Heterodera Schachtii Schm Research Articles

BvcZR3 and BvHs1pro-1 Genes Pyramiding Enhanced Beet Cyst Nematode (Heterodera schachtii Schm.) Resistance in Oilseed Rape (Brassica napus L.).

Beet cyst nematode (Heterodera schachtii Schm.) is one of the most damaging pests in sugar beet growing areas around the world. The Hs1pro-1 and cZR3 genes confer resistance to the beet cyst nematode, and both were cloned from sugar beet translocation line (A906001). The translocation line carried the locus from B. procumbens chromosome 1 including Hs1pro-1 gene and resistance gene analogs (RGA), which confer resistance to Heterodera schachtii. In this research, BvHs1pro-1 and BvcZR3 genes were transferred into oilseed rape to obtain different transgenic lines by A. tumefaciens mediated transformation method. The cZR3Hs1pro-1 gene was pyramided into the same plants by crossing homozygous cZR3 and Hs1pro-1 plants to identify the function and interaction of cZR3 and Hs1pro-1 genes. In vitro and in vivo cyst nematode resistance tests showed that cZR3 and Hs1pro-1 plants could be infested by beet cyst nematode (BCN) juveniles, however a large fraction of penetrated nematode juveniles was not able to develop normally and stagnated in roots of transgenic plants, consequently resulting in a significant reduction in the number of developed nematode females. A higher efficiency in inhibition of nematode females was observed in plants expressing pyramiding genes than in those only expressing a single gene. Molecular analysis demonstrated that BvHs1pro-1 and BvcZR3 gene expressions in oilseed rape constitutively activated transcription of plant-defense related genes such as NPR1 (non-expresser of PR1), SGT1b (enhanced disease resistance 1) and RAR1 (suppressor of the G2 allele of skp1). Transcript of NPR1 gene in transgenic cZR3 and Hs1pro-1 plants were slightly up-regulated, while its expression was considerably enhanced in cZR3Hs1pro-1 gene pyramiding plants. The expression of EDS1 gene did not change significantly among transgenic cZR3, Hs1pro-1 and cZR3Hs1pro-1 gene pyramiding plants and wild type. The expression of SGT1b gene was slightly up-regulated in transgenic cZR3 and Hs1pro-1 plants compared with the wild type, however, its expression was not changed in cZR3Hs1pro-1 gene pyramiding plant and had no interaction effect. RAR1 gene expression was significantly up-regulated in transgenic cZR3 and cZR3Hs1pro-1 genes pyramiding plants, but almost no expression was found in Hs1pro-1 transgenic plants. These results show that nematode resistance genes from sugar beet were functional in oilseed rape and conferred BCN resistance by activation of a CC-NBS-LRR R gene mediated resistance response. The gene pyramiding had enhanced resistance, thus offering a novel approach for the BCN control by preventing the propagation of BCN in oilseed rape. The transgenic oilseed rape could be used as a trap crop to offer an alternative method for beet cyst nematode control.

Rozwój populacji mątwika burakowego (Heterodera schachtii Schm.) w glebie i plonowanie buraka cukrowego w następstwie uprawy odmian o zróżnicowanej tolerancji na nicienia

Rozwój populacji mątwika burakowego (Heterodera schachtii Schm.) w glebie i plonowanie buraka cukrowego w następstwie uprawy odmian o zróżnicowanej tolerancji na nicienia

Genetic behavior of resistance to the beet cyst nematode (Heterodera schachtii Schm) in radish (Raphanus sativus L.)

The genetic nature of resistance to the beet cyst nematode (BCN) (Heterodera schachtiiSchm) in oil radish was investigated. A segregating population consisted of 245 F2individuals derived from self-pollinated single F1 plant of the radish cross ‘Pegletta X Siletta nova’ was used. Four oil radish populations and one Brassica napus cv. “Madora” as susceptible control were examined for the resistance to the BCN. The number of cysts found on the root system of radish plants and the attached soil were used for resistance criteria. The results showed that all F1 plants appeared in the resistance parent classes, which reflected the dominant behavior of the resistant gene(s). A bimodal distribution was observed for the F2 population. The F2 individuals distributed in two discreet phenotype classes, complying with the assumption of monogenic dominant inheritance of the BCN resistance in oil radish. Key words: Genetic behavior, Raphanus sativus, dominant gene(s), Heterodera schachtiiSchm.

