ResCap: plant resistance gene prediction and probe generation pipeline for resistance gene sequence capture.

Summary: The discovery of novel resistance genes (R-genes) is an important component in disease resistance breeding. Nevertheless, R-gene identification from wild species and close relatives of plants is not only a difficult but also a cumbersome process. In this study, ResCap, a support vector machine-based high-throughput R-gene prediction and probe generation pipeline has been developed to generate probes from genomic datasets. ResCap contains two integral modules. The first module identifies the R-genes and R-gene like sequences under four categories containing different domains such as TIR-NBS-LRR (TNL), CC-NBS-LRR (CNL), Receptor-like kinase (RLK) and Receptor-like proteins (RLPs). The second module generates probes from extracted nucleotide sequences of resistance genes to conduct sequence capture (SeqCap) experiments. For the validation of ResCap pipeline, ResCap generated probes were synthesized and a sequence capture experiment was performed to capture expressed resistance genes among six spring barley genotypes. The developed ResCap pipeline in combination with the performed sequence capture experiment has shown to increase precision of R-gene identification while simultaneously allowing rapid gene validation including non-sequenced plants. Availability and implementation: The ResCap pipeline is available at http://rescap.ltj.slu.se/ResCap/. Contact: sandeep.kushwaha@slu.se or sandeep@niab.org.in. Supplementary information: Supplementary materials are available at Bioinformatics Advances online.

Mutations in Two Aphid-Regulated ß-1,3-Glucanase Genes by CRISPR/Cas9 Do Not Increase Barley Resistance to Rhopalosiphum padi L.

Callose deposition is induced in plants by various stress factors such as when plants are attacked by herbivores and pathogens. In the case of aphids, callose plugging of aphid-damaged phloem sieve tubes is expected to reduce aphid access to the phloem sap, while aphid-induced upregulation of callose-degrading ß-1,3-glucanase genes in the host plant might counteract this negative effect on aphid performance. We have tested this hypothesis with barley mutants in which one or both of two ß-1,3-glucanase genes (1636 and 1639) have been mutated by CRISPR/Cas9 technique in cv. Golden Promise. These two genes were previously found to be upregulated by the cereal pest Rhopalosiphum padi L. in susceptible barley genotypes. Four 1636/1639 double mutant, three 1636 single mutant and two 1639 single mutant lines were tested for aphid resistance along with control lines. All mutant lines had single base insertions, causing frame shifts and premature stop codons. Three of the four double mutant lines showed significantly reduced ß-1,3-glucanase activity, and bacterial flagellin-induction resulted in significantly more callose formation in the leaves of double mutant compared to control and single mutant lines. However, we found no effect of these modified plant traits on barley resistance to R. padi. Both genes were confirmed to be upregulated by R. padi in Golden Promise. The gene 1637 is another ß-1,3-glucanase gene known to be upregulated by R. padi in barley and was here found to be higher expressed in a double mutant line when compared with a control line. If this can compensate for the general reduction of ß-1,3-glucanase activity in the double mutants is difficult to discern since phloem concentrations of these proteins are unknown.

Plant Genes Benefitting Aphids-Potential for Exploitation in Resistance Breeding.

Aphids are phloem sap-feeding insects common as pests in various crops. Here we review 62 omics studies of aphid/plant interactions to search for indications of how aphids may manipulate the plants to make them more suitable as hosts, i.e. more susceptible. Our aim is to try to reveal host plant susceptibility (S) genes, knowledge which can be exploited for making a plant more resistant to its pest by using new plant breeding techniques to knock out or down such S genes. S genes may be of two types, those that are involved in reducing functional plant defense and those involved in further increasing plant factors that are positive to the aphid, such as facilitated access to food or improved nutritional quality. Approximately 40% of the omics studies we have reviewed indicate how aphids may modify their host to their advantage. To exploit knowledge obtained so far, we suggest knocking out/down candidate aphid S genes using CRISPR/Cas9 or RNAi techniques in crops to evaluate if this will be sufficient to keep the aphid pest at economically viable levels without severe pleiotropic effects. As a complement, we also propose functional studies of recessively inherited resistance previously discovered in some aphid-crop combinations, to potentially identify new types of S genes that later could be knocked out or down also in other crops to improve their resistance to aphids.

