Integrating Systems Biology with Marker Assisted Selection to Guide the Stacking of Powdery Mildew Resistance Genes

The long-term goal of this project is to develop grape varieties that possess effective and durable resistance to powdery mildew (PM). Stacking resistance genes from multiple resistant genetic backgrounds and with the least functional redundancy is a proven breeding strategy to improve both durability and level of resistance. This strategy requires (a) the identification of multiple sources of resistance, (b) the functional characterization of the mechanisms of resistance to prioritize optimal genetic combination, and, finally, (c) marker assisted breeding to introduce the selected genes into elite varieties.

In continuation of our multi-year breeding effort, with the awarded budget in the 2016-2017 funding period we have continued the functional characterization of resistance responses activated in presence of known powdery mildew resistance loci. The experiments for the functional characterization of Ren2, Ren3, and Ren4 were completed and data analysis is ongoing. We started the experiments for the functional characterization of Run1, Run1.1, Run2.1, and Run2.2. The manuscript describing the genetic analysis of Ren6 and Ren7 and the associated markers used for introgression and stacking was published in BMC Plant Biology (Pap et al, 2016).

Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods based on Next Generation Sequencing (NGS) technologies for detection, identification and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. In the 1st year of the project (2015 – 2016) we collected diseased wood material from commercial vineyards and characterized the associated fungal pathogen species using traditional methods, such as morphological and sequence-based identification of purified fungal colonies (see progress report for the 2015-2016 funding cycle). We used these samples to determine how effective ITS-sequencing, meta-genome sequencing and meta­transcriptome sequencing approaches are in identifying and quantifying pathogenic species directly in planta. Data simulations allowed us to determine what mapping algorithm was the most specific and sensitive in detecting trunk pathogens both qualitatively and quantitatively. All NGS methods we tested were in agreement with traditional diagnostic methods, but also allowed us to detect simultaneously multiple pathogen species with no need of hands-on sample culturing and colony purification. Additionally, unlike traditional diagnostics, which are strictly qualitative, NGS approaches allowed us to determine the relative abundances of the different infecting species. Among all methods tested, ITS-seq is still the most cost-effective until library preparation costs for RNA and DNA-seq do not decline significantly. For this reason, ITS-seq was chosen for further protocol optimization. Both sensitivity and specificity of the ITS-seq approach remain to be improved for diagnostics purposes. In the second year of the project (reported here), we (a) confirmed that NGS allows the detection with high specificity of actively infecting pathogens when vines are experimentally infected with individual pathogen strains; (b) established that NGS detection is quantitative and allows to differentiate between diseased and healthy vines; (c) developed a protocol for testing dormant cuttings and started testing cuttings provided by a commercial nursery. In the 2016-2017 funding cycle, we also developed a new DNA extraction protocol that reduced the time required for processing and the amounts of sample, reagents and waste.

Evaluating Grape Rootstocks for Nematode Susceptibility

Unfortunately the Phylloxera contamination could not be tolerated due to the risk that these pests could escape the microplots and become established at the Kearney Agricultural Center, and because Phylloxera could interact with plant-parasitic nematodes in damaging the vines, thus confounding tests designed to determine nematode resistance. To address these issues, all vines will be removed from their microplots, and the microplot soils will be steam-treated to kill any insect or insect eggs that may be remaining. After this sterilization procedure, the microplots will be prepared for replanting. New plant material will be collected and propagated, and the experiment re-initiated.

Breeding Rootstocks Resistant to Aggressive Root-Knot Nematodes

The USDA Agricultural Research Service (USDA-ARS) grape rootstock improvement program in Geneva, NY, has undergone significant changes in the past several years as a result of the resignation of Peter Cousins from his ARS rootstock breeder position and the abolishment of the vacated rootstock breeder position by ARS due to the federal budget cuts. However, a continued effort has been made to ensure that promising rootstock selections from previous years of breeding effort are maintained and carried through the evaluation process. To meet this challenge, a multi-discipline and –institution cooperative research team has been formed under encouragement and endorsement of the California grape industry. Matthew Fidelibus of University of California-Kearney Agricultural Research and Extension Center (UC-KARE) now leads the effort for evaluation of horticultural characteristics and graft performance of the rootstock selections generated from the Geneva rootstock breeding program. Gan-Yuan Zhong of the USDA-ARS Grape Genetics Research Unit (GGRU) in Geneva, NY, on the other hand, leads the effort for maintaining rootstock breeding populations and evaluating these populations and selections for root-knot nematode (RKN) resistance and propagation ability. The new team has been in operation since 2013 and demonstrated its success in evaluating more than 600 rootstock selections and identifying 5 leading rootstock selections for further grafting evaluation. During 2016-2017, the Geneva Team received $15,000 grant support for this project, which was only about 20{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of what was requested. The team maintained about 150 rootstock mothervines in Geneva, evaluated 22 for RKN resistance and propagation ability, and transferred 27 to  Matthew Fidelibus to grow at the UC KARE in Parlier, CA.

