Molecular Techniques for Root-Knot Nematode Identification.

Among plant-parasitic nematodes, root-knot nematodes (RKN), Meloidogyne spp., are the most important parasite infecting economically important crops globally and causing severe losses in crop production. The use of efficient nematode control methods against these parasites depends upon their correct detection in roots and soil samples. Currently, the use of integrated identification methods, including biochemical, molecular, and morphological-based characters, is preferred. But the techniques using morphology and phylogenetic analysis are time-consuming and not suitable for routine analysis. They have only been used for studies of cryptic species, which were identified using integrative taxonomy. Here we describe the enzymatic and molecular-based methods that have successfully been used in Brazil for more than 25 years in the Nematology Lab at Embrapa Genetic Resources and Biotechnology for routine analysis. This technique is a combination of isozyme esterase profiling and molecular markers, with the aim of having a rapid and correct diagnosis of Meloidogyne spp. populations from field and greenhouse.

Biochemical and molecular depictions to develop ech42 gene-specific SCAR markers for recognition of chitinolytic Trichoderma inhibiting Macrophomina phaseolina (Maubl.) Ashby.

Trichoderma isolates were inhibited variably in-vitro growth of soil-borne phytopathogen Macrophomina phaseolina (Maubl.) Ashby causes root rot in cotton. The growth inhibition of test-pathogen was found to be higher (90.36%) in T. viride NBAIITv23 followed by T. koningii MTCC796 (85.77%) under dual culture antagonism. The microscopic examination suggested that the antagonists Tv23 and MTCC796 adopted mycoparasitism as a strong mode of action to restrain pathogen growth. However, antagonists T. harzianum NBAIITh1 (77.89%) and T. virens NBAIITvs12 (61.74%) demonstrated strong antibiosis action for growth inhibition of the test pathogen. A significant positive correlation was established between the growth inhibition of M. phaseolina and the release of cell wall degrading enzymes- chitinase (p = 0.001), ß-1,3, glucanase (p = 0.01), and protease (p = 0.05) under the influence of pathogen cell wall. The chitinase and ß-1,3, glucanase activities were elevated 2.09 and 1.75 folds, respectively, in potent mycoparasitic Tv23 strain influenced by a pathogen cell wall compared to glucose as a carbon source. The three unique DNA-RAPD fragments OPA-07(1033), OPA-16(983), and OPO-15(239), amplified by potent mycoparasitic Tv23 strain, were subjected to DNA sequencing and derived functional 864 bp from OPA-16(983) and have sequence homology to ech42 gene with partial CDs of 262 amino acids (nucleotide accession No. KF723016.1 and protein accession No.AHF57046.1). Novel SCAR markers were developed from a functional sequence of OPA-16 fragments and validated across the genomic DNA of eleven Trichoderma antagonists. The novel SCAR markers evolved from the RAPD-SCAR interface to authenticate chitinolytic Trichoderma associated with mycoparasitic action for eco-friendly biocontrol activity.

Development of sequence-characterized amplified region (SCAR) markers for accurate and differential identification of multienzyme-producing and non-enzymatic Aspergillus strains of industrial importance.

Aspergillus strains are known to produce multiple enzymes of industrial importance. To screen Aspergillus isolates and select a strain with the ability to produce multiple enzymes and discriminate it from non-enzymatic strains, a rapid and accurate approach is required. With this background, a DNA fingerprinting-based study was conducted to develop a simple but accurate molecular detection method with the potential to discriminate multienzyme-producing Aspergillus strains from non-enzymatic strains, irrespective of species. To achieve this, Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR was employed to derive group-specific Sequence Characterized Amplified Region (SCAR) markers (i.e., markers corresponding to PCR amplicons of known DNA sequence). To this end, both group-specific (multienzyme-producing and non-enzymatic Aspergillus group) SCAR markers were sought by comparing the ERIC fingerprint profiles and used to develop primers for use in specific and differential identification of multienzyme-producing Aspergillus isolates. As an outcome, the two SCAR-PCR formats were developed. One format is for specific identification of multienzyme-producing Aspergillus strains (SCAR-PCR1), and the other for identifying non-enzymatic Aspergillus strains (SCAR-PCR2). Both SCAR-PCRs were able to discriminate between these two contrasting groups. These formats are simple but accurate and rapid compared to the time-consuming and laborious conventional methods. Therefore, they could be efficient as an alternative strategy for the high-throughput screening of industrially important Aspergillus strains.

