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Establishment of a wild rocket (Diplotaxis tenuifolia) germplasm collection and its preliminary assessment for downy mildew resistance and genetic diversity

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Abstract

Rocket is a popular baby leaf vegetable from Brassicaceae family native from Mediterranean region appreciated for its organoleptic characteristics. Wild rocket (Diplotaxis tenuifolia L.) is characterized by distinct lobate leaves with a pungent aroma and typical bitter and spicy taste due to the presence of flavonoids and glucosinolates. In the last two decades this crop has been largely diffused and its economic relevance is increasing rapidly worldwide. Downy mildew is a rocket devastating disease, especially in temperate and humid climate regions. Caused by the oomycete Hyaloperonospora sp. this disease reduces drastically the quality and yield of the rocket leaves and, in multiple cases, leads to the complete annihilation of the crop. The severe legislation adopted in Europe on the use and registration of new pesticides, and the general environmental concerns, imply the need to develop new production strategies, less dependent on chemical products. The REMIRUCULA project (ref. PTDC/ASP-PLA/28963/2017) aims to improve rocket downy mildew resistance and to contribute for its sustainable production, assuring a healthier and safer product and environmental protection. The project consortium involves three scientific and academic organizations, INIAV, UAlg, and ITQB/NOVA, working in close commitment with the baby leaf producer Vitacress. The project integrates four main scientific tasks: 1) Establishment of collections of wild rocket germplasm and Hyaloperonospora sp. isolates; 2) Characterization of the rocket collection regarding the response to downy mildew infection under controlled environment and field conditions; 3) Biomolecular characterization of wild rocket accessions and Hyaloperonospora sp. isolates; and 4) Metabolomic characterization of rocket accessions with different downy mildew responses. Here we communicate the first preliminary results regarding the establishment of the wild rocket germplasm collection and the characterization of the (over 100) accessions regarding their response to mildew infection and molecular features.
ESTABLISHMENT OF A WILD ROCKET (Diplotaxis tenuifolia) GERMPLASM
COLLECTION AND ITS PRELIMINARY ASSESSMENT FOR DOWNY MILDEW
RESISTANCE AND GENETIC DIVERSITY
Coelho, PS1; Carranca, C1; Scotti, P1; Semedo, J1; Loureiro, MF1; Pais, I1;
Gaspar, C2; Lopes, V2; António, C3; Diogo, V4; Leitão, J4
1INIAV, IP, Av. República, 2780-157 Oeiras, Portugal; 2BPGV/INIAV, S. Pedro de Merelim, 4700-859 Braga, Portugal;
3ITQB/NOVA, Av. República, 2780-157 Oeiras, Portugal; 4UAlg/FCT, Campus de Gambelas, 8005-139 Faro, Portugal
INTRODUCTION
RESULTS
Fig.1. Composition of the rocket
germplasm collection.
Biomolecular Characterization
Wild Rocket (Diplotaxis tenuifolia L.) is a baby leaf from Brassicaceae family, and its economic
relevance is increasing rapidly worldwide. Downy mildew (DM) caused by the oomycete
Hyaloperonospora sp. is a devastating disease, that reduces drastically the quality and yield of
the rocket leaves.
The aims of the project correspond to 4 scientific tasks: 1) Establishment of collections of
wild rocket germplasm and Hyaloperonospora sp. isolates; 2) Characterization of the rocket
collection regarding the response to DM infection under controlled and field conditions;
3) Biomolecular characterization of wild rocket and Hyaloperonospora sp. isolates; and
4) Metabolomic characterization of rocket with different DM responses.
Fig. 2. Preliminary evaluation of resistance response to downy mildew in rocket cotyledons, 1st and 2nd leaves accessions.
The accessions were divided in 4 groups according to DM resistance response: R=Resistant, PR=Partially Resistant,
S=Susceptible, and HS=Highly Susceptible.
106 accessions were tested: Seed Banks=13, Breeding Companies=81, Commercial Varieties=3, and Eruca sp.=9.
Plants were inoculated by spraying with the Hyaloperonospora sp. isolate D5 at 15d, and were evaluated at 21d (Fig. 2).
Cotyledons were more susceptible to DM than the 1st and 2nd leaves. Diplotaxis sp. showed different cotyledon and
leaves responses: 2acc. were cotyledon Resistant (from a Seed Bank and Breeding Company) and maintained a R leaf
response; 7acc. were cotyledon Partially Resistant and had a PR/R leaf response; 18 acc. were cotyledon Susceptible
and had a S/PR 1st leaf response; and 70 acc. were cotyledon Highly Susceptible, of which only 1 acc. was R (from a
Seed Bank), 11 acc. were PR, 39 acc. were S, and 19 acc. maintained the HS response on leaves (Fig. 2).