Grapevine Rootstock Selections

A team of academic, government, and industry partners developed a plan to evaluate rootstock selections from a USDA­ARS rootstock breeding program. In winter 2013-14, potential rootstock selections were assessed for desirable mother vine traits, including the production of abundant, well-matured canes of adequate diameter, length, and internode spacing, with minimal lateral shoot growth, powdery mildew scars, freeze damage, or fruit production. Based on these criteria, 240 vines having very poor traits were identified and discarded, and 30 selections with very good traits were prioritized for further evaluation. In spring 2014, cuttings from the high-priority selections were distributed to several academic and industry labs, where their rooting ability, nematode resistance, and virus status were tested. These tests narrowed the high priority list to six selections (PC0349-11, PC0349-30, PC04153-4, PC0597-13, PC0784-334, and PC0790-37) which rooted adequately and were resistant to aggressive strains of root-knot nematodes (RKNs). PC0790­37 tested positive for SyV-1 and RSP viruses, so it was forwarded to Foundation Plant Services for virus elimination; testing on that selection will resume after clean plant material becomes available. Most of the remaining selections were eliminated because they were insufficiently resistant to RKN, or they rooted poorly in both labs. Four additional rootstock selections (PC0333-5, PC0366-27, PC03155-47, and PC0495-51) were added to the high priority list based on the performance of Syrah when grafted to those stocks. In 2015, cuttings from the highest-priority selections were distributed to the cooperators to confirm rootability and RKN resistance. Based on those results, cuttings from six superior selections (PC0333-5, PC0349-30, PC0349-11, PC04153-4, PC0495-51, and PC0597-13) were judged worthy of advancing to field trials to assess their performance as grafted plants in commercial vineyards.

 

Deep Sequencing for Trunk Disease Diagnostics

The aim of this multi-year project is to develop rapid and cost-effective diagnostic methods for detection, identification and quantification of trunk pathogens in asymptomatic and symptomatic grape wood. In the 2015 -2016 grant cycle, we have tested different NGS-based methods for the detection of trunk pathogen in symptomatic infected field material and compared the results with traditional diagnostic approaches. All methods we tested were in agreement with traditional diagnostic methods. NGS-based methods also detected simultaneously multiple pathogen species with no need of tedious and hands-on sample culturing and colony purification. Additionally, unlike traditional qualitative diagnostics, deep sequencing also allowed to determine the relative abundance of the different species. Among all methods tested, ITS deep sequencing (ITS-seq) remains the most cost effective until library prep costs for DNA and RNA sequencing do not decline significantly. For this reason, ITS-seq will be the subject of protocol optimization of the second year of the project. Both sensitivity and specificity of the method need to be improved for diagnostics purposes. The results of this first phase of the project demonstrate that unlike traditional approaches NGS-based detection delivers rapid simultaneous identification and quantification of multiple species in infected tissue with no need of culturing and isolation.

Evaluation of Grapevine Rootstock Selections

A team of academic, government, and industry partners developed a plan to evaluate rootstock selections from a USDA­ARS rootstock breeding program. In winter 2013-14, potential rootstock selections were assessed for desirable mother vine traits, including the production of abundant, well-matured canes of adequate diameter, length, and internode spacing, with minimal lateral shoot growth, powdery mildew scars, freeze damage, or fruit production. Based on these criteria, 240 vines having very poor traits were identified and discarded, and 30 selections with very good traits were prioritized for further evaluation. In spring 2014, cuttings from the high-priority selections were distributed to several academic and industry labs, where their rooting ability, nematode resistance, and virus status were tested. These tests narrowed the high priority list to six selections (PC0349-11, PC0349-30, PC04153-4, PC0597-13, PC0784-334, and PC0790-37) which rooted adequately and were resistant to aggressive strains of root-knot nematodes (RKNs). PC0790­37 tested positive for SyV-1 and RSP viruses, so it was forwarded to Foundation Plant Services for virus elimination; testing on that selection will resume after clean plant material becomes available. Most of the remaining selections were eliminated because they were insufficiently resistant to RKN, or they rooted poorly in both labs. Four additional rootstock selections (PC0333-5, PC0366-27, PC03155-47, and PC0495-51) were added to the high priority list based on the performance of Syrah when grafted to those stocks. In 2015, cuttings from the highest-priority selections were distributed to the cooperators to confirm rootability and RKN resistance. Based on those results, cuttings from six superior selections (PC0333-5, PC0349-30, PC0349-11, PC04153-4, PC0495-51, and PC0597-13) were judged worthy of advancing to field trials to assess their performance as grafted plants in commercial vineyards.

