Dr. Andy Walker; Unraveling an Olmo Mystery
In 1989, Dr. Harold Olmo gave Dr. Andy Walker a collection of grape seeds that he had produced the previous year. Walker believed they had the potential for resistance to both Pierces’ Disease (PD) and the dagger nematode (Xiphinema index) that vectors fanleaf virus. They were the offspring of two V. rupestris females and six M. rotundifolia males. The rotundifolia is known to confer resistance to PD, X. index and fanleaf virus. Walker’s lab began investigating the actual value of these rootstocks as parents in breeding new genotypes with effective resistance across a broad spectrum of applications, including rootstocks, raisin, table and winegrape production. For the next fifteen years, they tested and retested the populations, both in the laboratory and in actual field situations. The trials are continuing, but the results with the dagger nematode and PD are very promising. At least one big question has been answered, however; the high level of resistance is not coming from rotundifolia.
In initial testing, the rootstock crosses, called the 89 populations, were found to have very good resistance to phylloxera, a necessary attribute of any commercial rootstock. When they were tested for resistance to the root knot nematode, some were found also to have strong resistance to Meloidogyne incognita.
These seedlings were then tested for resistance to the dagger nematode. However, when the plants were tested, the resistance test results made no sense to the researchers; the actual segregation ratios were inconsistent with the expected resistant:susceptible ratios. Many of these selections were very resistant and were put into commercial rootstock trials across the state.
The next step was to cross siblings of the 89 populations, which created the 96 populations. When these “F2” progeny were tested, the segregation ratios implied that resistance to dagger nematode was controlled by a single gene. The researchers then began their studies on resistance to PD, caused by the bacterium Xylella fastidiosa (Xf), hoping that these dagger-nematode-resistant selections based on “rotundifolia” would also resist PD. If so, they could be crossed with high quality resistant vinifera table, raisin and wine grapes to form the basis of a PD breeding program. The 89 populations had strong resistance to PD and resistance segregated as a single locus in the same population being studied for dagger nematode resistance.
The researchers then began compiling a genetic map for resistance to both dagger nematode and PD in the 9621 population, a cross of two “siblings” from the 89 populations – D8909-15 x F8909-17. This project was initiated to create DNA markers capable of optimizing breeding and to launch an effort to find the genes responsible for resistance to Xi and PD. They first began mapping with AFLP DNA markers and made good progress positioning these resistance traits. But, AFLP markers do not establish parentage. All along, the researchers had encountered actual physical inconsistencies in these populations that nagged at them, leading them to question their source material. For instance, neither V. rupestris or M. rotundifolia have hair on their stems or leaves, yet some of the 89 progeny and the 9621 progeny had light amounts of hair on their leaves. And, very importantly, the progeny are fertile, even though crosses between Vitis species and rotundifolia should have 39 chromosomes and be sterile. But the progeny did have combined resistance to both Xf and Xi, which is a rotundifolia characteristic.
Dr. Summaira Riaz joined the Walker lab and began adding SSR markers to the study, and greatly increased the number of individuals in the 9621 map. SSR markers are capable of establishing parentages and were used to clarify the origin of Chardonnay and Cabernet Sauvignon by Dr. Carole Meredith. When they were added to the 9621 map, it became clear that there were important flaws in the original assumptions: the M. rotundifolia ‘Cowart’ was not the male parent and the two F2 progeny chosen for the sibling cross were not actually even siblings!!
The researchers found the original pollination records and checked with Dr. Olmo, but no further clues to the unknown male parents were uncovered. They looked at the original 89 progeny and found at least four different leaf types. In the spring they went back to the original, and now abandoned, field where the crosses were made. They found that most of the plants that were in bloom at the same time as the V. rupestris female vines and nearby were from a collection Dr. Olmo made during a trip to Mexico in 1961. At the UCD Herbarium, they found good records of the original Mexican collection. The pressed plants matched the living plants well. And they found Dr. Olmo’s trip journal.
They then tested a total of 85 Mexico collection genotypes and all the original rotundifolia parents of the 89 populations with 18 SSR markers to determine which if any may have inadvertently pollinated the rupestris females. The results found that many of these Mexican species were pollen parents of the 89 populations, rather than the rotundifolia parents that Dr. Olmo had attempted to use in the crosses. Many of the most resistant 89 progeny were in fact crossed to V. arizonica and its hybrids with V. candicans. There were a few of the intended V. rupestris x M. rotundifolia crosses, but the majority of the progeny were unintentional outcrosses. This is very important because these little-known, unintentional parents have the same level of disease resistance as the supposed parent, M. rotundifolia, a species with extreme resistance to Xi and PD. This leaves the researchers with an entirely new direction to pursue. They can focus on arizonica as a new source of disease resistance, and complete testing on the entire Mexican collection. These findings also imply that PD may have evolved with grape species in Mexico, since the resistance is so strong there, and that nematode resistance in these species needs to be further studied.
Nature can still have her way, sometimes in spite of our own best efforts. It may take her a little longer, but she has good sense………. The researchers now have DNA markers for both Xiphinema and PD resistance from V. arizonica, and the markers will be utilized in the breeding program this year, improving its efficiency.
Dr. Walker and his staff still have a lot more work to do. Their current focus includes determining whether rootstocks with high dagger nematode resistance can prevent infection by the fanleaf virus that the nematodes vector; whether the DNA markers they have identified function in the entire range of Xi and PD resistant species; and whether the genes controlling resistance to Xi and PD can be identified and characterized. Progress continues on the classical breeding of PD-resistant raisin, table and wine grapes and on nematode resistant rootstocks. The eventual hope is to transform grape varieties and rootstocks once the genes controlling resistance are identified. Meanwhile, the Walker lab continues to benefit from Dr. Olmo’s past intentional and unintentional efforts.