The Northern Root-Knot Nematode Meloidogyne hapla: New Host Records in Portugal.

Root-knot nematodes (RKNs), Meloidogyne spp., are a group of plant-parasitic nematodes (PPNs) of great economic significance worldwide. The northern root-knot nematode, Meloidogyne hapla, is one of the most important species of RKNs occurring in cold regions. In Portugal so far, M. hapla has been found parasitizing potato and fig trees. During surveys carried out in 2019-2022 in fields for horticultural and ornamental production, soil and root samples were collected. Roots were observed under a stereomicroscope to determine the presence of galls. Nematodes were extracted from the soil. Morphological features showed a high similarity and consistency with previous descriptions of the genus. For molecular analysis, total genomic DNA was isolated from single nematodes and used to amplify in multiplex reaction using the species-specific primers JMV1, JMV2 and JMVhapla, and for sequencing of the ITS region with the primers TW81/AB28. Multiplex PCR amplification produced a specific fragment of 440 bp and PCR amplification of the ITS region yielded a single fragment of 550 bp, as expected. The obtained sequences showed a similarity ranging from 99.8% to 100% with the sequences of M. hapla available in the database. The phylogenetic tree revealed that the isolates grouped with M. hapla isolates. From the 690 samples collected, M. hapla was detected in three different hosts (grapevine, eucalyptus and potato) in four districts of mainland Portugal and on Madeira Island. To our knowledge, this is the first report of M. hapla infecting the grapevine and eucalyptus in Portugal.

Development of a SCAR marker linked to fungal pathogenicity of rice blast fungus Magnaporthe Oryzae.

PCR-based molecular approaches including RAPD (random amplified polymorphic DNA), ISSR (inter-simple sequence repeat), and SRAP (sequence-related amplified polymorphism) are commonly used to analyze genetic diversity. The aims of this study are to analyze genetic diversity of M. oryzae isolates using PCR-based molecular approaches such as RAPD, ISSR, and SRAP and to develop SCAR marker linked to the pathogenicity of rice blast fungus. Twenty Magnaporthe oryzae isolates were collected mainly from the south of Vietnam and assessed for genetic variation by RAPD, ISSR, and SRAP methods. The comparison of those methods was conducted based on the number of polymorphic bands, percentage of polymorphism, PIC values, and phylogenetic analysis. Then, sequenced characterized amplified region (SCAR) markers were developed based on specific bands linked to fungal pathogenicity of rice blast fungus, M. oryzae. The results indicated that SRAP markers yielded the greatest number of polymorphic bands (174) and occupied 51.7% with polymorphism information content (PIC) value of 0.66. Additionally, the SRAP approach showed stability and high productivity compared with RAPD and ISSR. The SCAR marker developed from the SRAP method identified the presence of the avirulence AVR-pita1 gene involving fungal pathogenicity that can break down blast resistance in rice cultivars. The consistency of SCAR marker obtained in this study showed its efficiency in rapid in-field detection of fungal pathogenicity. SCAR marker developed from SRAP technique provides a useful tool for improving the efficiency of blast disease management in rice fields.

In vitro propagation of three mosaic disease resistant cassava cultivars.

