Background Zymoseptoria tritici is a hemibiotrophic ascomycete fungus causing leaf blotch of wheat that often decreases yield severely. Populations of the fungus are known to be highly diverse and poorly differentiated from each other. However, a genotyping tool is needed to address further questions in large collections of isolates, regarding regional population structure, adaptation to anthropogenic selective pressures, and dynamics of the recently discovered accessory chromosomes. This procedure is limited by costly and time-consuming simplex PCR genotyping. Recent development of genomic approaches and of larger sets of SSRs enabled the optimization of microsatellite multiplexing. Findings We report here a reliable protocol to amplify 24 SSRs organized in three multiplex panels, and covering all Z. tritici chromosomes. We also propose an automatic allele assignment procedure, which allows scoring alleles in a repeatable manner across studies and laboratories. All together, these tools enabled us to characterize local and worldwide populations and to calculate diversity indexes consistent with results reported in the literature. Conclusion This easy-to-use, accurate, repeatable, economical, and faster technical strategy can provide useful genetic information for evolutionary inferences concerning Z. tritici populations. Moreover, it will facilitate the comparison of studies from different scientific groups.

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

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 atram 2 for local assemblies, especially for read sets with less than 20× coverage. slag is freely available at https://github.com/cfcrane/SLAG.

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.

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.

The greenbug aphid (Schizaphis graminum (Rondani)) is a major pest of wheat and an important vector of wheat viruses. An RNA-seq study was conducted to investigate the microbial effects of two greenbug genotypes, the presence or absence of cereal yellow dwarf virus, and the condition of the wheat host over a 20-day time course of unrestricted greenbug feeding. Messenger RNA reads were mapped to ca. 47,000 bacterial, 1218 archaeal, 14,165 viral, 571 fungal, and 94 protozoan reference or representative genomes, plus greenbug itself and its wheat host. Taxon counts were analyzed with QIIME2 and DESeq2. Distinct early (days 1 through 10) and late (days 15 and 20) communities differed in the abundance of typical enteric genera (Shigella, Escherichia, Citrobacter), which declined in the late community, while the ratio of microbial to greenbug read counts declined 50% and diversity measures increased. The nearly universal aphid endosymbiont, Buchnera aphidicola, accounted for less than 25% of the read counts in both communities. There were 302 differentially expressed (populated) genera with respect to early and late dates, while 25 genera differed between the greenbug genotypes and nine differed between carrier and virus-free greenbugs. The late community was likely responding to starvation as the wheat host succumbed to aphid feeding. Our results add to basic knowledge about aphid microbiomes and offer an attractive alternative method to assess insect microbiomes.