Unique black spot resistance gene from 'George Vancouver' is better characterized

Technique Embryo Culture
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Hi Everyone!

It has been a long path to get to this point. We learned that the black spot resistance gene in ‘George Vancouver’ is in a unique portion of the rose genome compared to the other characterized genes to date, we developed some DNA markers to test for Rdr3, and tested some additional roses to see which roses seem to share this resistance gene. Rosa disease resistance genes (Rdr) 1&2 are on linkage group/chromosome 1 and Rdr4 is on 5. This one is Rdr3 and is on chromosome 6. Having them on different chromosomes can make it easier to combine them through crossing parents each with a different resistance gene as the genes should segregate independently. Thankfully these genes are dominant and all we need is one copy of an allele of a gene to confer resistance.

Rdr3 confers resistance to a number of races of black spot, but not others. We originally characterized the gene in ‘George Vancouver’ building off of Dr. Vance Whitaker’s Ph.D. thesis completed over a decade ago. We had to recreate the cross of GV with ‘Morden Blush’ to get a population of seedlings again that we could characterize each seedling of for resistance. We used the currently popular and more powerful marker system called SNPs compared to what was available to Vance years ago to find were in the genome the resistance gene is located and find some DNA markers that were close/maybe within the gene to easily test other roses with to see if they share the same gene. We actually found 3 markers in that area of the genome that if present helps confirm the presence of this unique gene to help provide greater confidence.

This gene does not provide resistance to all races of black spot, but 9 of the 13 we have in the collection. This gene in combination with other genes would increasing provide resistance to more and more races in the overall collection. In fact, Rdr4 and Rdr3 together would theoretically confer resistance to all the races we currently have.

Here are rose cultivars that tested positive for Rdr3 and could be possible parents/sources for Rdr3 to try to include in ones breeding program- Caldwell Pink, Frontenac, Champlain, Folksinger, Louis Jolliet, Quadra, Rainbow Knock Out, Sunsprite, and William Booth.

It seems like there are three distinct sort of groups of roses here. Material from Svejda’s Explorer program, ‘Sunsprite’ (parent of ‘Folksinger’ and ‘Sunsprite’ is from Kordes), and also ‘Caldwell Pink’, which seems like it may be a setigera hybrid perhaps of some sort from its morphology.

It is interesting that L83 (mother of GV and many other Explorer roses) does not have Rdr3. There is an open pollinated seedling of L83 (called L83-1), which is a triploid and shared with be by a friend to test for ploidy. The male parent must have been the source of Rdr3 and it was also perhaps maybe diploid or triploid to generate a triploid seedling.

We have a few extra roses tested that are in the background of the above roses that have Rdr3 to try to better understand its source across pedigrees. We plan to continue this work and present some of that extra information in an article later this year in the American Rose.

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Really good news. I wonder where RKO got the resistance. Not apparent from its short-range family tree I suppose? The notes I have on that say RKO is the same parentage as (Radsweet) Alaska. and a half-sib to Brite Eyes. Namely Radtee x Radral. But Radral (Carefree Celebration) is Radorg x Rader which leads me to a dead end and I don’t know parentage of Radtee either. If Radsunny is Brite Eyes x Radsweet (Alaska), I can see how Sunny KO and Brite Eyes (Radtee x Abraham Darby ) are both resistant to the B.S. in my garden, as is RKO, if Radtee is the source of Rdr3. This is consistent with my observation that many RKO offspring, whether OP (self?) seedlings or crosses using its pollen, seem to have B.S. resistance too.

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Let’s put this in the RHA newsletter!
Stephen

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Stephen, I agree!

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Thank you for this, David.

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So, how exactly can we amateur breeders capitalize on this news, David? Obviously we can use the cultivars you lists as having RDR3 as breeders and try to combine them with roses that have the other types of resistance but I’m not able to tell from the abstract which other cultivars those might be. Is there a list somewhere of which cultivars have which type of resistance so we can build a crossing matrix?

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Good question Don. There isn’t a good complied list of roses for each Rdr. That would be great to develop as we use the markers developed for each gene to test more cultivars. This list for Rdr3 I suspect is the most extensive list for any of the Rdr genes to date as we surveyed a number of roses to validate the markers and also challenged the roses with race 8 to confirm the markers are associated with the resistance. For Rdr1 Love and PeaceTM (‘BAIpeace’) is a rose we can access and Brite EyesTM (‘RADbrite’) for Rdr4. Perhaps in Debener’s work with his colleagues, they’ve surveyed more roses for Rdr1 that we may have access to in the US.