BACKGROUND: Cassava is a staple food for over 800 million people globally providing a cheap source of carbohydrate. However, the cultivation of cassava in the country is facing to viral diseases, particularly cassava mosaic disease (CMD) which can cause up to 95% yield losses. With aim to supply farmers demand for clean planting materials, there is need to accelerate the production of the elite cultivars by use of tissue culture in order to cope with the demand. METHODS: Nodal explants harvested from the greenhouse grown plants were sterilised using different concentrations of a commercial bleach JIK (3.85% NaOCl) and varying time intervals. Microshoots induction was evaluated using thidiazuron (TDZ), benzyl amino purine (BAP), and kinetin. Rooting was evaluated using different auxins (Naphthalene acetic acid NAA and Indole-3-butyricacid IBA). PCR-based SSR and SCAR markers were used to verify the presence of CMD2 gene in the regenerated plantlets. RESULTS: The highest level of sterility in explants (90%) was obtained when 20% Jik was used for 15 min. The best cytokinin for microshoots regeneration was found to be kinetin with optimum concentrations of 5, 10 and 20 µM for Agric-rouge, Atinwewe, and Agblehoundo respectively. Medium without growth regulators was the best for rooting the three cultivars. A survival rate of 100, 98, and 98% was recorded in the greenhouse for Agric-rouge, Atinwewe, and Agblehoundo respectively and the plantlets appeared to be morphologically normal. The SSR and SCAR analysis of micropropagated plants showed a profile similar to that of the mother plants indicating that the regenerated plantlets retained the CMD2 gene after passing through in vitro culture, as expected with micropropagation. CONCLUSION: The nodal explants was established to be 20% of Jik (3.85% NaOCl) with an exposure time of 15 min. Kinetin was proved to be the best cytokinins for microshoot formation with the optimum concentration of 5, 10 and 20 µM for Agric-rouge, Atinwewe, and Agblehoundo respectively. The protocol developed during this study will be useful for mass propagation of the elite cassava cultivars.

[Genomic Markers Associated with Cold-Hardiness in Brassica rapa L.]

Brassica rapa L. is a valuable and widespread species, but its cultivation in risk farming areas requires high-quality cold-hardy varieties to be developed. Mechanisms of the cold stress response in plants involve expression of numerous genes, including ribosomal ones, and are related to plant chromosome variability. FISH- and PCR-based methods were used to study intraspecific chromosome variability in the number and localization of 45S and 5S rDNA clusters and also to examine a set of molecular markers associated with cold-hardiness in winter B. rapa cultivars from high-risk farming areas. Several SSR (Na10-CO3 and BrgMS5339-1) and SCAR (BoCCA1-F/BoCCA1-1R1 and BoCCA1-F/BoCCA1-2R1) markers were identified as suitable for diagnosing cold-resistant and cold-susceptible genotypes in B. rapa. Compared with fodder cultivars, oilseed and leaf cultivars were shown to have more molecular markers associated with cold-hardiness and a higher level of polymorphism for the chromosomal distribution of 45S and 5S rDNAs, including chromosome heteromorphism. Thus, the least cold-resistant genotypes were found to display the lowest level of chromosome variability in the distribution of the 45S and 5S rDNA clusters and vice versa. The findings could be useful for the development of new cold-tolerant B. rapa varieties.

Recombination fraction and genetic linkage among key disease resistance genes (Co-42 /Phg-2 and Co-5/"P.ult") in common bean.