Introgression of resistance to Rhopalosiphum padi L. from wild barley into cultivated barley facilitated by doubled haploid and molecular marker techniques.

KEY MESSAGE: Long-term pre-breeding using Hordeum vulgare ssp. spontaneum as a donor of bird cherry-oat aphid resistance has resulted in agronomically improved resistance sources of barley along with easy-to-use molecular markers. Bird cherry-oat aphid (Rhopalosiphum padi L.) is a pest and a virus vector in barley to which there are no bred-resistant cultivars. The present study describes how resistance from Hordeum vulgare ssp. spontaneum has been introgressed in cultivated barley via five successive crosses with the same cultivar Lina (BC) and in parallel with other more modern barley cultivars. Most of the selections for resistance are based on measurements of individual aphid growth in the laboratory. This very slow phenotyping method has been complemented by molecular marker evaluation and application in part of the breeding material. Doubled haploid production in each generation has been crucial for more precise selection of lines with the quantitatively expressed resistance. A field trial of selected "BC3"-generation lines essentially confirmed the laboratory results, so did genotyping of the whole pedigree of parents and selected "BC2" and "BC4" offspring lines. The Infinium iSelect 50 K SNP assay confirmed relationships between lines and discerned several new markers for a resistance QTL on chromosome 2H.

A Novel QTL for Powdery Mildew Resistance in Nordic Spring Barley (Hordeum vulgare L. ssp. vulgare) Revealed by Genome-Wide Association Study.

The powdery mildew fungus, Blumeria graminis f. sp. hordei is a worldwide threat to barley (Hordeum vulgare L. ssp. vulgare) production. One way to control the disease is by the development and deployment of resistant cultivars. A genome-wide association study was performed in a Nordic spring barley panel consisting of 169 genotypes, to identify marker-trait associations significant for powdery mildew. Powdery mildew was scored during three years (2012-2014) in four different locations within the Nordic region. There were strong correlations between data from all locations and years. In total four QTLs were identified, one located on chromosome 4H in the same region as the previously identified mlo locus and three on chromosome 6H. Out of these three QTLs identified on chromosome 6H, two are in the same region as previously reported QTLs for powdery mildew resistance, whereas one QTL appears to be novel. The top NCBI BLASTn hit of the SNP markers within the novel QTL predicted the responsible gene to be the 26S proteasome regulatory subunit, RPN1, which is required for innate immunity and powdery mildew-induced cell death in Arabidopsis. The results from this study have revealed SNP marker candidates that can be exploited for use in marker-assisted selection and stacking of genes for powdery mildew resistance in barley.

A systematic review of rye (Secale cereale L.) as a source of resistance to pathogens and pests in wheat (Triticum aestivum L.).

Wheat is globally one of the most important crops. With the current human population growth rate, there is an increasing need to raise wheat productivity by means of plant breeding, along with development of more efficient and sustainable agricultural systems. Damage by pathogens and pests, in combination with adverse climate effects, need to be counteracted by incorporating new germplasm that makes wheat more resistant/tolerant to such stress factors. Rye has been used as a source for improved resistance to pathogens and pests in wheat during more than 50 years. With new devastating stem and yellow rust pathotypes invading wheat at large acreage globally, along with new biotypes of pest insects, there is renewed interest in using rye as a source of resistance. Currently the proportion of wheat cultivars with rye chromatin varies between countries, with examples of up to 34%. There is mainly one rye source, Petkus, that has been widely exploited and that has contributed considerably to raise yields and increase disease resistance in wheat. Successively, the multiple disease resistances conferred by this source has been overcome by new pathotypes of leaf rust, yellow rust, stem rust and powdery mildew. However, there are several other rye sources reported to make wheat more resistant to various biotic constraints when their rye chromatin has been transferred to wheat. There is also development of knowledge on how to produce new rye translocation, substitution and addition lines. Here we compile information that may facilitate decision making for wheat breeders aiming to transfer resistance to biotic constraints from rye to elite wheat germplasm.

