Comparing species detection success between molecular markers in DNA metabarcoding of coastal macroinvertebrates

DNA metabarcoding has great potential to improve marine biomonitoring programs by providing a rapid and accurate assessment of species composition in zoobenthic communities. However, some methodological improvements are still required, especially regarding failed detections, primers efficiency and incompleteness of databases. Here we assessed the efficiency of two different marker loci (COI and 18S) and three primer pairs in marine species detection through DNA metabarcoding of the macrozoobenthic communities colonizing three types of artificial substrates (slate, PVC and granite), sampled between 3 and 15 months of deployment. To accurately compare detection success between markers, we also compared the representativeness of the detected species in public databases and revised the reliability of the taxonomic assignments. Globally, we recorded extensive complementarity in the species detected by each marker, with 69% of the species exclusively detected by either 18S or COI. Individually, each of the three primer pairs recovered, at most, 52% of all species detected on the samples, showing also different abilities to amplify specific taxonomic groups. Most of the detected species have reliable reference sequences in their respective databases (82% for COI and 72% for 18S), meaning that when a species was detected by one marker and not by the other, it was most likely due to faulty amplification, and not by lack of matching sequences in the database. Overall, results showed the impact of marker and primer applied on species detection ability and indicated that, currently, if only a single marker or primer pair is employed in marine zoobenthos metabarcoding, a fair portion of the diversity may be overlooked.

FUSARIUM-ID v.3.0: An updated, downloadable resource for Fusarium species identification

Species within Fusarium are of global agricultural, medical, and food/feed safety concern and have been extensively characterized. However, accurate identification of species is challenging and usually requires DNA sequence data. FUSARIUM-ID (http://isolate.fusariumdb.org/) is a publicly available database designed to support the identification of Fusarium species using sequences of multiple phylogenetically informative loci, especially the highly informative ~680 bp 5' portion of the translation elongation factor 1-alpha (TEF1) gene that has been adopted as the primary barcoding locus in the genus. However, FUSARIUM-ID v.1.0 and 2.0 had several limitations, including inconsistent metadata annotation for the archived sequences and poor representation of some species complexes and marker loci. Here, we present FUSARIUM-ID v.3.0, which provides the following improvements: (i) additional and updated annotation of metadata for isolates associated with each sequence, (ii) expanded taxon representation in the TEF1 sequence database, (iii) availability of the sequence database as a downloadable file to enable local BLAST queries, and (iv) a tutorial file for users to perform local BLAST searches using either freely-available software, such as SequenceServer, BLAST+ executable in the command line, and Galaxy, or the proprietary Geneious software. FUSARIUM-ID will be updated on a regular basis by archiving sequences of TEF1 and other loci from newly identified species and greater in-depth sampling of currently recognized species.

Identification of individual root-knot nematodes using low coverage long-read sequencing

Root-knot nematodes (RKN; genus Meloidogyne) are polyphagous plant pathogens of great economic importance to agriculturalists globally. These species are small, diverse, and can be challenging for accurate taxonomic identification. Many of the most important crop pests confound analysis with simple genetic marker loci as they are polyploids of likely hybrid origin. Here we take a low-coverage, long-read genome sequencing approach to characterisation of individual root-knot nematodes. We demonstrate library preparation for Oxford Nanopore Technologies Flongle sequencing of low input DNA from individual juveniles and immature females, multiplexing up to twelve samples per flow cell. Taxonomic identification with Kraken 2 (a k-mer-based taxonomic assignment tool) is shown to reliably identify individual nematodes to species level, even within the very closely related Meloidogyne incognita group. Our approach forms a robust, low-cost, and scalable method for accurate RKN species diagnostics.

