Phytophthora cinnamomi: screening trials, progeny tests, and genetics of resistance

Transcription

1 Phytophthora cinnamomi: screening trials, progeny tests, and genetics of resistance P.H. Sisco TACF Annual Meeting Portland, ME 6 Oct 2017

2 Phytophthora root rot (ink disease) Primary pathogen is a soil-borne Oomycete Phytophthora cinnamomi Rands The North American and European Castanea species are susceptible, while the Asian species are resistant

3 Definitions Screen: Expose plants to the pathogen and subsequently plant in the field the ones that meet pre-determined criteria (e.g., survive one year, do not wilt, have less damage to their root systems). Progeny Test: Evaluate trees for resistance by screening their progeny for disease resistance according to the pre-determined criteria. Screened progeny are usually discarded after the test. Parents whose progeny pass the test are retained. The key metric is the family mean. Genetic Analysis: Carefully label and evaluate each seedling of a progeny set, extract DNA, and look for correlations between chromosome segments and resistance to the pathogen according to the pre-determined criteria.

4 As long as resistance can be inherited from the Asian ancestors of our BC families (i.e., is heritable), we can screen for resistance or progeny test with minimal knowledge of the genetics of resistance. However, the greater the environmental influence on disease resistance, the more difficult it is to successfully screen or progeny test. Genotype x Environment = Phenotype (appearance)

5 Timeline Year Hebard Frampton Genetics Kubisiak - Olukolu Genetics TZ Genetics Westbrook- Holliday Genetics James - Jeffers BC screening and progeny tests Craddock BC screening and progeny tests James - Jeffers B3F3 progeny tests Frampton B3F3 progeny Tests USFS Bent Creek B3F3 progeny tests

6 James/Jeffers Screening for Resistance to P. cinnamomi Since 2004: Phytophthora root rot phenotyping (PRRtest) of hybrid chestnut families at the Chestnut Return Farm in Seneca, SC by Steve Jeffers, Joe James, Paul Sisco, along with colleagues and volunteers April: Seed are planted outside in plastic tubs with a soilless container mix; plants are grouped by families and replicates distributed randomly in several tubs. Pure American and Chinese chestnut controls in each tub. planting in April June: Seedlings are counted, labeled, and leaf samples are taken 2 months old plants, June July: Inoculation with two isolates of P. cinnamomi September: Counts of dead vs. living seedlings December or January: Phenotyping of roots of fully dormant plants for severity of root rot symptoms First symptoms after inoculation Photos by P. Sisco and S. Jeffers

7 James/Jeffers Screening for Resistance to P. cinnamomi A team effort

8 Plants in December Ready for Evaluation

9 James/Jeffers Screening: Evaluation Plants evaluated in late Dec or early January Each plant dug & scored individually 0 = healthy, no visible lesions on roots 1 = root rot on lateral roots 2 = root rot on tap root or severe lateral root necrosis 3 = plant dead Survivors transplanted into pots for 1 yr and then moved to the orchard primarily plants rated 0 & 1 evaluated for durable resistance in the field

10 Phenotyping based on severity of root rot symptoms (established by Steve Jeffers and Joe James) Scoring lesions on dormant roots (December-January) Photos by S.N. Jeffers

11 Major Result of James/Jeffers Screening Resistance was found mostly in families with Graves/Mahogany background

12 Chapter contributions to James/Jeffers Screening Chapter Number of Families Sent for Screening AL 2 CC 64 GA 9 IN 5 KY 9 MA/RI 31 + Chinese controls (Mirick) MD 19 ME 1 MO 1 VT/NH 2 PA Chapter 28 PA Leffel 13 TN 12 VA Chapter 6 VA Meadowview 23 BC + > 50 B3F3 families