 

Development of Next Generation Rootstocks for California Vineyards

2015 Screening of crosses for salt resistance – chloride exclusion in experimental hybrids – A sampling of individuals from 10 hybrid populations (Table 1) was screened for salt tolerance and chloride exclusion.  These hybrid populations were 1) crosses of wild genotypes earlier found to be strong chloride excluders, 2) crosses of commercial rootstocks, or 3) crosses of rootstocks to strong-excluding wild genotypes. Plants were assayed using components of the rapid screen method: herbaceous cuttings grown in fritted clay media in 1-gallon pots, and with a standard growth period prior to salt exposure. However, a higher (75 mM) NaCl solution was used rather than the standard 25 mM NaCl solution because of anticipated strong chloride exclusion derived from one or both parents and the accompanying need to distinguish between individuals using an unusually high chloride concentration. Parentage of hybrid groups is listed in Table 2.  Harvest date was based on the death of the most susceptible individuals, which in this case took nearly two months of high salt exposure.

Salt stress phenotypes at the time of harvest are presented in Figure 1. Although visual symptoms are less informative than chloride concentration in the leaves, which are currently being assayed, such symptoms do generally correlate with chloride concentration and so provide a rough preliminary estimate of results. Most notable from Figure 1 are the poor performance of all hybrids derived exclusively from commercial rootstocks: Ramsey x St. George and Dog Ridge x St. George, despite St. George being an established strong chloride excluder. This result implies that the seven wild genotypes used in this study are imparting a superior chloride exclusion relative to that found in commercial rootstocks. In support of this result is the very strong performance of V. girdiana x V. arizonica hybrids (Figure 1), several of which were completely asymptomatic. Surprisingly, V. vinifera cv. Thompson Seedless also performed relatively well. Several possibilities can account for this, including higher leaf succulence in Thompson Seedless that could mask the ordinary phenotypic effects of high chloride accumulation in the leaves, but the actual performance will not be known until the leaf chloride concentrations have been determined. It is also possible, though unlikely, that V. vinifera has weak chloride exclusion at low concentrations and strong chloride exclusion at high concentrations, perhaps in response to the high osmotic stress of 75 mM NaCl. Forthcoming data on leaf chloride concentration will provide a clarified and robust ranking of the genotypes in this study, and promises to provide direction for salt tolerance screening of hybrid populations in 2016.

2015 screening of seedling populations for resistance to nematodes and salt – Testing of recent seedling populations for nematode resistance (HarmA and HarmC, ring and dagger nematodes) and salt resistance continues. Progress here was held up a bit with the hiring of Becky Wheeler and departure of Liang Zheng. Liang retired with Howard Ferris last summer and we have been trying to ensure a smooth transition. We have completely revamped the greenhouse space for nematode screening and have well-established populations of three root-knot strains (HarmA, HarmC and Race3) and ring nematodes. We are also building our X. index populations too.

In 2015 we scored 41 populations (662 individuals) that were made to combine broad nematode resistance with salt and drought resistance, for horticultural appearance – lack of brushy growth, long internodes, long canes and good vigor. Of these 662 seedlings 18 were excellent and an additional 54 had good horticultural characters. These seedlings and others that passed horticultural screening this year were moved to rooting studies. Last year we tested 571 seedlings from crosses to combine broad nematode resistance with salt and drought resistance for rooting ability; 60 of these seedlings rooted well and will be advancing to nematode testing and root assays for depth and fibrosity