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You can find some via google “rose rdrX resistance”

Rdr1 is often mentioned coming from multiflora

Rdr2 took me down a rabbit hole but I’m sure something specific is said but with the linkage may or may not be a similar list to Rdr1.

Something to look in to when I’m not on a train to work.

With Brite Eyes not being in Au, need to look into Rdr4 more for alternatives.

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Good point about R. multiflora. That is where Debener first got Rdr1 from and characterized it. They followed the gene through generations.
There was chromosome doubling along the way as they got the gene introgressed into some tetraploid modern rose hybrid genetics. Due to the pattern of infection it shared with Love and Peace (and maybe some other reasons), Vance suggested that they shared Rdr1. He spent 6 months in Debener’s lab during his Ph.D. The region of where Rdr1 is, is complicated. There seems to be multiple resistance gene patterns in tandem there and they weren’t sure which sequence is conferring the resistance. Recently Rouet et al, 2019 Identification of a polymorphism within the Rosa multiflora muRdr1A gene linked to resistance to multiple races of Diplocarpon rosae W. in tetraploid garden roses (Rosa × hybrida) | Semantic Scholar describe this region a bit more. I’m confused with Rdr2, but if I remember right it was within that region of tandem genes and I haven’t heard much since about it.

There are some offspring now of Brite EyesTM. I don’t think Sunny Knock Out inherited Rdr4 (we tested it for resistance to races 3, 8, and 9 and it was susceptible to 3, but Brites Eyes is resistant and it seems like there is just one key major gene resistance gene in Brite Eyes), but maybe its other seedlings Grand Champion Sunblush Rose or Milwaukee Calatrava inherited Rdr4?

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Possibly just asking the obvious…but if the genes are dominant and someone breeds a plant with both Rdr4 and Rdr3 they’d be able to breed it with something that gets a lot of blackspot, at the tetraploid level, getting an approx 1/6 inheritance for both genes and another 2/3 having at least 1 of the two genes?

So a spotty plant isn’t necessarily something to avoid like the plague, just need to pair it with something that’s resistant and select the seedlings that have whatever you want from the spotty parent and show resistance. Seems counter to the “cull any seedling with spots” mantra that floats around…maybe I’m missing something.

just did another punnet even if only 1 copy of either RdrX with the other parent doing nothing, it’s 18/36 (so 1/2) inheriting it…

Not that it super helps me, no Brite Eyes or Brite Eyes descendants here (or RADtee for that matter), hopefully it get’s searched for in other places.

Do have Sunsprite from the Rdr3 list though.

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That’s a great question Plazbo. Yes, that sounds like a good plan and what Will I think has done in his work in some of his lines. As he takes his healthier key parents that likely have multiple dominant genes and cross them with standard modern roses that for instance have unique colors (like to breed Orchid RomanceTM), it is possible to generate nice hybrids that inherit the resistance factors and bring in additional traits. There is another way to consider it as well. There are both vertical resistance (like what we are talking about here) and horizontal resistance (once a rose gets infected with black spot how well it slows its growth). Will’s Knock Out and Double Knock Out and their sports especially have really good horizontal resistance (as well as likely some vertical resistance/dominant single gene resistances even though it hasn’t been characterized because of some challenges to generate large numbers of offspring from them). With Will bringing home lots of black spot he was able to overcome the vertical resistance and then see black spot in many of his roses to build select for horizontal resistance over generations. When a rose like Knock Out/Double Knock Out comes out, most people don’t have the diversity of black spot races he has and the vertical genes hold, but when they are broken down, there is still strong horizontal resistance. That is a winning combination. When we cross and try to stack multiple vertical genes with roses without much horizontal resistance (or vertical resistance), the challenge is that if/when the vertical resistance genes are overcome, there is little horizontal resistance to hold back the black spot. FOr instance, ‘George Vancouver’ held up well in our area for awhile, but when a race came to the area that could overcome Rdr3, GV defoliates kind of fast. By combining more resistance factors, it makes it harder to have a race that is able to overcome both.