Identification of a putative cation transporter gene from sugar beet ( Beta vulgaris L.) by DDRT-PCR closely linked to the beet cyst nematode resistance gene Hs1pro-1

The beet cyst nematode ( Heterodera schachtii Schm.) is a widespread pest that causes agronomic losses in sugar beet cultivation. Since no nematode resistance genes have been found in Beta vulgaris L. cultivars, a chromosomal fragment from Beta procumbens has been bred into cultivated sugar beet in order to provide resistance to Heterodera schachtii. The introgressed translocation carries the nematode resistance gene Hs1 pro-1 and may also contain genes that are linked to the defense response and/or signal transduction. A novel cDNA designated Bp23 was isolated from the translocation in this work by comparing resistant and susceptible sugar beets of a segregating F 2 population by differential display. Bp23 shows similarity to Arabidopsis thaliana BAC clones and to diverse plant ESTs with similarity to the Escherichia coli cation transporter ChaC. The open reading frame could encode a hydrophilic protein of 25 kDa that is similar to cation transporters and has a presumptive K + selective motif at the N-terminus.

Sporamin-mediated resistance to beet cyst nematodes (Heterodera schachtii Schm.) is dependent on trypsin inhibitory activity in sugar beet (Beta vulgaris L.) hairy roots.

Sporamin, a sweet potato tuberous storage protein, is a Kunitz-type trypsin inhibitor. Its capability of conferring insect-resistance on transgenic tobacco and cauliflower has been confirmed. To test its potential as an anti-feedant for the beet cyst nematode (Heterodera schachtii Schm.), the sporamin gene SpTI-1 was introduced into sugar beet (Beta vulgaris L.) by Agrobacterium rhizogenes-mediated transformation. Twelve different hairy root clones expressing sporamin were selected for studying nematode development. Of these, 8 hairy root clones were found to show significant efficiency in inhibiting the growth and development of the female nematodes whereas 4 root clones did not show any inhibitory effects even though the SpTI-1 gene was regularly expressed in all of the tested hairy roots as revealed by northern and western analyses. Inhibition of nematode development correlated with trypsin inhibitor activity but not with the amount of sporamin expressed in hairy roots. These data demonstrate that the trypsin inhibitor activity is the critical factor for inhibiting growth and development of cyst nematodes in sugar beet hairy roots expressing the sporamin gene. Hence, the sweet potato sporamin can be used as a new and effective anti-feedant for controlling cyst nematodes offering an alternative strategy for establishing nematode resistance in crops.

Painting of parental chromatin in Beta hybrids by multi-colour fluorescent in situ hybridization.

Sugar beet (Beta vulgaris L.) is a relatively young crop and has a narrow gene pool. In order to introduce genetic variability into the crop, interspecific hybrids, selected from crosses with wild beets of the sections Corollinae and Procumbentes, have been generated. The introgressed B. procumbens chromatin carries resistance genes to beet cyst nematode Heterodera schachtii Schm. These lines are important for breeding of nematode-resistant sugar beet, while Corollinae species are potential donors of tolerance to biotic and abiotic stresses such as drought or saline soils. We have used in situ hybridization of genomic DNA to discriminate the parental chromosomes in these interspecific hybrids. Suppression of cross-hybridization by blocking DNA was not necessary indicating that the investigated Beta genomes contain sufficient species-specific DNA enabling the unequivocal determination of the genomic composition of the hybrids. Interspecific hybrid lines with an additional chromosome (2n = 18 + 1), chromosome fragment (2n = 18 + fragment) or translocation of B. procumbens (2n = 18) were analysed by genomic in situ hybridization (GISH) at mitosis and meiosis. Species-specific satellites and ribosomal genes used in combination with genomic DNA or in rehybridization experiments served as landmark probes for chromosome identification in hybrid genomes. The detection of a B. procumbens translocation of approx. I Mbp demonstrated the sensitivity and resolution of GISH and showed that this approach is a powerful method in genome analysis projects of the genus Beta.