Optimizing Crops for Biocontrol of Pests and Disease.

Volatile compounds and extrafloral nectar are common defenses of wild plants; however, in crops they bear an as-yet underused potential for biological control of pests and diseases. Odor emission and nectar secretion are multigene traits in wild plants, and thus form difficult targets for breeding. Furthermore, domestication has changed the capacity of crops to express these traits. We propose that breeding crops for an enhanced capacity for tritrophic interactions and volatile-mediated direct resistance to herbivores and pathogens can contribute to environmentally-friendly and sustainable agriculture. Natural plant volatiles with antifungal or repellent properties can serve as direct resistance agents. In addition, volatiles mediating tritrophic interactions can be combined with nectar-based food rewards for carnivores to boost indirect plant defense.

Targeted gene mutation in tetraploid potato through transient TALEN expression in protoplasts.

Potato is the third largest food crop in the world, however, the high degree of heterozygosity, the tetrasomic inheritance and severe inbreeding depression are major difficulties for conventional potato breeding. The rapid development of modern breeding methods offers new possibilities to enhance breeding efficiency and precise improvement of desirable traits. New site-directed mutagenesis techniques that can directly edit the target genes without any integration of recombinant DNA are especially favorable. Here we present a successful pipeline for site-directed mutagenesis in tetraploid potato through transient TALEN expression in protoplasts. The transfection efficiency of protoplasts was 38-39% and the site-directed mutation frequency was 7-8% with a few base deletions as the predominant type of mutation. Among the protoplast-derived calli, 11-13% showed mutations and a similar frequency (10%) was observed in the regenerated shoots. Our results indicate that the site-directed mutagenesis technology could be used as a new breeding method in potato as well as for functional analysis of important genes to promote sustainable potato production.

Does insecticide application in a winter oilseed rape field influence the abundance of pollen beetle Meligethes aeneus in nearby ornamental flowers and vegetables?

BACKGROUND: Pollen beetle is a pest that attacks oilseed rape as well as many other brassicaceous crops, garden vegetables and ornamental flowers. The present study was primarily carried out to investigate whether insecticide application in brassicaceous field crops might influence the abundance of pollen beetles in nearby private garden flowers and vegetables. RESULTS: At peak emergence of the new generation of pollen beetles, a significantly higher number of beetles were found in flowers, and in window traps, alongside untreated as opposed to alongside treated sections of the winter oilseed rape (WOSR) field. However, the type of flower played a role in the number of pollen beetles found in the flowers. The presence of pollen beetles in both ornamental and wild flowers was also significantly influenced by the direction of placement of the flowers. No pollen beetle, neither overwintering nor newly emerged, was observed in any of the brassicaceous vegetables placed along the field. CONCLUSION: The number of pollen beetles in the WOSR field strongly influenced the number of pollen beetles in nearby flowers of preference to the beetles, and insecticide treatment with Biscaya (thiacloprid) against pollen beetle in oilseed rape may thus help, indirectly, to protect nearby garden flowers from damage.

Perception of pesticide use by farmers and neighbors in two periurban areas.

Public concern about pesticide use is high although varying with social factors. Individual differences in 'perception' and attitude to pesticide use might be particularly evident in periurban regions where farmers and other people live together. This was investigated using a questionnaire sent to 600 farmers and 600 non-farmers in two periurban areas of Sweden. 'Neighbors'(1) were found to have a more negative attitude to pesticides than farmers, who were slightly positive to the use. Neighbors perceived pesticide use in agriculture to be more harmful to the environment than did farmers and also to reduce the quality of products. Both farmers and neighbors thought that farmers are the predominant users of pesticides. However, reported pesticide users within the home setting were just as common among the neighbors as among the farmers. Perceptions of pesticide use were also found to differ between periurban regions within the country. Attitudes and perceptions of pesticide use, as well as of who is the user, differ based on the group of people in a periurban environment and between different regions. Such differences might play a role for tensions and conflicts in the periurban area of a society and also for regulations of pesticide use.