Identification of QTL Combinations that Cause Spikelet Sterility in Rice Derived from Interspecific Crosses

Background The exploitation of useful genes through interspecific and intersubspecific crosses has been an important strategy for the genetic improvement of rice. Postzygotic reproductive isolation routinely occurs to hinder the growth of pollen or embryo sacs during the reproductive development of the wide crosses. Result In this study, we investigated the genetic relationship between the hybrid breakdown of the population and transferred resistance genes derived from wide crosses using a near-isogenic population composed of 225 lines. Five loci (qSS12, qSS8, qSS11, ePS6-1, and ePS6-2) associated with spikelet fertility (SF) were identified by QTL and epistatic analysis, and two out of five epistasis interactions were found between the three QTLs (qSS12, qSS8 and qSS11) and background marker loci (ePS6-1 and ePS6-2) on chromosome 6. The results of the QTL combinations suggested a genetic model that explains most of the interactions between spikelet fertility and the detected loci with positive or negative effects. Moreover, the major-effect QTLs, qSS12 and qSS8, which exhibited additive gene effects, were narrowed down to 82- and 200-kb regions on chromosomes 12 and 8, respectively. Of the 13 ORFs present in the target regions, Os12g0589400 and Os12g0589898 for qSS12 and OS8g0298700 for qSS8 induced significantly different expression levels of the candidate genes in rice at the young panicle stage. Conclusion The results will be useful for obtaining a further understanding of the mechanism causing the hybrid breakdown of a wide cross and will provide new information for developing rice cultivars with wide compatibility.

PpMYB36 Encodes a MYB-Type Transcription Factor That Is Involved in Russet Skin Coloration in Pear (Pyrus pyrifolia)

Fruit color is one of the most important external qualities of pear (Pyrus pyrifolia) fruits. However, the mechanisms that control russet skin coloration in pear have not been well characterized. Here, we explored the molecular mechanisms that determine the russet skin trait in pear using the F1 population derived from a cross between russet skin (‘Niitaka’) and non-russet skin (‘Dangshansu’) cultivars. Pigment measurements indicated that the lignin content in the skin of the russet pear fruits was greater than that in the non-russet pear skin. Genetic analysis revealed that the phenotype of the russet skin pear is associated with an allele of the PpRus gene. Using bulked segregant analysis combined with the genome sequencing (BSA-seq), we identified two simple sequence repeat (SSR) marker loci linked with the russet-colored skin trait in pear. Linkage analysis showed that the PpRus locus maps to the scaffold NW_008988489.1: 53297-211921 on chromosome 8 in the pear genome. In the mapped region, the expression level of LOC103929640 was significantly increased in the russet skin pear and showed a correlation with the increase of lignin content during the ripening period. Genotyping results demonstrated that LOC103929640 encoding the transcription factor MYB36 is the causal gene for the russet skin trait in pear. Particularly, a W-box insertion at the PpMYB36 promoter of russet skin pears is essential for PpMYB36-mediated regulation of lignin accumulation and russet coloration in pear. Overall, these results show that PpMYB36 is involved in the regulation of russet skin trait in pear.

Diagnostic value of Rf1 gene molecular markers in sunflower

Background. Modern production of sunflower seeds is currently based on the cultivation of high-yielding heterotic F1 hybrids from cross-breeding of lines with cytoplasmic male sterility (CMS) of PET1-type and fertility restorer lines. The paternal parent serves as a donor of the nuclear Rf1 gene functional allele, which is responsible for pollen fertility restoration in F1 plants. The detection of carriers of the Rf1 locus recessive and dominant alleles using diagnostic molecular markers accelerates breeding of female and male parental lines for creating hybrids. Materials and methods. The material for the study included 75 lines of various origins from the VIR sunflower genetic collection as well as hybrids from crosses of VIR 116A sterile line with fertile lines differing in the type of cytoplasm (fertile or sterile) and the presence of molecular markers, most of which were linked to the Rf1 locus. For marker validation, two different approaches were used: either by analyzing associations between the ability of a line to restore pollen fertility and the presence of molecular markers in its genotype, or by estimating recombination frequency between the Rf1 locus and marker loci in four segregating hybrid populations. Results. According to the obtained results, no markers demonstrated 100% efficiency in the analysis of the sample of genotypes. The ORS511 marker was most frequently observed among the lines presumably carrying the dominant allele Rf1. Pollen fertility of F1 hybrids from interline crossings was 89-99%. The segregation for fertility/sterility in F2 fitted the theoretical ratio of 3:1 expected in case of the monogenic control of the trait. The markers HRG01, HRG02 and ORS511 were linked to the fertility restoration trait, with recombination rates between Rf1 locus and markers varying in different cross combinations. The analysis of VIR 116А × VIR 740 and VIR 116А × RIL 130 hybrids showed that among the marker loci studied, the ORS511 was closest to the Rf1 locus Rf1 (recombination frequency of 2.2 and 3.3%, respectively). The recombination rate between the Rf1 and ORS511 loci equaled 7.5% in the cross VIR 116А × VIR 210 and 8.9% in VIR 116 × VIR 195. Conclusion. The markers ORS511, HRG01 and HRG02 are the most efficient for the identification of alleles of the Rf1 gene and for the marker assisted selection in hybrid populations produced involving sunflower lines from the VIR collection.