Genetic localization of four genes for nematode (Heterodera schachtii Schm.) resistance in sugar beet (Beta vulgaris L.)

Sugar beet (Beta vulgaris L.) is highly susceptible to the beet cyst nematode (Heterodera schachtii Schm.). Three resistance genes originating from the wild beets B. procumbens (Hs1 (pro-1)) and B. webbiana (Hs1 (web-1), Hs2 (web-7)) have been transferred to sugar beet via species hybridization. We describe the genetic localization of the nematode resistance genes in four different sugar beet lines using segregating F2 populations and RFLP markers from our current sugar beet linkage map. The mapping studies yielded a surprising result. Although the four parental lines carrying the wild beet translocations were not related to each other, the four genes mapped to the same locus in sugar beet independent of the original translocation event. Close linkage (0-4.6 cM) was found with marker loci at one end of linkage group IV. In two populations, RFLP loci showed segregation distortion due to gametic selection. For the first time, the non-randomness of the translocation process promoting gene transfer from the wild beet to the sugar beet is demonstrated. The data suggest that the resistance genes were incorporated into the sugar beet chromosomes by non-allelic homologous recombination. The finding that the different resistance genes are allelic will have major implications on future attempts to breed sugar beet combining the different resistance genes.

Physical mapping and cloning of a translocation in sugar beet (Beta vulgaris L) carrying a gene for nematode (Heterodera schachtii) resistance from B. procumbens

Two diploid (2n=18) sugar beet (Beta vulgaris L.) lines which carry monogenic traits for nematode (Heterodera schachtii Schm.) resistance located on translocations from the wild beet species Beta procumbens were investigated. Short interspersed repetitive DNA elements exclusively hybridizing with wild beet DNA were found to be dispersed around the translocations. The banding pattern as revealed by genomic Southern hybridization was highly conserved among translocation lines of different origins indicating that the translocations are not affected by recombination events with sugar beet chromosomes. Physical mapping revealed that the entire translocation is represented by a single Sal I fragment 300 kb in size. A representative YAC (yeast artifical chromosome) library consisting of approximately 13,000 recombinant clones (2.2 genome equivalents) with insert sizes ranging between 50 and 450 kb and an average of 130kb has been constructed from the resistant line A906001. Three recombinant YACs were isolated from this library using the wild beet-specific repetitive elements as probes for screening. Colinearity between YAC inserts and donor DNA was confirmed by DNA fingerprinting utilizing these repetitive probes. The YACs were arranged into two contigs with a total size of 215 kb; these represent a minimum of 72% of the translocation.

Long-range organization of a satellite DNA family flanking the beet cyst nematode resistance locus (Hs1) on chromosome-1 of B. patellaris and B. procumbens

New members of a satellite DNA family (Sat 121), specific for wild beets of the section Procumbentes of the genus Beta, were isolated. Sequence analysis showed that the members of Sat-121 fall into two distinct classes. The organization of Sat-121 in the vicinity of a beet cyst nematode (Heterodera schachtii Schm.) resistance locus (Hs1) in B. patellaris and B. procumbens was investigated by pulsed-field gel electrophoresis (PFGE) using DNA from a series of resistant monosomic fragment additions, each containing an extra chromosome fragment of B. patellaris chromosome-1 (pat-1) in B. vulgaris. In this way several clusters of Sat-121 flanking the Hs1 (pat-1) locus were identified. In nematode resistant diploid introgressions (2n=18), which contain small segments of B. procumbens chromosome-1 (pro-1) in B. vulgaris, only two major Sat-121 clusters were detected near the Hs1 (pro-1) locus.

