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Ilyonectria pseudodestructans, a plant pathogen that is known to cause root rot on fruit trees such as grapevine and apple, has recently been reported to also cause tuber decay in potato. The increasing risk of this pathogen on various horticultural crops makes it essential to develop a rapid and accurate detection method. In this study, an RNase H-dependent PCR (rhPCR) protocol and a modified probe-based rh-quantitative PCR (rhqPCR) protocol for I. pseudodestructans detection were developed. Both the forward and reverse primers for rhPCR and rhqPCR carry an RNA nucleotide at the site where a single-nucleotide polymorphism between I. pseudodestructans and strains of other Ilyonectria spp. is located, and the rhqPCR also contains a fluorescent-labeled target-specific probe. The primers were designed based on the sequence of the histone H3 gene and could amplify a DNA fragment of 73 bp. In the specificity test, by alignment via the BLASTn tool, the RNA nucleotide bases on both the forward and the reverse primers were identical to the corresponding genomic site of 16 of 17 (94.1%) database-available I. pseudodestructans strains, and different from 43 of 44 (97.7%) database-available strains of other Ilyonectria spp. When the rhPCR and rhqPCR protocols were applied on 11 I. pseudodestructans strains and 46 other strains of different species of plant pathogens, all of the I. pseudodestructans strains generated positive reactions whereas all of the other strains were negative, which indicated an excellent specificity of the primers. In the sensitivity test, the lowest DNA template amount for a positive reaction using the rhPCR and rhqPCR methods was 2 pg for I. pseudodestructans genomic DNA. When testing the rhqPCR method on gBlock, the lowest number of molecules for a positive reaction was six. These results indicated a high sensitivity of the protocol for I. pseudodestructans detection. To our knowledge, this is the first report of a probe-based rhqPCR to be applied to plant disease diagnosis; in addition, this is also the first rapid molecular protocol to detect I. pseudodestructans. The new rhPCR and rhqPCR methods have a potential to be applied by plant disease diagnostic labs for their routine work.

Reliable estimates of genetic diversity among the accessions in a breeding population is important knowledge for use in breeding. Among the different types of molecular markers, single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) are largely used by breeders; however, our knowledge of the reliability of the estimates of genetic diversity based on these two types of markers in multiple populations is limited. In this study, a doubled haploid (DH) and an inbred population developed from Brassica napus × Brassica oleracea interspecific crosses were used for comparative analysis of these two types of markers. The estimates based on SNP and SSR markers showed a stronger correlation in the inbred population which was expected to carry greater genetic diversity as compared to the DH population. This inference was also evident from the analysis of different diversity groups (least, intermediate, and most similar) of these two populations for significant difference between the groups for six agronomic and seed quality traits, where this analysis failed to differentiate the diversity groups of the DH population for any of the traits. However, both marker types could differentiate the diversity groups of the inbred population for several traits. Furthermore, the six sub-populations of the inbred population could also be differentiated by both marker types. Thus, the results demonstrate the greater utility of the SSR and SNP markers in a genetically diverse population. This knowledge can be used while grouping a breeding population for diversity groups; however, caution needs to be taken while using the markers in a genetically narrow population.

Candidatus Liberibacter solanacearum (Lso) is the causal agent of zebra chip of potato (Solanum tuberosum), which can significantly reduce potato yield. In this study, a loop-mediated isothermal amplification (LAMP) method for the detection of Lso haplotypes A and B was developed and evaluated. Two sets of LAMP primers named LAMP-A and LAMP-B were designed and tested for specificity and sensitivity. Both LAMP-A and LAMP-B were specific to Lso in in silico analysis using the Primer-Blast tool. The LAMP-A and LAMP-B could only produce positive signal from DNA mixtures of Lso-infected tomato but not from the genomic DNA of 37 non-target plant pathogens. The sensitivity of LAMP-A and LAMP-B on Lso haplotypes A and B were tested on gBlocks and genomic DNA from Lso-infected tomato. On the genomic DNA, for LAMP-A, the lowest amount of template DNA for a positive LAMP reaction was 2 to 20 ng on four haplotype A strains and 20 to 80 ng on four haplotype B strains; for LAMP-B, the lowest amount of template DNA for a positive LAMP reaction was 0.02 to 2 ng on four haplotype B strains and 20 ng to no amplification on four haplotype A strains. On gBlocks, for LAMP-A, the lowest number of copies for a positive LAMP reaction was 60 on haplotype A and 600 on haplotype B; for LAMP-B, the lowest number of copies for a positive LAMP reaction was 60 on haplotype B and 600 on haplotype A. Therefore, considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, the LAMP-A and LAMP-B primers can be used together to test the probable Lso-infected plant or psyllid samples to rapidly, accurately and directly differentiate haplotypes A and B. We highly recommend this LAMP system to plant pathology practitioners and diagnostic labs for routine detection of Lso and confirmation of zebra chip disease on potato or tomato.