Breeding Rootstocks Resistant to Aggressive Root-Knot Nematodes

The USDA Agricultural Research Service (USDA-ARS) grape rootstock improvement program in Geneva, NY has undergone some significant changes in the past two years as a result of the resignation of Dr. Peter Cousins from his ARS rootstock breeder position and the abolishment of the vacated rootstock breeder position by ARS due to the federal budget cuts. However, a consistent effort has been made to ensure that promising rootstock selections from previous years of breeding effort are maintained and carried through the evaluation process. To meet this challenge, a multi-discipline and –institution cooperative research team has been formed under the encouragement and endorsement of the California grape industry. Matthew Fidelibus of University of California-Kearney Agricultural Research and Extension Center (UC-KARE) has lead the effort for evaluation of horticultural characteristics and graft performance of the rootstock selections generated from the Geneva rootstock breeding program. Gan-Yuan Zhong of the USDA-ARS Grape Genetics Research Unit (GGRU) in Geneva, NY, on the other hand, has lead the effort for creating rootstock breeding populations and evaluating these populations and advanced selections for root-knot nematode (RKN) resistance and propagation ability.

By forming such a team, we have effectively leveraged our complementary expertise and capabilities to meet the overall project objectives in a timely and efficient manner. The new team has been in operation since 2013. During the 2014-2015 funding year, we maintained several hundreds of resistant rootstock selections/mothervines at the UC KARE in Parlier, CA. With the help of nursery representatives, Andy Walker of UC-Davis, and others, we evaluated the horticultural characteristics of these rootstock mothervines and identified 30 of them for further evaluation. We evaluated the rooting ability and RKN resistance of the 30 selections and identified 6 with desirable rooting ability and resistance to RKNs. These 6 selections will be further evaluated in 2015-2016. During 2014-2015, we have also maintained about 200 rootstock mothervines in the USDA-ARS rootstock breeding program in Geneva, NY. These mothervines will need to be further evaluated and transferred to grow in UC KARE in Parlier, CA. We also screened a population for nematode resistance marker development. We continued the effort in evaluating 22 selections, grafted to Syrah, in replicated rootstock trials at the UC-KARE. 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.

 

Development of Next Generation Rootstocks for California Vineyards

2014 Pollinations – The 2014 crosses are presented in Table 1. They focused on combining PD resistant rootstocks with nematode resistance from arizonica forms with XiR1 X. index resistance and the GRN rootstocks; using excellent forms of chloride exclusion from Claire Heinitz’ work in crosses with GRN nematode resistance; using double chloride exclusion (shoot and root exclusion – most forms sequester chloride in the roots but prevent it from moving to the shoots, these prevent chloride from building up in the roots); combining drought resistance with chloride exclusion and GRN nematode resistance; combining deep rooting (Dog Ridge and 14uRu) with GRN nematode resistance; and combine vinifera x rotundifolia (VR) ring nematode resistance and potential for virus tolerance with GRN nematode resistance.

2014 Screening of Crosses for Nematode Resistance – Nina Romero and I walked about 1,100 of the 2010-2012 progeny and scored them for horticultural characteristics (cane length and brushiness and internode lengths. About 20{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of these progeny were advanced to rooting tests with ten 2-3 node cuttings. Those that rooted well and scored highly for horticultural characters were advanced to nematode testing against a combined inoculum of HarmA and HarmC (Harmony and Freedom aggressive root-knot nematode strains) and then against ring nematode. Unfortunately, not all selections were tested for both nematodes, but we have selections that will be tested to confirm either resistance. We also tested these selections for salt tolerance in a quick screen to select those with strong resistance and potential for breeding and selection as rootstocks Table 2 presents the best of the ring nematode resistant selections in comparison to nematode numbers and nemas/g or root obtained for O39-16, our highly resistant control. Plants were propagated by Nina and grown in 4 inch pots for testing. They were inoculated with 1,500 ring or 500 root-knot nematodes and evaluated for population development (ring) or egg masses (root-knot) after 3 months of growth. None were as highly resistant as either of our two standards the rotundifolia-based rootstocks O39-16 and GRN-1, but we will select the best in terms of rooting and root-knot and ring resistance to advance to further nematode testing against citrus and dagger nematodes. Table 3 and 4 present the results of testing with the combined HarmA/HarmC root-knot nematode inoculum. The breeding objective for Table 3 progeny was to improve the rooting of the GRN series (particularly GRN-5) and moderate vigor by crossing with 101-14Mgt. Thirty of these with egg mass / g of root data below 2 will be advanced to further testing. The selections tested in Table 4 were hoped to combine salt tolerance, deeper rooting and broad nematode resistance. Thirty-three of these will be advanced to salt and additional nematode testing. They include a broad range of resistance backgrounds and have good promise. Table 5 presents the parentage and number of selections that survived a severe salt screen that Nina devised. About 300 of the 1,100 we scored for horticultural characters and that rooted at 50{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} and above were tested for salt resistance by submerge them in 150 mM NaCl (about 30{aed9a53339cdfc54d53cc0c4af03c96668ab007d9c364a7466e3349a91bf0a23} of seawater) for 2 weeks to eliminate as many selections as possible prior to additional testing – 61 did not develop any salt burn symptoms, although they had reduced root and shoot growth; 2 or 30 V. rupestris selections from Missouri also passed this test. All of these 2 selections will be rested with our established screen to compare the effectiveness of this rapid screen. Selections that include the GRN rootstocks as parents will be advanced to screening against all the nematode strains.