13 Data tabulated over the years P. cinnamomi resistance screen Chestnut Return Plantation Test Results Evaluation of Survivors PRRS Score Family Code Generatio n of seed tested Source of Resistance Year tested Family Line Tested Sent by Notes No. Plants 3 dead no lesions % Survival at end of 1st yr HYKO B3 Mahogany 2005 Hyko x JB575 JB575 PA controlled pollination % SB1 B3 Mahogany 2005 Boston SB1 x JB575 JB575 MA controlled pollination % SUDA B3 Graves 2005 Sudbury x AB247 AB247 MA controlled pollination % UXB B3 Graves 2005 Uxbridge x GL356 GL356 MA controlled pollination % SUDB B3 Graves 2005 Sudbury x BE138 BE138 MA controlled pollination % FITCH B3 Clapper 2005 Fitchburg x GL96 GL96 MA controlled pollination % AND B3 Mahogany 2005 Andover x BX39 BX39 MA controlled pollination % PRNT Chin Princeton, MA, Chinese 2006 Princeton, MA (John Mirick) Princeton, MA, Chinese MA OP % Gry B3 Mahogany 2006 Franklin EMC x B2214 B2214 MA controlled pollination % RN88 B2 Douglas Hybrid 2006 Rn88-38 x Hind Douglas B1 PA PA controlled pollination % Frye B4 Graves 2006 Frye Mtn. x B3176 B3176 ME controlled pollination % Nwt B4 Clapper 2006 Newton CS19 x HE416 HE416 MA controlled pollination % UxB B3 Graves 2006 Uxbridge KD-1 x GL356 GL356 MA controlled pollination % VKS B4 Clapper 2006 VKS x CB582 CB582 IN controlled pollination % Pke B3 Clapper 2006 Pike Co Marinero x GL367 GL367 KY controlled pollination %

14 Screening was a Progeny Test of BC 2 and BC 3 parents Project to test Graves/Mahogany trees for PRR resistance -- Results as of Feb 2015 BC3 tree BC2 tree BC1 tree Line Code Gr or Mah MA49 GL94 Graves BH2A8G Graves Color Coding MA64 GL443 Graves BH2D1G2 Graves tested - resistant MA63 GL443 Graves BH2D1G2 Graves infer resistance MA97 GL443 Graves BH2D1G2 Graves tested - susceptible GA34 GL30 Graves BH2D2G2 Graves infer susceptibility MD34 GL30 Graves BH2D2G2 Graves SC1 GL185 Graves Bu3F2G Graves SC329 GL185 Graves Bu3F2G Graves GL356 Graves Bu3F3G Graves GB110 Be134 Graves Bu3F3G Graves DV130 Be52 Graves Bu3F3G Graves GB269 Be395 Graves Bu3F4G Graves MA9 (repl) WV167 Graves Bu3F5G Graves SC267 VA183 Graves BH2D1G Graves SC926 VA183 Graves BH2D1G Graves MD15 GL187 Graves BH2D3G Graves

15 Hill Craddock and Taylor Perkins set up screening at UT-Chattanooga 2015

16 Screening for resistance to P. cinnamomi is much easier than screening for blight resistance Seedlings can be screened their first year Many seedlings can be screened in a small space Relatively less environmental influence on resistance compared to blight So far all isolates of P. cinnamomi tested have given the same results

17 2012: Progeny tests of B 3 F 3 families from Meadowview at James farm Question from the TACF Staff: Which B 3 F 2 trees at the Duncan (Clapper) and Wagner (Graves) farms had at least some resistance to P. cinnamomi? Joe James planted half in tubs and half in a new field plot, hoping to develop a seed orchard with resistance to both chestnut blight and P. cinnamomi

18 2014: Progeny tests of B 3 F 3 families from Meadowview at NCSU Question from the TACF Staff: Which B 3 F 2 trees at the Duncan (Clapper) and Wagner (Graves) farms had at least some resistance to P. cinnamomi? Seedlings planted in individual pots and inoculated with twice with P. cinnamomi on rice grains

19 Screening for resistance to Phytophthora cinnamomi Chestnut Return/Clemson NCSU Blocked tub inoculations V8-vermiculite inoculum Chestnut Return isolates of P. cinnamomi Single tree inoculations Rice grain inoculum Frasier fir isolates of P. cinnamomi Evaluate below ground symptoms: root rot severity and mortality Measure above ground symptoms: % crown wilt and mortality

20 Rate of progeny testing BC3-F2 mothers Source Clapper Graves Total N BC3 F3 families tested ChestnutReturn NCSU Year of inoculation N BC3-F3 families tested as of Graves 33 Clapper 161 Graves 106 Clapper

21 Heritability of stem wilt and mortality after inoculation with P. cinnamomi greater for Graves than for Clapper trait Stem 8 weeks Stem 16 weeks 8 weeks 16 weeks h2 ± 1 SE Clapper Graves

22 Similar results in James/Jeffers trials: Resistance highly heritable in Graves, weakly heritable in Clapper trait Mortality Root Infection Rating h2 ± 1 SE Clapper Graves