Association of genetic markers with sex determination in Thai red tilapia

The objectives of this study were to verify the polymorphism on sex-linked marker loci and to assess their associations with phenotypic sex characteristics in red tilapia. Four sex-linked genetic markers of Amh, SCAR4, SCAR5, and Oni3161 were genotyped in the Thai red tilapia population. The Amh marker was significantly associated with the phenotypic sex of red tilapia with an accuracy of 46.2%. No significant association of SCAR4, SCAR5, and Oni3161 marker polymorphisms with phenotypic sex characteristics was observed in this study. However, the combinations of these two, three, and four markers were increasingly associated with phenotypic sex characteristics for red tilapia with an accuracy of 62.8, 68.4, and 72.4%, respectively. These results indicate that these combined genetic markers were associated with the phenotypic sex of red tilapias. These findings confirmed the importance of these genetic markers as candidate markers for sex determination in the Thai red tilapia population.

​Association of ISSR and ISSR-RGA Markers with Powdery Mildew Resistance Gene in Mungbean

Background: Powdery mildew (PM) possesses a wide range of host plants, including mungbean that causes severe yield reductions. Using resistant varieties is an economically and environmentally effective approach in controlling the disease. Methods: In this study, the genetic basis of inheritance and marker loci for PM resistance were investigated using 126 F2:9 recombinant inbred lines (RILs) raised from resistant and susceptible parents, V4785 and CN72, respectively. PM reaction in these RILs was visually scored from natural infection in the field and the segregation pattern was determined by the chi-square test (χ2). Result: The results revealed the segregation ration of 1:1, indicative of a dominantly inherited resistance gene controlling resistance against PM in V4785. Observation of potential polymorphism in two parents and two different bulks, each containing 10 RIL individuals showing either the highest resistance or susceptibility, identified 2 and 37 putatively linked inter-simple sequence repeat (ISSR) and ISSR-anchored resistance gene analog (ISSR-RGA) markers, respectively. Of these, 1 ISSR and 3 ISSR-RGA markers were stably inherited and linked to PM resistance (P less than 0.01). These linked marker systems may prove useful for facilitating the improvement of a durable resistant variety through marker-assisted selection in mungbean breeding programs.

Characterizing Microbiomes via Sequencing of Marker Loci: Techniques To Improve Throughput, Account for Cross-Contamination, and Reduce Cost

New methods to characterize microbiomes reduce technology-imposed limitations to study design, but many new approaches have not been widely adopted. Here, we present techniques to increase throughput and reduce contamination alongside a thorough review of current best practices.

Diversity and relationships of Miscanthus sinensis from 20 geographical distributions in China based on SSR markers

Miscanthus is a C4 herbaceous perennial species and it was chosen as a bioenergy crop due to high biomass yield. Miscanthus sinensis has many phenotypes which are adaptated to various environments in China. In this study, 421 accessions of M. sinensis were collected from 22 provinces, and the genetic polymorphisms amongst these germplasm collections were identified using 20 primer pairs designed against 10 each from eSSR and gSSR transferable markers from barley. A total of 95 SSR loci were detected producing a 100% polymorphic rate among these M. sinensis accessions. The gSSR markers showed a richer genetic polymorphism than eSSR markers. Based on the UPGMA clustering, there was a distinct sub-population separation in M. sinensis, which indicates that geographical differences and natural selection are the driving forces for genetic variation and evolution in the species. The 20 pairs of barley markers matched to 26 loci associated with date of heading, plant height, leaf weight, stem weight, leaf/stem ratio, and total biomass yield. Eleven marker loci were associated with the date of heading, 4 with plant height, 10 with leaf weight, 7 with stem weight, 3 with leaf/stem ratio, and 10 with biomass yield.