Ilyonectria pseudodestructans is a plant pathogen causing root rot on fruit trees such as grapevine and apple. Recently, it was reported to be a pathogen of potato. The increasing risk of this pathogen on plants makes it essential to develop a rapid and accurate detection method. In this study, an RNase H-dependent PCR (rhPCR) protocol and a modified probe based rh-quantitative PCR (rhqPCR) protocol for I. pseudodestructans detection were developed. Both the forward and reverse primers for rhPCR and rhqPCR carry a RNA nucleotide at the site where a single-nucleotide polymorphism between I. pseudodestructans and strains of other Ilyonectria species is located and the rhqPCR also contains a fluorescent-labeled target-specific probe. The primers were designed based on the sequence of the Histone H3 gene and could amplify a DNA fragment of 73 base pairs (bp). In the specificity test, by alignment via the Blastn tool, the RNA nucleotide bases on both the forward and reverse primers were identical to the corresponding genomic site of 16 of 17 (94.1%) database-available I. pseudodestructans strains, and different to 43 of 44 (97.7%) database-available strains of other Ilyonectria species. When the rhPCR/rhqPCR protocols were applied on 11 I. pseudodestructans strains and 46 other strains of different species of plant pathogens, all the I. pseudodestructans generated positive reactions but all the other strains were negative, which indicated an excellent specificity of the primers. In the sensitivity test, the lowest DNA template amount for a positive reaction using the rhPCR/rhqPCR methods was 2 pg for I. pseudodestructans genomic DNA. When testing the rhqPCR method on gBlock, the lowest number of molecules for a positive reaction was six. These results indicated a high sensitivity of the protocol for I. pseudodestructans detection. This is the first report of a probe-based rhqPCR to be applied on plant disease diagnosis; in addition, this is also the first rapid molecular protocol to detect I. pseudodestructans, and therefore, the new rhPCR/rhqPCR methods are recommended to plant disease diagnostic labs for their routine work.

Spongospora subterranea is the causal agent of powdery scab of potato (Solanum tuberosum), which can significantly reduce potato quality. In this study, we developed and evaluated a loop-mediated isothermal amplification (LAMP) method for the detection of S. subterranea. A set of LAMP primers named PS-LAMP was designed and tested for specificity and sensitivity. In the specificity test, in silico analysis using the NCBI Primer-BLAST tool indicated that PS-LAMP was specific to S. subterranea. The in vitro tests confirmed specificity, showing that PS-LAMP could produce positive signals from DNA isolated from each of three potato tubers with powdery scab symptoms but did not produce positive signals from DNA isolated from 38 nontarget plant pathogens. The sensitivity of PS-LAMP was tested on both gBlocks and DNA isolated from potato samples with powdery scab symptoms. On gBlocks, the lowest number of copies for a positive LAMP reaction was six, which was similar to results obtained via qPCR, but it was 10 times more sensitive than conventional PCR. On a DNA sample from S. subterranea-infected potato, the lowest amount of template DNA for a positive LAMP reaction was 2 pg, which was incomparable with the sensitivity of qPCR. Considering the convenience of the LAMP technique, as well as the high specificity and sensitivity, this assay can be very useful for plant pathology practitioners and diagnostic labs interested in rapid, accurate, and routine detection of S. subterranea and confirmation of powdery scab disease.

Eyes opening and closing status is one of the most important components to monitor the driver's fatigue.d The current research mainly considers eyes blink frequency and the closing duration to judge the driver's fatigue. To identify driver's fatigue level, eyes opening and closing ratio (EOCR) is a critical factor and therefore, it is desirable to detect EOCR for driver's fatigue monitoring. The proposed method aims to simultaneously segment images and measure the parameters of the EOCR. A BiSeNet‐based iris and pupil segmentation network is first proposed and the Visual Geometry Group (VGG) ConvNet‐based model to detect the EOCR value is provided by considering the main features rounding eyes area and the iris‐pupil size for building test dataset. The comparison experiments are conducted with the proposed method and the other existing work in different datasets, such as CASIA‐Iris‐Thousand, CASIA‐Iris‐Interval, and UBIRIS.v2. The results demonstrate that the proposed method has superior detection effects on both infrared images and colour images than other existing approaches. Furthermore, the experiments of detecting the EOCR and iris and pupil segmentation are carried out with the test dataset and the results show that the proposed method can reliably identify driver's eye opening and closing degree.

