Source: https://www.avf.org/category/rootstocks/breeding-rootstocks/
Timestamp: 2019-04-26 04:31:23+00:00

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Recent developments in molecular technology have led to significant improvements in detection and control of many pathogens. The use of those techniques for pathogen detection in quarantine and certification programs has not yet been universally accepted. This is primarily because of the need to validate these techniques and determine their limitations. We are proposing to supply the data for that validation for the case of grapevine registration and certification. We will make a side-by-side comparison of 1) the classical, currently used technique for the analysis of viral pathogens of grapevine, with 2) the more recently developed technology of Next Generation. Sequencing (NGS). In both cases, we will test the same set of fifty selected grapevine accessions infected with one or multiple viruses of importance to the grape industry. In the first case, viral pathogens will be analyzed using biological assays on a standard herbaceous and woody index panel of host plants, as is required by APHIS and CDFA for certification. We will compare the results from that bioassay with a second analysis based on NGS of the total grapevine viruses in each of the selected accessions. The two tests will run concurrently. We expect to show that, for the evaluation of the disease status of grapevine stocks, NGS is superior to biological assay, as well as to ELISA, RT-PCR and real time RT-qPCR in sensitivity, reliability, speed and labor intensity, and cost. We will make the case for the replacement of biological assays with NGS for the certification of novel grapevine accessions. Our data will be useful to federal and state regulatory agencies as evidence supporting the revision of the existing mandated protocols for the testing and release of novel grapevine accessions from quarantine. The improvements brought with the up-date to NGS technology for this application will be of significant benefit to the grape growing industry.
The first year objectives of this project have been met. We have identified the first batch of 20 grapevine accessions that carry infections of agronomic importance, for use in the comparative demonstration of the effectiveness of the two techniques evaluated in this project. We have chipbud grafted material from each of those to the standard four bioassay index hosts, and begun their two year incubation period toward symptom scoring. We have also made total RNA extractions from those infected plants and begun NGS analysis, using BLAST sequence comparisons to subtract the host coded sequences from those of the pathogens of interest. This progress will generate the data for the comparisons between the techniques, which will meet the subsequent objectives of this proposal.
The 2013 crosses focused on developing rootstocks with deeper root systems, the genetics of root architecture traits, and introgressing the excellent soil pest resistance from rotundifolia into rootstocks using semi-fertile vinifera x rotundifolia (VR) hybrids (see Table 1). This may also be a way to incorporate fanleaf tolerance and allow improvement of O39-16. VR hybrids are normally sterile but a few were selected by Olmo to have some fertility. Unfortunately they are also crosses with vinifera so we must be assured of their phylloxera resistance (studies underway).
GRN Field Trials – This was the first year data was gathered from GRN rootstock trials; most of which are being overseen by farm advisers and Constellation. We took crop yields at a trial in Dunnigan with Franzia and another in Lodi with Gallo. This data will be combined with pruning weights (not yet taken) and presented with the next report and as a bulletin to nurseries and cooperators.
Nematode testing – We work closely with Howard Ferris and his technician to evaluate the nematode resistance of rootstock breeding populations. Nin Romero (my chief greenhouse and field technician) propagated and assisted with the nematode resistance screening of hundreds of seedlings this year. Nina and I first examined the populations and evaluated them for brushy growth, internode length, and vigor. Most were also evaluated for their ability to root from dormant cuttings. They were tested for resistance to the Harmony/Freedom aggressive root-knot strains (HarmA and HarmC) and Xiphinema index, and many were also screened for ring nematode resistance. The best 21 are shown in Table 2 and will be advanced to field testing on the UC Davis campus with 101-14 and 1103P comparison controls.
Fanleaf – We continue to make progress on identifying and verifying the function of the Xiphinema index resistance gene from V. arizonica b42-26, and it resistance locus XiR1. Two gene candidates are members of the NB-LRR (nucleotide binding-leucine rich repeat) resistance gene family that control recognition of pests and diseases and the triggering of a defense reaction. These two candidates were transformed into St. George and Thompson Seedless and they reduced susceptibility to X. index resistance, but the transformed plants were still susceptible. There are more lines to test and we are examining gene expression with qPCR and will pursue native promoters to determine if they can increase resistance.