Anthracnose (Colletotrichum lindemuthianum), Angular leaf spot (Pseudocercospora griseola) and Pythium root rot are important pathogens affecting common bean production in the tropics. A promising strategy to manage these diseases consists of combining several resistance (R) genes into one cultivar. The aim of the study was to determine genetic linkage between gene pairs, Co-42 /Phg-2, on bean-chromosome Pv08 and Co-5/"P.ult" on-chromosome Pv07, to increase the efficiency of dual selection of resistance genes for major bean diseases, with molecular markers. The level of recombination was determined by tracking molecular markers for both BC3F6 and F2 generations. Recombination fraction r, among gene pairs, the likelihood of linkage, L(r), and logarithm of odds (LOD) scores were computed using the statistical relationship of likelihood which assumes a binomial distribution. The SCAR marker pair SAB3/PYAA19 for the gene pair Co-5/"P.ult" exhibited moderate linkage (r = 32 cM with a high LOD score of 9.2) for BC3F6 population, but relatively stronger linkage for the F2 population (r = 21 cM with a high LOD score of 18.7). However, the linkage among SCAR marker pair SH18/SN02, for the gene pair Co-42 /Phg-2 was incomplete for BC3F6 population (r = 47 cM with a low LOD score of 0.16) as well as F2 population (r = 44 cM with a low LOD score of 0.7). Generally, the weak or incomplete genetic linkage between marker pairs studied showed that all the four genes mentioned earlier have to be tagged with a corresponding linked marker during selection. The approaches used in this study will contribute to two loci linkage mapping techniques in segregating plant populations.

Race- and Isolate-specific Molecular Marker Development through Genome-Realignment Enables Detection of Korean Plasmodiophora brassicae Isolates, Causal agents of Clubroot Disease.

Clubroot is one of the most economically important diseases of the Brassicaceae family. Clubroot disease is caused by the obligate parasite Plasmodiophora brassicae, which is difficult to study because it is non-culturable in the laboratory and its races are genetically variable worldwide. In Korea, there are at least five races that belongs to four pathotype groups. A recent study conducted in Korea attempted to develop molecular markers based on ribosomal DNA polymorphism to detect P. brassicae isolates, but none of those markers was either race-specific or pathotype-specific. Our current study aimed to develop race- and isolate-specific markers by exploiting genomic sequence variations. A total of 119 markers were developed based on unique variation exists in genomic sequences of each of the races. Only 12 markers were able to detect P. brassicae strains of each isolate or race. Ycheon14 markers was specific to isolates of race 2, Yeoncheon and Hoengseong. Ycheon9 and Ycheon10 markers were specific to Yeoncheon isolate (race 2, pathotype 3), ZJ1-3, ZJ1-4 and ZJ1-5 markers were specific to Haenam2 (race 4) isolate, ZJ1-35, ZJ1-40, ZJ1-41 and ZJ1-49 markers were specific to Hoengseong isolate and ZJ1-56 and ZJ1-64 markers were specific to Pyeongchang isolate (race 4, pathotype 3). The PCR-based sequence characterized amplified region (SCAR) markers developed in this study are able to detect five Korean isolates of P. brassicae. These markers can be utilized in identifying four Korean P. brassicae isolates from different regions. Additional effort is required to develop race- and isolate-specific markers for the remaining Korean isolates.

Development of Species-Specific SCAR Markers, Based on a SCoT Analysis, to Authenticate Physalis (Solanaceae) Species.

Physalis is an important genus in the Solanaceae family. It includes many species of significant medicinal value, edible value, and ornamental value. However, many Physalis species are easily confused because of their similar morphological traits, which hinder the utilization and protection of Physalis resources. Therefore, it is necessary to create fast, sensitive, and reliable methods for the Physalis species authentication. Intended for that, in this study, species-specific sequence-characterized amplified region (SCAR) markers were developed for accurate identification of the closely related Physalis species P. angulata, P. minima, P. pubescens, and P. alkekengi var. franchetii, based on a simple and novel marker system, start codon targeted (SCoT) marker. A total of 34 selected SCoT primers yielded 289 reliable SCoT loci, of which 265 were polymorphic. Four species-specific SCoT fragments (SCoT3-1404, SCoT3-1589, SCoT5-550, and SCoT36-520) from Physalis species were successfully identified, cloned, and sequenced. Based on these selected specific DNA fragments, four SCAR primers pairs were developed and named ST3KZ, ST3MSJ, ST5SJ, and ST36XSJ. PCR analysis of each of these primer pairs clearly demonstrated a specific amplified band in all samples of the target Physalis species, but no amplification was observed in other Physalis species. Therefore, the species-specific SCAR primer pairs developed in this study could be used as powerful tools that can rapidly, effectively, and reliably identify and differentiate Physalis species.