Potato (Solanum tuberosum) wart, caused by the biotrophic fungal pathogen Synchytrium endobioticum, is a serious disease of cultivated potatoes. A rapid and accurate method for wart diagnosis is needed. In this study, we described the development and evaluation of a loop-mediated isothermal amplification (LAMP) method for the detection of S. endobioticum. Four sets of LAMP primers were designed. Their sensitivity was tested on a gBlock and the specificity of the most sensitive primer set (LAMP1) was verified by in silico analysis and on DNA of non-target species. The LAMP1 primer set showed a detection limit at 7-8 gBlock DNA molecules per reaction. On sensitivity, the LAMP was comparable to qPCR (SYBR green- and probe-based) and about 10 times more sensitive than conventional PCR. Considering the convenience of operation, the LAMP protocol is accurate and robust and should be recommended for the routine lab testing for potato wart.

Gene expression profiling offers a means of understanding pathogenesis in clubroot of crucifers caused by the biotrophic pathogen Plasmodiophora brassicae. In this study, a canola (Brassica napus) cultivar 45H29 was inoculated with P. brassicae pathotypes 5I (P5I) and 5X (P5X), and the pathogen:plant DNA biomass ratio in roots was determined at 7, 14, and 21 days after inoculation (dai) by quantitative PCR (qPCR) analysis. The P5X:plant biomass ratio increased across the time course, while the P5I:plant biomass ratio decreased. To investigate genes differentially expressed during the infection process, the expression of 205 P. brassicae genes encoding putative secreted proteins was analysed from the inoculated samples at 14 dai. One of the 205 genes was expressed at a higher level in P5X than in P5I, while 15 genes were expressed at a higher level in P5I than in P5X. Of these 16, 13 genes encoded proteins with high cysteine content, while three genes encoded proteins with an RXLR motif. The expression of the 16 genes was analysed further in the 7‐ and 21‐dai samples. No transcripts of the 16 genes were detected from either pathotype at 7 dai. In contrast, 11 genes were differentially expressed between the two pathotypes at 21 dai, with nine being more highly expressed in P5X and two in P5I. One gene showed homology to PbSUNK2 found in P. brassicae‐infected Arabidopsis thaliana root tissues. These 16 genes may offer important information to improve the current understanding of canola–P. brassicae interactions.

The planting of clubroot resistant (CR) canola (Brassica napus) is the most effective method to manage clubroot. Since 2013, many Plasmodiophora brassicae isolates capable of overcoming resistance have been detected, often in mixtures with avirulent isolates. To improve understanding of the effect of low concentrations of virulent isolates on host resistance, three CR canola cultivars (45H29, L135C, and L241C) were inoculated with pairs of isolates representing virulent/avirulent pathotypes (2*/2, 3*/3, and 5*/5) collected after or before the introduction of CR canola, respectively. Seven-day-old seedlings of each cultivar were incubated for 2 days in low concentrations (1 × 103 spores/ml) of the virulent isolates, followed by a second inoculation with a high concentration (1 × 107 spores/ml) of the avirulent isolates. Positive controls comprised seedlings inoculated with low concentrations of the virulent isolates followed by high concentrations of the virulent isolates (PC1) or only with high concentrations of virulent isolates (PC2). Negative controls comprised seedlings inoculated only with high concentrations of the avirulent isolates (NC1) or only with low concentrations of the virulent isolates (NC2). Clubroot severity was significantly higher in all nine experimental treatments (low virulent plus high avirulent) than in the negative control NC1 (high avirulent) but was lower in the experimental treatments than in the positive controls (PC1 and PC2). Low concentrations of virulent isolates alone (NC2) caused moderate clubroot. Disease severity correlated well with P. brassicae biomass in canola as determined by quantitative PCR analysis 28 to 35 days after inoculation. This study revealed that low concentrations of virulent isolates compromised canola resistance for infection by avirulent isolates.