All Eruca sp. (control) cotyledon and leaves inoculated with isolate D5 were Resistant.
Genomic DNA was, so far, extracted from 40 D. tenuifolia accessions. A higher quantity of genomic DNA was extracted
from previously partially purified cell nuclei of a single plant and sent for next generation sequencing using an Illumina
platform.
The bioinformatics analysis of the genomic and transcriptomic data available online (ncbi.nlm.nih.gov) allowed the
identification of 20 microsatellite loci among the uploaded Diplotaxis cDNA sequences (genomic data are still very scanty)
and 18 genomic microsatellite loci of Hyaloperonospora sp.
Primers were obtained for all these loci and the respective microsatellite markers are presently being validated.
Hyaloperonospora sp. sporulation
on infected wild rocket (21d).
The first results regard the Establishment of the germplasm collection (Fig. 1), and the characterization of the
accessions to their Response to DM (Fig. 2) and Molecular features.
CONCLUSION
In most accessions at 21 days evaluation, the 2nd leaf was very young and hence very resistant. The increase of
DM susceptibility with leaf aging was observed in other Brassica, such as cabbage, broccoli and radish.
Downy Mildew Response
This work is funded by national funds through the FCT - Foundation for Science and
Technology, I.P., in the context of the project «Refª PTDC/ASP-PLA/28963/2017».
cot.
cot.
1st leaf
2nd leaf
... A doença do míldio na rúcula selvagem, causado pelo oomiceta Hyaloperonospora sp., é uma ameaça crescente à produção em regiões de clima temperado e húmido [13] e conduz a perdas elevadas de produtividade e qualidade das folhas (Diplotaxis tenuifolia (L.) DC.), sendo normalmente a rúcula cultivada (Eruca sativa (L.) Cav.) mais resistente ao míldio. O potencial da resistência varietal no controlo ao míldio foi demonstrado em plantas jovens de Diplotaxis e Eruca testadas em condições de crescimento controladas [14,15] . O género Hyaloperonospora reúne mais de 100 espécies que afetam culturas economicamente importantes da família das Brassicáceas. ...
Technical Report
Full-text available
A rúcula (Diplotaxis spp. e Eruca spp.) é uma hortícola de folhas “baby-leaf” muito apreciada pelas suas características organoléticas e benefícios para a saúde. Pertence à família das Brassicáceas e a rúcula selvagem (Diplotaxis tenuifolia (L.) DC.) é a mais apreciada pelosconsumidores. Nas últimas décadas, tem tido uma grande expansão e, atualmente, apresenta uma relevância económica crescente a nível mundial devido à utilização na indústria de saladas minimamente processadas. Apesar de ser uma cultura bem-adaptada ao clima mediterrânico, a sua produção intensiva, ao ar livre ou em estufa, favorece a ocorrência de míldio. Esta doença causa perdas de produção e prejuízos avultados, pelo que a identificação de variedades resistentes à infeção e a adoção de práticas agronómicas sustentáveis são importantes para o aumento de rendimento dos produtores.
... A doença do míldio na rúcula selvagem, causado pelo oomiceta Hyaloperonospora sp., é uma ameaça crescente à produção em regiões de clima temperado e húmido [13] e conduz a perdas elevadas de produtividade e qualidade das folhas (Diplotaxis tenuifolia (L.) DC.), sendo normalmente a rúcula cultivada (Eruca sativa (L.) Cav.) mais resistente ao míldio. O potencial da resistência varietal no controlo ao míldio foi demonstrado em plantas jovens de Diplotaxis e Eruca testadas em condições de crescimento controladas [14,15] . O género Hyaloperonospora reúne mais de 100 espécies que afetam culturas economicamente importantes da família das Brassicáceas. ...
Article
Full-text available
A rúcula (Diplotaxis spp. e Eruca spp.) é uma hortícola de folhas “baby-leaf” muito apreciada pelas suas características organoléticas e benefícios para a saúde. Pertence à família das Brassicáceas e a rúcula selvagem (Diplotaxis tenuifolia (L.) DC.) é a mais apreciada pelos consumidores. Nas últimas décadas, tem tido uma grande expansão e, atualmente, apresenta uma relevância económica crescente a nível mundial devido à utilização na indústria de saladas minimamente processadas. Apesar de ser uma cultura bem-adaptada ao clima mediterrânico, a sua produção intensiva, ao ar livre ou em estufa, favorece a ocorrência de míldio. Esta doença causa perdas de produção e prejuízos avultados, pelo que a identificação de variedades resistentes à infeção e a adoção de práticas agronómicas sustentáveis são importantes para o aumento de rendimento dos produtores.
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