Transfer of resistance to the beet cyst nematode (Heterodera Schachtii Schm.) from Sinapis alba L. (white mustard) to the Brassica napus L. gene pool by means of sexual and somatic hybridization

Sexual and somatic hybrid plants have been produced between Sinapis alba L. (white mustard) and Brassica napus L. (oil-seed rape), with the aim to transfer resistance to the beet cyst nematode Heterodera schachtii Schm. (BCN) from white mustard into the oil-seed rape gene pool. Only crosses between diploid accessions of S. alba (2n = 24, Sa1Sa1) as the pistillate parent and several B. napus accessions (2n = 38, AACC) yielded hybrid plants with 31 chromosomes. Crosses between tetraploid accessions of S. alba (2n = 48, Sa1Sa1Sa1Sa1) and B. napus were unsuccessful. Somatic hybrid plants were also obtained between a diploid accession of S. alba and B. napus. These hybrids were mitotically unstable, the number of chromosomes ranging from 56 to more than 90. Analysis of total DNA using a pea rDNA probe confirmed the hybrid nature of the sexual hybrids, whereas for the somatic hybrids a pattern identical to that of B. napus was obtained. Using chloroplast (cp) and mitochondrial (mt) DNA sequences, we found that all of the sexual F1 hybrids and somatic hybrids contained cpDNA and mtDNA of the S. alba parent. No recombinant mtDNA or cpDNA pattern was observed. Three BC1 plants were obtained when sexual hybrids were back-crossed with B. napus. Backcrossing of somatic hybrids with B. napus was not successful. Three sexual hybrids and one BC1 plant, the latter obtained from a cross between a sexual hybrid and B. napus, were found to show a high level of BCN resistance. The level of BCN resistance of the somatic hybrids was in general high, but varied between cuttings from the same plant. Results from cytological studies of chromosome association at meiotic metaphase I in the sexual hybrids suggest partial homology between chromosomes of the AC and Sa1 genomes and thus their potential for gene exchange.

Random Amplified Polymorphic DNA (RAPD) Markers in Sugar Beet (Beta vulgaris L.): Mapping the Genes for Nematode Resistance and Hypocotyl Colour

AbstractThe random amplified polymorphic DNA (RAPD) technique was adapted for segregation analysis in sugar beet. 83 IO/I individuals were scored with a set of 20 arbitrary decamer primers. 4 preliminary linkage groups could be established, enclosing 9 RAPD markers, 2 isozyme loci, a gene for the hypocotyl colour and a gene for resistance to the root knot nematode (Heterodera schachtii Schm.).

Transfer of resistance to the beet cyst nematode (Heterodera schachtii Schm.) into the Brassica napus L. gene pool through intergeneric somatic hybridization with Raphanus sativus L.

An intergeneric somatic hybrid was obtained through PEG-induced protoplast fusion between Brassica napus L. (oil-seed rape, AACC, 2n=38) and a beet cyst nematode resistant genotype of Raphanus sativus L. (fodder radish, RR, 2n=18). The hybrid nature of the regenerated plant was confirmed by flow cytometric analysis, RFLP-analysis, and chromosome counts. Southern blot analysis of total DNA using pPhcPS1 (rbc-L) as probe indicated that the somatic hybrid contains chloroplasts of B. napus. The mitochondrial genome of the somatic hybrid was studied more extensively using several probes and restriction enzymes. The results indicate inter- or intraspecific mitochondrial DNA recombination. Resistance to the beet cyst nematode (Heterodera schachtii Schm., BCN) was expressed in the hybrid at a high level.

DNA markers closely linked to nematode resistance genes in sugar beet (Beta vulgaris L.) mapped using chromosome additions and translocations originating from wild beets of the Procumbentes section.

Genes conferring resistance to the beet cyst nematode (Heterodera schachtii Schm.) have been transferred to sugar beet (Beta vulgaris L.) from three wild species of the Procumbentes section using monosomic addition and translocation lines, because no meiotic recombination occurs between chromosomes of cultured and wild species. In the course of a project to isolate the nematode resistance genes by strategies of reverse genetics, probes were cloned from DNA of a fragmented B. procumbens chromosome carrying a resistance gene, which had been isolated by pulsed-field gel electrophoresis. One probe (pRK643) hybridized with a short dispersed repetitive DNA element, which was found only in wild beets, and thus may be used as a molecular marker for nematode resistance to progeneis of monosomic addition lines segregating resistant and susceptible individuals. Additional probes for the resistance gene region were obtained with a polymerase chain reaction (PCR)-based strategy using repetitive primers to amplify DNA located between repetitive elements. One of these probes established the existence of at least six different chromosomes from wild beet species, each conferring resistance independently of the others. A strict correlation between the length of the wild beet chromatin introduced in fragment addition and translocation lines and the repeat copy number has been used physically to map the region conferring resistance to a chromosome segment of 0.5-3 Mb.