I suspect my Oso Easy Petit Pink has 2 vertical genes and Rdr3 may be one (GV is its grandfather). In crosses of it with a more typical rose that may not have any vertical resistance genes, it seems like about 25% of the seedlings stand up well to black spot in my plots. I think I have different races of black spot in the garden each able to overcome one of the genes, but not both. So, for each gene and assuming they are segregating independently and there is just one copy of the dominant allele of each gene- there is a 50% chance a gamete would inherit each of the one dominant allele copies. So, the chance for a gamete to carry and transmit both is (0.5)*(0.5) = .25 or 25% of the time.

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vertical vs horizontal that’s what I was missing.

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Any idea if “Pink Lady” aka “Alaska” has Rdr4? I believe it should has Rdr4 since it shares Radtee as a mother? Unless the resistance comes from Abraham Darby, which I doubt.

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Just from observations today and checking notes from previous years. Sunsprite isn’t resistant to at least one of the strains here in Sydney as it’s spotty now and has been in previous years so that’s a pity.

Thornless multiflora (grew from root stock) is entirely spotless. So may indicate Rdr1 and/or Rdr2 is of more value here in Au…possibly aligns well with Warren being in favour of Hybrid Musks for health given the multiflora heritage.

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We have a race here in MN/WI that overcomes Rdr3 (‘Sunsprite’ and ‘George Vancouver’) too and is very widespread now throughout the region. Years back I used ‘George Vancouver’ a lot, but then when the race swept through it and most of the seedlings went down pretty hard. For Rdr4 Brite EyesTM gets infected periodically in our region, but this race that gets it seems much less widespread. With races moving with commerce of roses, etc. they can come in and take hold quick and it is unpredictable which race(s) come and go. Baby Love and its descendants too have looked great in our region, but then came down pretty hard with black spot within a relatively short itme. Hopefully as we learn more about the resistance factors and strategically combine different resistance genes in single roses as well as work to elevate hortizontal resistance, breeders can develop roses with more longer term durability for black spot resistance. It feels like even though good progress has been made the past 20 years especially for more roses that stand up to black spot well, we are still pretty early in recognizing the genes and mechanisms of resistance.

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Is it possible that focusing TOO much on eliminating strains of BS, cerco, etc, we’re ignoring a larger good? It appears it will take decades to make significant progress on this while at the same time creating a commercially wonderful plant? Are we not losing sight of Sam McGredy’s urging that you should simply cross great roses with great roses? So, fine, Sunsprite is only partly resistant to BS strains but it has so many good qualities, is a proven female that passes on many good characteristics. If we simply crossed roses like Sunsprite with other fine, reasonably resistant roses, like my current fave here in the SF Bay Area Oso Easy Italian Ice (works well as a female, ZERO disease of any sort here but cerco in TX), and for broader value, roses rated tops for garden AND exhibition by ARS that have worked, especially as a female, plus ADR winners (e.g., Grand Amore–Haven’t used it yet), would we not make more progress and certainly progress in time for us to see more commercially introduceable roses, thus bringing more pleasure to thousands of people. After all, isn’t that our ultimate goal? My own bias is toward breeding for HT-form healthy-enough-not-to-need-spraying roses that would work in a windowbox–geranium competitor. In addition to Italian Ice, I’ve earlier had success with Happy Trails as a male. I’m planning to use Rainbow’s End (It’s a proven male. Is it a decent female?) I tried Grand AMore last year for the first time as a female and didn’t get takes. I may try a bit again and try it as a male. Any thoughts?

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Hi Marty,

I’m looking through my notes for roses I tried to do chromosome counts on. I want to redo Grand Amore to make sure, but out of what I could tell from the better spread cells it looked like Grand Amore was triploid. Maybe that could be contributing to it being a challenging female. I’d love to learn others experiences with it too. It seems like a nice hybrid tea. With moving a few years ago I lost my plant of it. I see there is some dormant bareroot plants of it at Home Depot and maybe I should get a new one.

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Is the cosmic scheme of things, the $10 is worth it triploid or not. What we need now, perhaps more than anything, is hope of a big win. It’s a little like betting $2 on a relatively long-shot horse: a little thrill at the possibility of a big win, and if you lose, no biggie. At minimum, if you can resist pollinating a couple, the cut bloom is a lovely gift.

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David, I love the work that you have done in characterizing black spot resistance genes. It is a great service to all of us!

Working somewhat “blind”, in the mix of black spot races, since we only get mild cases in the spring and fall in our climate, I have been adding cleaner roses to the breeding program over the years including many of Will Radler’s varieties. Do you know whether RADsweet (AKA ‘Alaska’) has the Rdr3 gene as suggested above? Also, does ‘Baby Love’ have one of the 3 genes that you mentioned?