Diferenciación genética de tilapia roja y gris (Oreochromis niloticus) mediante microsatélites y marcadores SCAR como indicadores del sexo genético

El objetivo del trabajo fue realizar la estandarización de los protocolos moleculares, diferenciar los linajes de la tilapia roja y gris mediante microsatélites de ADN y evaluar los marcadores SCAR5F-X/5R y SCAR-5F/5R-Y como indicadores del sexo genético de O. niloticus. El microsatélite UNH106 permitió diferenciar genéticamente los linajes de la tilapia roja de la gris. Los microsatélites UNH136, UNH115 y UNH995 presentaron loci monomórficos tanto en la tilapia roja como en la gris en el lote poblacional del Centro Experimental de Genética de la Universidad Nacional de Trujillo. Se describen los tamaños de fragmentos para los microsatélites y del gen de referencia β actin. También se confirmó la efectividad de los marcadores SCAR como informativos en la determinación del sexo genético evaluados en hembras XX y YY y machos XY y YY de O. niloticus roja y hembras XX y machos XY de O. niloticus gris.

The aims of this work was to develop a standardized molecular protocols, to differentiate red and gray tilapia lineages using DNA microsatellites and to evaluate SCAR-5F-X/5R and SCAR-5F/5RY markers associated with the phenotypic sex of O. niloticus. The UNH106 microsatellite allowed differentiating genetically the lineages of red tilapia from gray. The markers UNH136, UNH115 and UNH995 presented monomorphic loci in both the red and gray tilapia in the population stock of the Centro Experimental de Genética of the Universidad Nacional de Trujillo. Fragment sizes for microsatellites and the reference gene β actin are described. The effectiveness of SCAR markers as informative in the determination of the genetic sex in XX and YY females and XY y YY males of red tilapia and XX females and XY males of gray tilapia was also confirmed.

Morphological and chemical characteristics of doubled haploids of flue-cured tobacco combining resistance to Thielaviopsis basicola and TSWV.

Thielaviopsis basicola and Tomato spotted wilt virus (TSWV) are the most important problems in a moderate climate zone. Previously obtained doubled haploids (DH) of F1 hybrids of the flue-cured line WGL3 resistant to Th. basicola and the dark-cured line PW-834 carrying RTSW-al gene provided the research material. Biological tests and SCAR markers linked with TSWV were applied to confirm resistance of DH. Lines combining resistance to TSWV and Th. basicola were evaluated for morphological and chemical characteristics. Most of DH were significantly shorter than parents but two lines, 31/A/2 and 31/B/3, were close to the flue-cured WGL3. Usually DH possessed fewer leaves while one of them 31/B/3, exceeded parental forms. The doubled haploids flowered later than their parents. The most negative effect was reduced area of mid-position leaves of DH. It might be explained by a recombination during microsporogenesis in F1, however the influence of 'Polalta'-derived RTSW-al gene cannot be excluded. Extensive line to line variation for nicotine and sugars content was not associated with the genes for TSWV and Th. basicola resistance. Biological tests and field performance of DH revealed potential to overcome the negative effect of coupling between the RSTV-al gene and genes responsible for the morphological deformations.

Sequence characterized amplified region marker as a tool for selection of high-artemisinin containing species of Artemisia.

