data indicate a general deficiency for heterozygotes in Hessian fly. The North American population structure is consistent with multiple introductions, isolation by distance, and human-abetted dispersal by bulk transport of puparia in infested straw or on harvesting equipment.

Finished genome of the fungal wheat pathogen Mycosphaerella graminicola reveals dispensome structure, chromosome plasticity, and stealth pathogenesis.

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed "mesosynteny" is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.

Patterned sequence in the transcriptome of vascular plants.

BACKGROUND: Microsatellites (repeated subsequences based on motifs of one to six nucleotides) are widely used as codominant genetic markers because of their frequent polymorphism and relative selective neutrality. Minisatellites are repeats of motifs having seven or more nucleotides. The large number of EST sequences now available in public databases offers an opportunity to compare microsatellite and minisatellite properties and evaluate their evolution over a broad range of plant taxa. RESULTS: Repeated motifs from one to 250 nucleotides long were identified in 6793306 expressed sequence tags (ESTs) from 88 genera of vascular plants, using a custom data-processing pipeline that allowed limited variation among repeats. The pipeline processed trimmed but otherwise unfiltered sequence and output nonredundant loci of at least 15 nucleotides, with degree of polymorphism and PCR primers wherever possible. Motifs that were an integral multiple of three in length were more abundant and richer in G/C than other motifs. From 80 to 85% of minisatellite motifs represented repeats within proteins, up to the 228-nucleotide repeat of ubiquitin, but not all of these repeats preserved reading frame. The remaining 15 to 20% of minisatellite motifs were associated with transcribed repetitive elements, e.g., retrotransposons. Relative microsatellite motif frequencies did not correlate tightly to phylogenetic relationship. Evolution of increased microsatellite and EST GC content was evident within the grasses. Microsatellites were less frequent in the transcriptome of genera with large genomes, but there was no evidence for greater dilution of the transcriptome with transposable element transcripts in these genera. CONCLUSION: The relatively low correlation of microsatellite spectrum to phylogeny suggests that repeat loci evolve more rapidly than the surrounding sequence, although tissue specificity of the different EST libraries is a complicating factor. In-frame motifs are more abundant and higher in GC than frame-shifting motifs, but most EST minisatellite loci appear to represent repeats in translated sequence, regardless of whether reading frame is preserved. Motifs of four to six nucleotides are as polymorphic in EST collections as the commonly used motifs of two and three nucleotides, and they can be exploited as genetic markers with little additional effort.

Identification and genetic mapping of highly polymorphic microsatellite loci from an EST database of the septoria tritici blotch pathogen Mycosphaerella graminicola.

A database of 30,137 EST sequences from Mycosphaerella graminicola, the septoria tritici blotch fungus of wheat, was scanned with a custom software pipeline for di- and trinucleotide units repeated tandemly six or more times. The bioinformatics analysis identified 109 putative SSR loci, and for 99 of them, flanking primers were developed successfully and tested for amplification and polymorphism by PCR on five field isolates of diverse origin, including the parents of the standard M. graminicola mapping population. Seventy-seven of the 99 primer pairs generated an easily scored banding pattern and 51 were polymorphic, with up to four alleles per locus, among the isolates tested. Among these 51 loci, 23 were polymorphic between the parents of the mapping population. Twenty-one of these as well as two previously published microsatellite loci were positioned on the existing genetic linkage map of M. graminicola on 13 of the 24 linkage groups. Most (66%) of the primer pairs also amplified bands in the closely related barley pathogen Septoria passerinii, but only six were polymorphic among four isolates tested. A subset of the primer pairs also revealed polymorphisms when tested with DNA from the related banana black leaf streak (Black Sigatoka) pathogen, M. fijiensis. The EST database provided an excellent source of new, highly polymorphic microsatellite markers that can be multiplexed for high-throughput genetic analyses of M. graminicola and related species.

A nearest-neighboring-end algorithm for genetic mapping.

MOTIVATION: High-throughput methods are beginning to make possible the genotyping of thousands of loci in thousands of individuals, which could be useful for tightly associating phenotypes to candidate loci. Current mapping algorithms cannot handle so many data without building hierarchies of framework maps. RESULTS: A version of Kruskal's minimum spanning tree algorithm can solve any genetic mapping problem that can be stated as marker deletion from a set of linkage groups. These include backcross, recombinant inbred, haploid and double-cross recombinational populations, in addition to conventional deletion and radiation hybrid populations. The algorithm progressively joins linkage groups at increasing recombination fractions between terminal markers, and attempts to recognize and correct erroneous joins at peaks in recombination fraction. The algorithm is O (mn3) for m individuals and n markers, but the mean run time scales close to mn2. It is amenable to parallel processing and has recovered true map order in simulations of large backcross, recombinant inbred and deletion populations with up to 37,005 markers. Simulations were used to investigate map accuracy in response to population size, allelic dominance, segregation distortion, missing data and random typing errors. It produced accurate maps when marker distribution was sufficiently uniform, although segregation distortion could induce translocated marker orders. The algorithm was also used to map 1003 loci in the F7 ITMI population of bread wheat, Triticum aestivum L. emend Thell., where it shortened an existing standard map by 16%, but it failed to associate blocks of markers properly across gaps within linkage groups. This was because it depends upon the rankings of recombination fractions at individual markers, and is susceptible to sampling error, typing error and joint selection involving the terminal markers of nearly finished linkage groups. Therefore, the current form of the algorithm is useful mainly to improve local marker ordering in linkage groups obtained in other ways. AVAILABILITY: The source code and supplemental data are http://www.iubio.bio.indiana.edu/soft/molbio/qtl/flipper/ CONTACT: ccrane@purdue.edu.