Association of a nematode resistance bearing addition chromosome with a recurring leaf intumescence somaclonal variation in sugar beet

Intumescent leaf variants of sugar beet (Beta vulgaris L.) were obtained through callus culture of a monosomic addition that carried resistance to Heterodera schachtii Schm. The frothy pockmarked appearance of the leaf surface was due to hyperplastic growth of the mesophyll and epidermal cells. The epidermis had many malformed stomata. Veins were underdeveloped, but protrusions beneath were pronounced. Intumescence occurred in 20.3% of the regenerated plants and it was heritable to F1 and later progeny. Leaf intumescence is a new phenotype for Beta. About 73.5% of regenerants contained the donor somatic chromosome number, the remainder were doubled or mixoploids, with no chromosome losses apparent. The 38-chromosome intumescent plant represents a dual somaclonal variation, chromosome doubling and leaf intumescence. Progeny of the 19- and 38-chromosome intumescent plants intercrossed or pollinated by diploids or tetraploids had 9, 18, 19, 27, 28, 29, 36, 37, 38, or 39 chromosomes. All intumescent plants were aneuploids with the monosome addition. There were linkages for leaf intumescence (Li), resistance to H. schachtii (Hs), and hypocotyl color (Rpro) on the addition chromosome. The efficacy of Hs remained intact through the in vitro culture and succeeding crosses. The Li-bearing plants manifested depressed growth and markedly reduced seed set. Leaf intumescence was thought to be the alternative expression of galling potential of Beta procumbens Chr. Sm. germ plasm.Key words: somaclonal variation, leaf intumescence, nematode resistance, monosomic addition, Beta vulgaris L.

Sugar beets homozygous for resistance to beet cyst nematode (Heterodera schachtii Schm.), developed from monosomic additions of Beta procumbens to B. vulgaris

Monosomic additions of Beta vulgaris x B. procumbens with resistance to beet cyst nematode (Heterodera schachtii Schm.) were used for the production of resistant diploids through incorporation of the B. procumbens chromosome fragment bearing the resistance gene(s) into one of the sugar beet chromosomes. The heterozygotes obtained accordingly were selfed for producing homozygotes. These homozygotes differed morphologically from commercial sugar beet varieties, but produced reasonable amounts of pollen. Female transmission of resistance was 100%, whereas male transmission, apart from some exceptions, was more than 90%. The number of hypersensitivity reactions to penetrated larvae was related to the degree of susceptibility. Larval development was severely retarded in the resistant plants, preventing most of them to produce cysts. If cysts were formed, their content was considerably less as compared to those in the susceptible plants.

Reproduction of the beet cyst nematode Heterodera schachtii Schm. on transformed root cultures of Beta vulgaris L.

Transformed hairy root cultures of Beta vulgaris L., grown in petri-dishes, were inoculated with a suspension of surface-sterilized larvae of the beet cyst nematode Heterodera schachtii Schm. Larvae developed into both male and female adults. Juvenile larvae were hatched from the newly-formed cysts, indicating that fertilization had occurred. Results of a glasshouse test showed that H. schachtii did not lose its pathogenicity after being cultured on transformed roots. This technique can be developed further for the mass-propagation of sedentary nematodes and for the in vitro storage of isclates.

Resistance to Heterodera schachtii in patellares section of the genus Beta

Fifty-two accessions of the section Patellares wild beet (including 26 Beta patellarisMoq., 13 B. procumbensChr. Sm and 13 B. webbianaMoq.) and 14 progeny families were selected and tested against sugarbeet cyst nematode, Heterodera schachtiiSchm. All Patellares species tested were highly resistant, but not immune, to the development of H. schachtii, after infection. This is the first report that mature female nematodes developed in the roots of B. webbiana plants. The occasional development of nematode cysts in roots of juvenile wild beets was, however, not a heritable genetic factor.