Malaria is currently one of the most important causes of mortality in developing countries. High resistance to available antimalarial drugs has been reported frequently, thus it is crucial to focus on the discovery of new antimalarial drugs. Artemisinin, an effective antimalarial medication, is isolated from various Artemisia species. To identify the Artemisia species producing high quantity of artemisinin, eight species of Artemisia were screened with the genetic sequence characterized amplified region (SCAR) marker for higher quantity of artemisinin. The DNA band corresponding to SCAR marker was cloned into pGEM®-T Easy vector and sequenced. The content of artemisinin in tested species was also measured using high-performance liquid chromatography (HPLC) assay. The primers designed for high-artemisinin SCAR marker could amplify a specific band of approximately 1000 bp which was present in two Artemisia annua and Artemisia absinthium species. These SCAR marker sequences for two selected species were submitted into the GenBank databases under KC337116 and KC465952 accession numbers. HPLC analysis indicated that two selected Artemisia species, genetically recognized as high-artemisinin yielding plants, had higher artemisinin content in comparison to other examined species. Therefore, in this study, we propose developed SCAR marker as a complementary tool for confidently detection of high-artemisinin content in Artemisia species.

Homoeologous GSL-ELONG gene replacement for manipulation of aliphatic glucosinolates in Brassica rapa L. by marker assisted selection.

Aliphatic glucosinolates are the predominant sulfur-rich plant secondary metabolites in economically important Brassica crops. Glucosinolates and their hydrolysis products are involved in plant-microbe, plant-insect, plant-animal, and plant-human interactions. It is, therefore, important to manipulate glucosinolate profiles and contents in Brassica species. In this study, aliphatic glucosinolates were genetically manipulated through homoeologous recombination in backcross lines followed by marker assisted selection in B. rapa. A resynthesized B. napus line, from a cross between B. rapa and B. oleracea, was backcrossed with Chinese cabbage doubled haploid line, RI16. Marker assisted selection for non-functional gene was performed in each backcross generations. Advanced backcross progenies (BC3F2) were developed to identify homoeologous gene replacement and/or introgression. Reduction in 5C aliphatic glucosinolates (gluconapoleiferin, glucoalyssin, and glucobrassicanapin) was observed in BC3F2 progenies of the recurrent parent that carried the GSL-ELONG (-) gene. The GSL-ELONG (-) positive backcross progenies were also screened by the A-genome and BraGSL-ELONG gene specific marker, which linked with 5C aliphatic glucosinolates. The A-genome specific marker was absent in the plants of advanced backcross progenies which showed reduction in 5C aliphatic glucosinolates. The results suggest that the functional allele had been replaced by the non-functional GSL-ELONG (-) allele from B. oleracea. Some advanced backcross progenies (BC3F2) positive for the GSL-ELONG (-) allele and the A-genome specific SCAR marker BraMAM1-1 did not show reduction in 5C aliphatic glucosinolates, suggesting that GSL-ELONG (-) allele is recessive. Replacement of the functional locus in the A-genome by non-functional counterpart in the C-genome reduced the content of 5C aliphatic glucosinolates in B. rapa seeds with 20 µmol/g.

Effectiveness of combining resistance to Thielaviopsis basicola and Tomato spotted wilt virus in haploid tobacco genotypes.

Black root rot (BRR) caused by Thielaviopsis basicola as well as Tomato spotted wilt virus (TSWV) are the most serious problems in tobacco growing regions. We crossed the breeding line WGL 3 carrying BRR resistance derived from N.glauca with the line PW-834 the resistance of which to TSWV was transferred from cultivar Polalta. Anthers obtained from F(1) hybrid plants were cultured to induce haploids combining resistance to Th. basicola and TSWV. Flow cytometry analysis revealed 242 haploids and 2 spontaneous doubled haploids among regenerants. All haploids were cloned and then evaluated for BRR as well as TSWV resistance. The presence of pathogens was detected by microscopic evaluation of roots or using DAS-ELISA test. Microscopic assessment showed that, 132 haploids had no symptoms of Th. basicola which, together with the absence of symptoms in the F(1) hybrids, indicated a dominant monogenic mode of inheritance. At the same time only 30 haploids demonstrated resistance to TSWV. SCAR markers associated with TSWV resistance gene detection was applied. The results indicate that small proportion of TSWV-resistant haploids is probably due to the influence of deleterious genes flanking the resistance factor that reduced vitality of gametophytes. Altogether, 24 haploids showed multiple resistance to Th. basicola and TSWV.