Genetic mapping for faba bean lags far behind other major crops. Density enhancement of the faba bean genetic linkage map was carried out by screening 5,325 genomic SSR primers and 2033 expressed sequence tag (EST)‐SSR primers on the parental cultivars '91825' and 'K1563'. Two hundred and fifteen genomic SSR and 133 EST‐SSR primer pairs that detected polymorphisms in the parents were used to screen 129 F₂ individuals. This study added 337 more SSR markers and extended the previous linkage map by 2928.45 cM to a total of 4516.75 cM. The number of SSR markers in the linkage groups varied from 12 to 136 while the length of each linkage group ranged from 129.35 to 1180.21 cM. The average distance between adjacent loci in the enhanced genetic linkage map was 9.71 cM, which is 2.79 cM shorter than the first linkage map of faba bean. The density‐enhanced genetic map of faba bean will be useful for marker‐assisted selection and breeding in this important legume crop.

The size of the human eye opening and closing plays a certain role in the human-computer interaction platform, such as the PERCLOS parameter commonly used in driver fatigue detection. Considering that the image in the actual application scene is often accompanied by distance, blur, etc., this paper proposes an algorithm based on BiseNet and Vgg methods which can segment the pupil and iris while outputting the realtime opening ratio of the eye.

This study was conducted with the objective of finding genetic marker(s) to differentiate pathotypes of the clubroot pathogen Plasmodiophora brassicae. Ideally, such markers should be used as a diagnostic tool to identify or predict certain pathotypes. By using PCR with gene-specific primers, 117 non-housekeeping P. brassicae genes were investigated for their presence/absence in isolates representing pathotypes 2, 3, 5, 6 or 8 (Williams’ system), and in new strains of the pathogen recently identified from Alberta, Canada, which are highly virulent on clubroot resistant canola (Brassica napus) cultivars. One gene, named Cr811, was present in isolates of pathotype 5 and in a new strain of P. brassicae that also is classified as pathotype 5 on the system of Williams, but causes severe clubroot on resistant canola. Three additional newly identified virulent populations, similar to pathotype 3, also were tested and found to carry Cr811. In contrast, the Cr811 gene was absent in all isolates representing the original pathotypes 2, 3, 6 and 8. Quantitative real-time PCR analysis indicated greater copy numbers of Cr811 in the new virulent pathotype 3-like and pathotype 5-like populations versus the original pathotype 5, indicating a potential method to identify new pathotypes of P. brassicae before the breakdown of resistance could be detected in a canola crop. The association of Cr811 with infection by P. brassicae of previously resistant canola cultivars, as revealed by qPCR analysis, suggests that Cr811 plays an important role in the interaction between host resistance and P. brassicae pathogenicity.

Simple sequence repeat (SSR) markers have previously been applied to linkage mapping of the pea (Pisum sativum L.) genome. However, the transferability of existing loci to the molecularly distinct Chinese winter pea gene pool was limited. A novel set of pea SSR markers was accordingly developed. Together with existing SSR sequences, the genome of the G0003973 (winter hardy) × G0005527 (cold sensitive) cross was mapped using 190 F2 individuals. In total, 157 SSR markers were placed in 11 linkage groups with an average interval of 9.7 cM and total coverage of 1518 cM. The novel markers and genetic linkage map will be useful for marker-assisted pea breeding.

Simple sequence repeat (SSR) marker is a powerful tool for construction of genetic linkage map which can be applied for quantitative trait loci (QTL) and marker-assisted selection (MAS). In this study, a genetic map of faba bean was constructed with SSR markers using a 129 F2 individuals population derived from the cross of Chinese native variety 91825 (large seed) and K1563 (small seed). By screening 11 551 SSR primers between two parents, 149 primer pairs were detected polymorphic and used for F2 population analysis. This SSR-based genetic linkage map consisted of 15 linkage groups with 128 SSR. The map encompassed 1587 cM with an average genetic distance of 12.4 cM. The genetic map generated in this study will be beneficial for genetic studies of faba bean for identification of marker-locus-trait associations as well as comparative mapping among faba bean, pea and grasspea.

The study of genetic diversity between Lathyrus sativus L. and its relative species may yield fundamental insights into evolutionary history and provide options to meet the challenge of climate changes. 30 SSR loci were employed to assess the genetic diversity and population structure of 283 individuals from wild and domesticated populations from Africa, Europe, Asia and ICARDA. The allele number per loci ranged from 3 to 14. The average gene diversity index and average polymorphism information content (PIC) was 0.5340 and 0.4817, respectively. A model based population structure analysis divided the germplasm resources into three subgroups: the relative species, the grasspea from Asia, and the grasspea from Europe and Africa. The UPGMA dendrogram and PCA cluster also demonstrated that Asian group was convincingly separated from the other group. The AMOVA result showed that the cultivated species was quite distinct from its relative species, however a low level of differentiation was revealed among their geographic origins. In all, these results provided a molecular basis for understanding genetic diversity of L. sativus and its relatives.