Transfer of resistance to beet cyst nematode from Beta patellaris to sugar beet

The chromosome bearing the gene(s) for resistance to the beet cyst nematode (Heterodera schachtii Schm.), could be transferred from Beta patellaris to B. vulgaris by interspecific hybridization and repeated backcrossing. A tetraploidized B. vulgaris originating from Anatolia (Turkey) was used as an intermediate. After several backcrosses with a number of different tetraploid and diploid beet cultivars, monosomic additions could be obtained which had the resistance bearing chromosome of B. patellaris. Cytological techniques were used for the study of this chromosome in resistant hybrids and monosomic additions. The analysis of pairing behaviour of the B. patellaris chromosome was hampered by the occurrence of non-specific stickiness of the chromosomes in meiocytes at pachytene and diakinesis. Moreover, the nature of resistance in monosomic additions was studied in growth cabinet experiments, revealing that the penetration rates of the larvae into the root system did not differ from the susceptible control plants. Among the F1B6 and F1B7 backcross derivatives, no plants with intermediate levels of resistance were found.

INHERITANCE OF RESISTANCE TO HETERODERA SCHACHTII AND CHROMOSOME SEGREGATION IN TRIPLOID SUGAR BEETS

Reciprocal crosses of autotriploid nematode (Heterodera schachtii Schm.)-resistant sugar beet (Beta vulgaris L.) 6122 and diploid susceptible plants resulted in low seed set and germination. Chromosome numbers of the progeny of 6122 ranged from 18 to 38, with the majority of the progeny in 18 and 19 chromosome classes. Inheritance of nematode resistance through 6122 as female parent was 24.8% and that of the reciprocal was 5.1%. Plant 6122 probably bears only one dose of the resistance factor(s). The resistance has little effect on the survival of aneuploid gametes. The original triploid zygote was probably developed from fertilization of a first division restitution diploid megaspore bearing nematode resistance fertilized by a haploid gamete. At pachytene, trivalent associations of 6122 were not intact, with the occurrence of intercalary and terminal desynapses, heteromorphic pairings, and inversion loops. On an average 7.24III, 7.71III, and 0.09III were observed at metaphase I in microsporocytes of 6122, the autotriploid control, and triploid interspecific 4x B. vulgaris × 2x B. procumbens Chr. Sm. F1 hybrids, respectively. Meiotic irregularities occurred in over 19% of 6122 cells at AI. At TII up to six micronuclei, two AII bridges, carry-over AI bridges, and single and double restitution nuclei occurred.

Sugar Beets Homozygous for Nematode Resistance and Transmission of Resistance to their Progenies1

Heterozygous, cyst nematode (Heterodera schachtii Schm.)‐resistant, self‐compatible sugar beet (Beta vulgaris L.) plants were self‐pollinated in the greenhouse; self‐incompatible plants were interpollinated; and nematode‐susceptable plants derived from resistant parents were sib‐crossed in isolation chambers. Among the 184 S2 and F3 families from resistant parents, five were 100% nematode‐resistant. These were the first homozygous resistant families to be reported. The results indicated that 2.7% of the resistant S1 and F2 plants and only 1.1% of all plants, resistant and susceptible, were homozygous for nematode resistance. The remainder of the resistant S1 plants produced approximately 41.8% resistant progeny. Plants from these true‐breeding families transmitted resistance to 98.4% of their progeny in the following generation. The remaining 1.6% were moderately resistant. Two supposedly S2 progeny plants from a resistant homozygote transmitted resistance to less than 50% of their self‐pollinated progeny. These plants probably resulted from outcrosses.These findings suggested that resistant homozygotes as seed parents gave full transmission of nematode resistance. Resistant heterozygotes derived from testcrosses to these homozygotes acquired little enhancement in rate of transmission of resistance. Nematode resistance is probably conditioned primarily by complementary genes. In parental line breeding and development after the F1 generation, it would be necessary to use progeny testing to identify true‐breeding individuals. However, nematode‐resistant commercial hybrids could be developed when only one of the parental components is homozygous resistant.