23 Long upper tail of resistance among Graves BC3-F2s Scaled breeding values PRR resistance BC3-F3 hybrids are expected to inherit only ~ 4% of their genome from Chinese chestnut. Suggests major effect loci control resistance to P. cinnamomi in Graves American chestnut Clapper B3 F2s Graves B3 F2s Chinese chestnut

24 Long upper tail of resistance in Graves also observed in Chestnut Return Trials Scaled breeding values PRR resistance American chestnut Clapper B3 F2s Graves B3 F2s Chinese chestnut

25 Strong genetic correlation in root rot mortality in NCSU vs. James/Jeffers trials PRR mortality BLUPs NCSU Source Clapper r genetic = 0.95 ± 0.03 Graves 53 BC3-F3 families screened at both locations (44 Graves & 9 Clapper) Resistance screening at Chestnut Return/Clemson marginally more lethal PRR mortality BLUPs Chestnut Return

26 Resistance to Cryphonectria parasitica and Phytophthora cinnamomi uncorrelated PRR mortality BLUPs Progeny of 103 Graves BC3-F2 mothers screened for resistance to chestnut blight and Phytophthora root rot Genetic correlation PRR mortality v. chestnut blight canker size = ± Blight canker severity BLUPs

27 Progeny tests on a larger scale at USDA Forest Service s Bent Creek Greenhouse in Asheville, NC

28 Water from Phytophthora screening captured and sterilized

29 Potential Problem How many of our B 3 F 2 seed orchard trees will have adequate resistance to both chestnut blight and Phytophthora cinnamomi? In genetic terms, how large will be the Effective Population Size? Will the population of trees we reintroduce be genetically diverse enough to prevent harmful inbreeding in the future?

30 Combining blight and PRR resistance: breeding PRR resistant selections Intercross to expand effective population size and increase PRR resistance Cross with blight resistant transgenic tree Select for PRR resistance and deploy 2 generations of breeding and selection

31 Summary of results of studies on the genetics of resistance to P. cinnamomi

32 Determining genetics of resistance to P. cinnamomi in Asian chestnut species First Study: Fred Hebard TACF John Frampton NC State University

33 First Study Screened and genotyped two Mahogany BC 1 families Conclusion: The ratio of living vs. dead plants after one season was close to 1:1, suggesting a single gene for resistance, but genetic mapping efforts were not successful. BC 1 plants that survived the first year s screening continued to die in subsequent years.

34 Determining genetics of resistance to P. cinnamomi in Asian chestnut species Second study: Paul Sisco CC-TACF Mike French KY-TACF Joe James CC-TACF Laura Georgi Clemson Tom Kubisiak SIFG Bode Olukolu Clemson

35 Second Study: Nanking BC 1 family AdairKY1 (American chestnut in Kentucky) x pollen from GL158 ( Nanking F 1 at Meadowview) 2008: 48 BC 1 progeny screened in the center of a single tub in James/Jeffers PRR trials; edges of tub devoted to American and Chinese chestnut controls DNA collected from healthy plants before inoculation; Then plants inoculated with P. cinnamomi. Above-ground phenotyping by Joe James and Laura Georgi. segregation approximately 1:1 (27 alive vs. 21 struggling or dead) Low density genetic map constructed by Tom Kubisiak. 203 SNPs assigned to 12 linkage groups Pollination team at work Mother tree Large surviving Castanea dentata in Adair County, Kentucky Pollen was sent from Meadowview by Paul Sisco to Mike French, who did the pollinations in KY