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 some lines exhibited reduced susceptibility to X. index (Figure 1), but the transformed plants were still susceptible. There are more lines to test (Table 6) and we are examining gene expression with qPCR and will pursue native promoters to determine if they can increase resistance. Xiaoqing Xie and Cecilia Agüero have been producing green-grafted M. rotundifolia and GFLV infected Chardonnay plants to test resistance to the virus in different cultivars of M. rotundifolia. After initial success with Lucida and Trayshed (Figure 2), following experiments include five additional varieties and O39-16. Xiaoqing has also produced a number of tetraploid VR hybrids that we hope will be better able to hybridize with other rootstocks and allow us to introgress rotundifolia’s remarkable resistance, which is very difficult due to the differences in chromosome number (Table 7). The diploid and tetraploid forms of four VR genotypes have been established in the field for further analysis. Olmo was able to produce some fertile VR hybrids but because these are vinifera x rotundifolia some will susceptible to phylloxera. A new MS student Tarana Shaghazi is testing these to determine which have the best phylloxera and ring nematode resistance. Many of these were used in crosses in 2013, and a few were used in 2014, to provide breeding material if they have good phylloxera resistance. Cecilia Agüero is also conducting pre-bloom hormone treatments on clusters in the field to test the effect of candidate cytokinins on reducing fanleaf expression. These candidates were identified by our earlier studies of xylem constituents from O39-16 and associated with its ability to induce tolerance to fanleaf disease (Figure 3). O9-16’s potential to act as natural nematicide to X. index – Evan Goldman is finished his MS thesis on the ability of O39-16 to eliminate X. index from a vineyard. He sampled X. index numbers in a 22-year-old Oakville vineyard that as planted was a large replicated rootstock trial with 4 row x 50 vine blocks. He sampled over the season to compare X. index populations on O39-16, 110R and 3309C, the later two are susceptible to X. index.

His results are summarized in the abstract below from his MS. I am including Figures 4 and 5 from the June 2014 report. Potential to Eradicate Xiphinema index Using the Bioantagonistic Rootstock‘O39-16’ Evan Goldman MS Abstract. Abstract: Previous reproduction studies of Xiphinema index (the dagger nematode) on the grape rootstock ‘O39-16’ showed that populations decreased over time. In addition, the alternative host range of X. index is limited and does not seem to include many common vineyard weeds. This study was conducted to determine the most effective sampling method to recover X. index and to evaluate the possibility that the nematode can be eradicated over time from vineyards that have been planted with‘O39-16’ rootstock. Two sampling methods (shovel vs. Oakfield tube) were used, and the nematodes were extracted and identified. Pearson’s test determined that there was a poor correlation between the two methods and subsequent sampling used the shovel method. The populations of X. index and X. americanum on ‘O39-16’ were compared with adjacent populations on ‘3309C’ and ‘110R’ rootstocks, both susceptible to X. index feeding. Samples were collected from beneath drip emitters on three dates,and on each date the same drip zones were sampled. Nematodes were extracted and identified. Very few X. index were recovered from ‘O39-16’; most samples were devoid of X. index. Significantly fewer X. index were recovered from ‘O39-16’ than from either ‘3309C’ or ‘110R’. There was a tendency for ‘O39-16’ to have more X. americanum than either ‘3309C’ or ‘110R’, although the differences were usually not significant. To verify the absence of X. index on ‘O39-16’, soil pits were dug alongside previously sampled vines. Samples were collected at 25 cm, 50 cm, and 100 cm and nematodes wereextracted and identified. Although the differences were not significant, there was a trend for fewer 3 nematodes at increasing depths. In conclusion, the likelihood that X. index can be eradicated through the use of ‘O39-16’ is high. However, these results need to be verified in other vineyards, especially those planted solely on ‘O39-16’.