One of my favorite parents, ‘Darlow’s Enigma’, seems to confer some black spot resistance. Since it is a Multiflora type, would it be a reasonable guess that it has the Rdr1 gene?

Your suggestion to “stack” resistance gene seems like an excellent strategy. Then, bringing in strongly horizontal resistant roses like KO and DKO into the mix would cover much of the concern with regard to black spot… Ultimately, crossing the resultant seedlings with more attractive roses and combining for a couple generations with each other, might get something very nice.

Of course all of this would be made “easy” with field testing kits that could detect markers in seedlings…

How far off are we from such tools?!

Jim Sproul

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Hi Jim!

Thank you!! It is fun to do and be a part of the learning process, even though how a person can fully implement and use certain technologies can be a bit up in the air for awhile as other tools are advanced too.

Jason Zurn and Nahla Bassil have taken the lead on the molecular components and were able to be part of this work because it was a subcomponent of a larger fruit project (their lab works with fruit). With that grant over now, it may be difficult for awhile to get more roses tested with these more expensive and complicated SNP markers. They are wonderful collaborators and have constraints by USDA for what they can/should work on.

I wish there was a simpler PCR test I could do in my lab and run on an agarose gel. Maybe we can try to work towards that for these genes… It would be fun to test a lot more roses to see what we have out there now containing Rdr3 at least and do so affordably. There is a local company that does do SNP testing. After SNP discovery and there are these DNA markers linked to genes of interest, it can be streamlined to test seedlings for just those of interest. They typically multiplex them so during the run you can test for several at the same time on the same seedling. This company works with agronomic breeders primarily. This approach is routinely done now in some apple and strawberry breeding programs. Vance Whitaker who got his Ph.D. with rose black spot runs the strawberry breeding program at the U of FL. They use several markers for fruit quality and disease resistance. I forgot the numbers exactly now, but I think they used to plant out maybe 4,000 seedlings per year. Now they germinate maybe 5 to 10x that many seedlings and do the SNP tests on young seedlings and throw out everything that doesn’t have the key genes of interest linked to these SNPs. They plant out the same number, but basically preselect and only plant out those that have the key market limiting traits the SNPs are linked to. I think they can do it affordably in house or with a company with all their volume and it saves a lot of effort ultimately to help make sure the plants have the key traits and then they can focus on trying to select for additional traits of value. They have the benefit of strawberries being an annual crop generally for FL growers and growers buying new plants each year for a royalty stream to pay for continued development and really good advances for growers. I should see what options there are for this local company to test our 3 different SNPs linked to Rdr3. It may not be as expensive as I suspect and we can test a whole group of roses. I think it may be $4-5 per sample if I remember right, but I’d have to buy a whole 96 plate if I filled each sample spot or not. If that is the case it may be doable to work towards one plate and 96 roses. We passed along DNA of our Brite EyesTM population we characterized for Rdr4 to a grad student at another institution to fine tune marker development and characterize that gene more. Hopefully they’ll get some really good SNP markers identified within or very close to the gene.

I think Baby Love’s gene is different than the others. It may be a unique allele of the previous genes or a different gene somewhere else in the genome. I say this because of the pattern of which races it is resistant to. Some of its offspring (including your seedling you sent us) has the same pattern. We generated a new population now (the seedlings are young and under lights now at home) to someday chase and try to understand and characterize what is in Baby Love.

That’s interesting Rdr1 was first characterized out of R. multiflora by Debener’s group. Some multiflora descendants (‘Sven’, ‘Lena’, ‘Ole’, Candy Oh! Vivid Red, etc.) all seem to have high horizontal resistance (all the races infect these, but there is very limited lesion growth). It would be fun to put ‘Darlow’s Enigma’ through the race array to see if it has strong horizontal resistance or some race specific resistance. I forgot the details why, but Vance suggested Love and Peace has Rdr1, so the gene seems to be in some more modern material too besides just multiflora it seems.

I put all the races into storage and closed up the lab a week or so ago because of the pandemic and limitations / stay at home orders. It was really sad to do that. We have had the races growing continually for 5-6 years now putting them on fresh leaves every 2 weeks and they were growing very strong. I took leaves with each of the 13 races and put them in baggies and put them in the standard -20C freezer and double checked that there were at least a half a dozen or so little vials of each race in liquid nitrogen. Hopefully we’ll be able to wake them up again after the pandemic and be able to continue the work.

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