Pea (Pisum sativum L.) is an important food legume globally, and is the plant species that J.G. Mendel used to lay the foundation of modern genetics. However, genomics resources of pea are limited comparing to other crop species. Application of marker assisted selection (MAS) in pea breeding has lagged behind many other crops. Development of a large number of novel and reliable SSR (simple sequence repeat) or microsatellite markers will help both basic and applied genomics research of this crop. The Illumina HiSeq 2500 System was used to uncover 8,899 putative SSR containing sequences, and 3,275 non-redundant primers were designed to amplify these SSRs. Among the 1,644 SSRs that were randomly selected for primer validation, 841 yielded reliable amplifications of detectable polymorphisms among 24 genotypes of cultivated pea (Pisum sativum L.) and wild relatives (P. fulvum Sm.) originated from diverse geographical locations. The dataset indicated that the allele number per locus ranged from 2 to 10, and that the polymorphism information content (PIC) ranged from 0.08 to 0.82 with an average of 0.38. These 1,644 novel SSR markers were also tested for polymorphism between genotypes G0003973 and G0005527. Finally, 33 polymorphic SSR markers were anchored on the genetic linkage map of G0003973 × G0005527 F2 population.

Background Grasspea (Lathyrus sativus L., 2n = 14), a member of the family Leguminosae, holds great agronomic potential as grain and forage legume crop in the arid areas for its superb resilience to abiotic stresses such as drought, flood and salinity. The crop could not make much progress through conventional breeding in the past, and there are hardly any detailed molecular biology studies due to paucity of reliable molecular markers representative of the entire genome. Results Using the 454 FLX Titanium pyrosequencing technique, 651,827 simple sequence repeat (SSR) loci were identified and 50,144 nonredundant primer pairs were successfully designed, of which 288 were randomly selected for validation among 23 L. sativus and one L. cicera accessions of diverse provenance. 74 were polymorphic, 70 monomorphic, and 144 with no PCR product. The number of observed alleles ranged from two to five, the observed heterozygosity from 0 to 0.9545, and Shannon’s information index ranged from 0.1013 to 1.0980, respectively. The dendrogram constructed by using unweighted pair group method with arithmetic mean (UPGMA) based on Nei's genetic distance, showed obvious distinctions and understandable relationships among the 24 accessions. Conclusions The large number of SSR primer pairs developed in this study would make a significant contribution to genomics enabled improvement of grasspea.

Background Faba bean (Vicia faba L.) is an important food legume crop, grown for human consumption globally including in China, Turkey, Egypt and Ethiopia. Although genetic gain has been made through conventional selection and breeding efforts, this could be substantially improved through the application of molecular methods. For this, a set of reliable molecular markers representative of the entire genome is required. Results A library with 125,559 putative SSR sequences was constructed and characterized for repeat type and length from a mixed genome of 247 spring and winter sown faba bean genotypes using 454 sequencing. A suit of 28,503 primer pair sequences were designed and 150 were randomly selected for validation. Of these, 94 produced reproducible amplicons that were polymorphic among 32 faba bean genotypes selected from diverse geographical locations. The number of alleles per locus ranged from 2 to 8, the expected heterozygocities ranged from 0.0000 to 1.0000, and the observed heterozygosities ranged from 0.0908 to 0.8410. The validation by UPGMA cluster analysis of 32 genotypes based on Nei's genetic distance, showed high quality and effectiveness of those novel SSR markers developed via next generation sequencing technology. Conclusions Large scale SSR marker development was successfully achieved using next generation sequencing of the V. faba genome. These novel markers are valuable for constructing genetic linkage maps, future QTL mapping, and marker-assisted trait selection in faba bean breeding efforts.

UNLABELLED PREMISE OF THE STUDY Expressed sequence tag (ESTs)-derived microsatellite markers were developed in Lathyrus sativus by screening the National Center for Biotechnology Information (NCBI) database. The usefulness of these novel markers was validated for size polymorphism among grasspea accessions. • METHODS AND RESULTS Three hundred EST-simple sequence repeat (SSR) primer pairs were identified and loci characterized for size polymorphism among 24 grasspea accessions from worldwide sources. Among them 139 SSR loci produced no PCR product, 117 SSR loci were monomorphic, and 44 SSR loci were polymorphic. The mean number of alleles per locus ranged from two to 11. The observed heterozygosity and expected heterozygosity ranged from 0.000 to 1.000 and 0.042 to 0.836, respectively. • CONCLUSIONS These novel markers will be useful and convenient to study genetic mapping and molecular breeding in grasspea.