The USDA Agricultural Research Service grape rootstock improvement program, based at the Grape Genetics Research Unit, is breeding rootstocks resistant to aggressive root-knot nematodes. We define aggressive root-knot nematodes as those which feed on and damage the rootstocks Freedom and Harmony. We screened 5212 candidate grape rootstock seedlings (representing 34 different populations) for resistance to aggressive root-knot nematodes. We select only those seedlings which completely suppress nematode reproduction and show zero nematode egg masses. The nematode resistance evaluation total includes 494 seedlings of a genetic study population that also are qualified for consideration as rootstocks. Selected seedlings are propagated and then planted into the vineyard. We tested the propagation ability of 123 selections (already tested once for nematode resistance). We evaluated 22 selections, grafted to Syrah, in replicated rootstock trials at the University of California Kearney Agricultural Research and Extension center. We pollinated 412 clusters of crosses in 48 unique combinations specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes and collected 33,530 rootstock cross seeds. Matador, Minotaur, and Kingfisher rootstocks, released by this USDA ARS grape rootstock breeding program in 2010, are being distributed by Foundation Plant Services and planted by California nurseries.
We made more crosses to increase the number of Muscadinia rotundifolia-based progeny by crossing 101-14 Mgt, 161-49C (V. berlandieri x V. riparia) and 5BB with M. rotundifolia Trayshed pollen to create populations with broad pest resistance and the ability to induce fanleaf tolerance in scions. We have over 200 progeny in the 101-14 x Trayshed population and many are undergoing rooting and horticultural screening this Winter. We have screened subsets of the population for resistance to dagger, root-knot and ring nematodes; they segregate for dagger and root-knot resistance and all resist ring nematodes. To further our study of fanleaf tolerance, we also collected xylem sap from stronger progeny in the 101-14 x Trashed population. We will screen these saps with targeted metabolites from our fanleaf tolerance investigations. We also made crosses to produce PD resistant rootstocks with good nematode resistance and good rooting; better drought and salt tolerance; and crosses to make virus tolerant rootstocks. Cecilia Agüero successfully made constructs of two of the XiR1, Xiphinema index resistance genes we characterized from V. arizonica/girdiana b42-26 (Hwang et al. 2010. Theoretical and Applied Genetics 121:780-799). She has transformed them into St. George and Thompson Seedless, and into tomato (a host of X. index and very easy to transform and study). We should be able to inoculate these plants with X. index later this year to test the function of these gene candidates. This project is primarily funded by the American Vineyard Foundation, and it also supports our efforts to determine which metabolites are responsible for O39-16?s fanleaf tolerance. We will be testing for presence and levels of five metabolites and two cytokinins in saps from O39-16 and about 20 individuals from the 101-14 x Trayshed population. Kevin Fort?s excellent work on the mechanisms of, and screening for, salt tolerance are coming to fruition and four publications are ready for submission. He is now working as a post-doc on generous funding from E&J Gallo who funded a joint project between Andrew McElrone and myself. Kevin is studying interactions between drought and salinity, and working out experiments to accurately screen drought tolerance and to better understand root architecture. He is helping to supervise Claire Heinitz who is studying the eco-genetics of salt tolerance in southwestern Vitis, and Cecilia Osorio who is studying the anatomical basis of drought tolerance. Jean Dodson is also working on drought adaptation by studying the influence of rootstocks on phenological events such as root senescence, leaf drop and harvest dates, which will greatly impact vine water use. Karl Lund is analyzing the feeding behavior of eight phylloxera strains on the root tips of 11 rootstocks and Vitis species. He has also initiated the screening of a V. vinifera x V. berlandieri 9031 mapping population. This V. berlandieri accession has excellent phylloxera resistance and the progeny segregates for resistance, which allows the development of a genetic map for this source of resistance.
The USDA grape rootstock improvement program, based at the Grape Genetics Research Unit, is breeding grape rootstocks resistant to aggressive root-knot nematodes. We define aggressive root-knot nematodes as those which feed on and damage the rootstocks Freedom and Harmony. In 2009 we screened 5126 candidate grape rootstock seedlings (representing 69 different populations) for resistance to aggressive root-knot nematodes. We select only those seedlings which completely suppress nematode reproduction and show zero nematode egg masses. Selected seedlings are propagated and then planted into the vineyard. We screened an additional 420 seedlings for nematode resistance genetics studies. We tested the propagation ability of 190 selections (already tested once for nematode resistance) and of these retested 80 selections to confirm nematode resistance in replicated trials. We planted eleven selections, grafted to Syrah, into a new rootstock trial at the University of California Kearney Ag Center and identified eleven more selections to be grafted to Syrah for a rootstock trial to be planted in 2010. We pollinated 317 clusters of crosses in 55 unique combinations specifically aimed at the breeding of improved rootstocks with resistance to aggressive root-knot nematodes. Virus testing is complete for our most elite selections and several of these are candidates for possible variety release.

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