36 Second Study: Two non-overlapping QTLs identified on Linkage Group E GL158 LG_E Reference Chinese chestnut LG_E 0.0 CmSNP01520 AdairKY1 LG_E 4.8 CmSNP CmSI CmSNP00886 CmSNP00834 CmSNP00068 CmSNP01197 CmSI0944 CmSNP01378 CmSI0011 CmSI0380 CmSNP CmSNP01038 CmSNP CmSNP CmSNP01056 CmSNP00006 CmSNP CmSNP CmSNP01106 CmSNP CmSNP CmSNP CmSNP01031 CmSNP00214 CmSNP CmSNP CmSNP00244 CmSNP01522 CmSNP CmSNP01320 CmSNP CmSNP00921 CmSNP CmSNP CmSNP01199 CmSNP00371 CmSNP00822 CmSNP CmSNP CmSNP00907 CmSNP CmSNP CmSNP CmSI0541 CmSI CmSNP CmSNP CmSNP00268 CmSNP CmSNP CmSNP00875 CmSNP01530 CmSNP00762 CmSNP00495 CmSI0463 CmSNP CmSNP CmSNP CmSNP CmSNP00216 GL158 map: LG_E 46->62 cm LOD 5.39 R2= 40.4 ± 10.9 Positively correlated with PRR resistance CmSNP00122 CmSNP01486 CmSNP00968 Phyto CmSNP00736 Bode 0.0 CmSNP CmSNP CmSNP CmSNP CmSNP00964 CmSNP00140 CmSNP01498 Phyto 23.9 CmSNP00752 CmSNP00045 CmSNP00273 CmSNP00934 CmSNP01421 CmSNP00631 CmSI0467 CmSI0606 CmSNP00330 CmSNP01051 CmSNP01367 CmSI0939 CmSNP00249 CmSNP00166 CmSNP00422 CmSNP00540 CmSI0617 CmSI0761 CmSNP01320 CmSNP01415 CmSNP00158 CmSI0321 CmSI0076 CmSNP00715 CmSNP01343 CmSNP01364 CmSNP01515 CmSNP00650 CmSNP01131 CmSNP00140 CmSI0883 CmSI0870 CmSNP01324 CmSNP00984 CmSNP00239 CmSI0829 CmSI0908 CmSI0035 CmSNP01034 CmSNP00349 CmSNP00957 CmSNP01427 CmSNP01513 CmSNP00273 CmSNP00227 CmSI0558 CmSNP00271 CmSNP00505 CmSNP00955 CmSNP01458 CmSI0396 CmSNP01331 CmSI0681 CmSNP01499 CmSNP00615 CmSI0886 CmSNP01222 AdairKY1 map: LG_E 12->15 cm LOD 4.42 R2=34.6 ± 11.1 Negatively correlated with PRR resistance 47.5 CmSNP CmSNP CmSNP CmSNP CmSNP CmSI CmSNP01353 Tom

37 Determining genetics of resistance to P. cinnamomi in Asian chestnut species Third study: present Paul Sisco CC-TACF Tatyana Zhebentyayeva Clemson Taylor Perkins TN-TACF Inga Meadows Clint Neel TN-TACF Dana Nelson SIFG and U. Of KY Albert Abbott U. Of KY

38 Third study: 2012 present Analysis of early generation Mahogany and Nanking families Hybrid family Total Root rot symptom severity classes Type of code-year plants family HB BC1 HB BC1 HB BC1 HB BC1 JB BC3 JB BC3 Source of resistance C. mollissima 'Mahogany' NK1-2012* BC1 NK2-2012* BC1 NK BC1 NK BC1 NK5,6-2015** F2 NK5,6-2016** F2 * Half sib crosses ** Full sib crosses total C. mollissima Nanking'

39 Genomic loci associated with resistance to Phytophthora cinnamomi A B C D E F Location of genes for Phytophthora resistance based on analysis of Chinese chestnut cvs. Mahogany and Nanking G H I J K L

40 Genomic loci associated with resistance to Phytophthora cinnamomi Location of genes for blight resistance based on analysis of Chinese chestnut cv. Mahogany A B C D E F Location of genes for Phytophthora resistance based on analysis of Chinese chestnut cvs. Mahogany G H I J K L and Nanking

41 The Good News: Genetic mapping results were consistent over years, locations, and sources of inoculum JB1 Mahogany B3 family 2013 Clint Neel tub Pegram, Tennessee Neel Farm inoculum 2014 James/Jeffers tubs Seneca, South Carolina James Farm inoculum

42 Fourth Study Association Genetics Approach Look for correlations between genes/loci and resistance in our Graves B3F2 trees using genome-wide sequencing Advantage of having smaller bits of the Chinese genome easier to pinpoint genes

43 Determining genetics of resistance to P. cinnamomi in Asian chestnut species Fourth Study: 2016 present Jared Westbrook TACF Jason Holliday VA Tech

44 Our results supported by studies in Portugal by Rita Costa and colleagues working with European chestnut x Japanese chestnut BC families

45 Conclusions so Far About the Genetics of Resistance More than one gene/locus is correlated with resistance in Asian Castanea species. A different subset of genes/loci are important in different cultivars of the Asian species. Resistance to Phytophthora cinnamomi is present in some Graves/Mahogany families but not in pure Clapper families We do not yet know how many genes/loci are needed to give adequate long-term resistance to P. cinnamomi.

46 GOAL: To develop American chestnut trees with long-lasting disease resistance Hill Craddock and Taylor Perkins Ben Jarrett and Jared Westbrook Tom Saielli Martin Cipollini