Plant volatile-induced aphid resistance in barley cultivars is related to cultivar age.

Recent studies have shown that volatile chemical interaction between certain barley (Hordeum vulgare) cultivars can cause reduced host plant acceptance by the aphid Rhopalosiphum padi, and that certain cultivars can induce this effect while others can respond. In this study, we tested whether inducing and responding capabilities are linked to year of release in Swedish two-rowed spring barley. Eighteen cultivars released between 1897 and 1992 were tested in randomly selected subsets with pairwise combinations of volatile emitters and receivers. Significantly reduced aphid acceptance as a result of exposure to volatiles from plants of a different cultivar were found in 24% of the cultivar combinations. In general, older cultivars had a higher degree of aphid resistance after barley volatile treatment than did younger cultivars. The inducing effect of the emitter was also related to date of emitter cultivar release but the time relationship was reversed. Combinations with a younger volatile emitter and an older volatile receiver gave the strongest reduction in aphid acceptance of treated plants. Linear relationships between microsatellite diversity of emitting cultivars and their efficiency as inducers indicated that younger cultivars might have a more unique odour, whereas older cultivars may be more sensitive to induction.

Microarray analysis of the interaction between the aphid Rhopalosiphum padi and host plants reveals both differences and similarities between susceptible and partially resistant barley lines.

The bird cherry-oat aphid (Rhopalosiphum padi L.) is an important pest on cereals causing plant growth reduction without specific leaf symptoms. Breeding of barley (Hordeum vulgare L.) for R. padi resistance shows that there are several resistance genes, reducing aphid growth. To identify candidate sequences for resistance-related genes, we performed microarray analysis of gene expression after aphid infestation in two susceptible and two partially resistant barley genotypes. One of the four lines is a descendant of two of the other genotypes. There were large differences in gene induction between the four lines, indicating substantial variation in response even between closely related genotypes. Genes induced in aphid-infested tissue were mainly related to defence, primary metabolism and signalling. Only 24 genes were induced in all lines, none of them related to oxidative stress or secondary metabolism. Few genes were down-regulated, with none being common to all four lines. There were differences in aphid-induced gene regulation between resistant and susceptible lines. Results from control plants without aphids also revealed differences in constitutive gene expression between the two types of lines. Candidate sequences for induced and constitutive resistance factors have been identified, among them a proteinase inhibitor, a serine/threonine kinase and several thionins.

Pea lectin expressed transgenically in oilseed rape reduces growth rate of pollen beetle larvae.

In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T0-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T2-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T2-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.

Growth, herbivory and disease in relation to gender in Salix viminalis L.

Many species of dioecious plants show sex-related differences in growth rate and rates of attack by various herbivores and diseases. The common pattern is for males to grow faster than females and to be less well defended against herbivores. In willows (Salix spp.), the predominance of female-biased sex ratios has been ascribed in part to differential feeding by herbivores. In this study of Salix viminalis, seven families grown on agricultural land showed no gender-related variation in shoot biomass or rates of herbivory by insects (lepidopterans and cecidomyiids). However, Melampsora rust disease was found to be more severe on females than on males when the plants were in a non-reproductive stage. After flowering and seed-set females tended to be more affected in some families but less affected in others. Although, on average, there was a female bias in the sex ratio of S. viminalis, sex ratios differed significantly between families. These ratios were not related to any of the recorded biotic agents, but rather to relationships between families. These results are interpreted in terms of resource allocation between reproduction, growth and defence, and causes for divergence from the expected patterns are discussed. The results may have implications for S. viminalis breeding strategies where the aim is to produce biofuel. For instance, these findings suggest that gender can be ignored when selecting for a high growth rate and resistance to Melampsora and certain insect pests.