investigating the relationship between the i-3 locus and ...tgc.ifas.ufl.edu/2016/7. investigating...

Investigating the Relationship

Between the I-3 Locus and Increased

Bacterial Spot Sensitivity

S.F. Hutton and J. Li

[email protected]

VARIETY UPDATE

Table 1. Total and extra-large marketable yields, fruit size, and culls for hybrids for fruit harvested breaker and beyond at

the Gulf Coast Research and Education Center in fall 2014.

Yield (25lb boxes/A)

Fruit size

Marketable

(% by wt)

1st Harvest Total of 3 harvests

Hybrid Resistances1 Total Extra-large Total Extra-large

Fla. 8969 TSWV 649 a2 556 a 1738 a 1312 a 6.7 b 80 ab

Fla. 8970 TYLCV, FCR 271 b-d 246 b-d 1674 a 1239 ab 6.9 ab 80 a

Fla. 8971 TSWV-tol, TYLCV-tol 97 c-e 93 de 1646 a 1478 a 8.4 a 73 ab

Fla. 8972 479 ab 439 ab 1537 a 1172 ab 6.6 b 67 ab

Fla. 8973 TYLCV?, TSWV? 202 c-e 198 c-e 1407 ab 1162 ab 7.6 ab 72 Ab

Fla. 8866 TSWV 272 b-d 238 cd 1284 ab 1071 ab 7.4 ab 72 a-d

HM 1823 FCR 323 bc 311 bc 1247 ab 1092 ab 7.7 ab 73 ab

Fla. 8946 102 c-e 96 de 1234 ab 1027 a-c 7.2 ab 66 a-c

Fla. 8891 TSWV 275 b-d 230 cd 1203 ab 970 a-c 6.8 ab 63 a-d

HM 8849 FCR 164 c-e 158 c-e 1199 ab 947 a-c 7.3 ab 60 a-d

Fla. 8974 TYLCV-tol 215 c-e 209 cd 1189 ab 1028 a-c 7.6 ab 61 a-d

Ridge Runner TYLCV, FCR 194 c-e 185 c-e 789 bc 687 b-d 7.7 ab 56 b-d

Florida 91 4 e 4 e 478 c 415 cd 8.4 a 40 b-d

Dixie Red TSWV, N 59 de 59 de 460 c 419 cd 7.6 ab 41 cd

Florida 47 11 e 8 e 446 c 317 d 6.8 ab 42 cd 1TYLCV = Tomato Yellow Leaf Curl Virus; FCR = Fusarium crown and root rot; F3 = Fusarium wilt race 3; N = nematode; tol =

tolerance 2Mean separation by Duncan's multiple range test at P ≤ 0.05 based on a larger number of hybrids. Means within columns

followed by the same letter are not significantly different.

the Gulf Coast Research and Education Center in spring 2015.

Fruit size

Marketable

(% by wt)

Fla. 8972 358 a2 281 a 2273 a 1820 a 6.6 c-f 82 a-e

Fla. 8970 TYLCV, FCR 68 b 66 b 1818 ab 1620 ab 7.4 a-f 90 ab

Fla. 8942 TSWV, F3 105 b 95 b 1814 ab 1663 ab 7.6 a-f 89 ab

Fla. 8973 TYLCV, TSWV 176 b 149 b 1812 ab 1555 ab 7.0 b-f 81 b-e

Fla. 8971 TYLCV-tol, TSWV-tol 43 b 43 b 1669 bc 1630 ab 9.0 a-d 20 b-e

Fla. 8946B TYLCV 81 b 74 b 1661 bc 1574 ab 7.9 a-e 90 ab

Florida 91 39 b 39 b 1222 cd 1196 bc 8.6 a-d 82 a-e

Florida 47 56 b 50 b 1034 d 891 c 7.1 b-f 34 fg 1TYLCV = Tomato Yellow Leaf Curl Virus; FCR = Fusarium crown and root rot; F3 = Fusarium wilt race 3; N = nematode; tol =

Total and extra-large marketable yields, fruit size, and culls for hybrids for fruit harvested breaker and beyond at the

North Florida Research and Education Center in spring 2015.

Fruit size

Marketable

(% by wt)

Hybrid Total Extra-large Total Extra-large

FL 8969 441 ab 304 a-d 1166 a 547 abc 5.7 gh 86 a

Red Morning 482 a 422 ab 955 abc 698 a 6.9 ab 77 b-e

FL 8971 391 a-c 338 ab 918 ab 691 a 6.8 a-d 85 ab

FL 8970 258 a-e 211 b-f 886 abc 500 abc 6.2 a-g 84 abc

Ridge Runner 383 a-c 336 ab 872 abc 614 ab 6.9 abc 83 a-d

Red Bounty 373 a-d 309 a-c 855 bcd 497 abc 6.5 a-g 80 a-d

FTM 2259 403 a-c 331 ab 855 cde 552 abc 6.8 a-e 75 de

FL 8900 300 a-d 204 b-f 812 b-e 454 abc 6.2 a-g 76 cde

Mountain Majesty 346 a-e 305 a-d 802 b-e 612 ab 7.2 a 85 ab

FTM 1129 335 a-e 260 a-e 774 b-e 512 abc 6.6 a-g 83 a-d

Dixie Red 349 a-e 302 a-d 706 b-e 471 abc 6.7 a-e 84 abc

FL 8945 184 de 126 c-f 694 b-e 320 bcd 5.7 fgh 83 a-d

BHN 602 112 e 72 f 691 b-e 285 cd 6.0 b-g 77 b-e

FL 8955 337 a-e 210 b-f 689 b-e 315 bcd 5.6 gh 87 a

BSS 1056 194 b-e 103 ef 676 b-e 342 bcd 5.9 d-h 81 a-d

SV 7631 323 a-e 242 a-f 662 b-e 363 bcd 6.2 a-g 80 a-d

FL 47 161 c-e 100 ef 596 cde 258 cd 5.8 d-h 82 a-d

FTM 2260 163 c-e 116 c-f 581 cde 239 cd 5.8 e-h 78 b-e

Quincy 130 de 80 ef 538 de 255 cd 5.9 d-h 80 a-e

FL 8942 121 e 48 f 538 de 116 d 4.9 h 76 cde

FTM 2261 123 de 107 d-f 528 e 327 bcd 6.3 a-g 71 e 2Mean separation by Duncan's multiple range test at P ≤ 0.05 based on a larger number of hybrids. Means within

columns followed by the same letter are not significantly different.

the Gulf Coast Research and Education Center in fall 2015.

Fruit size

Marketable

(% by wt)

Early Harvests (1 & 2) Total of 4 harvests

HMX1823 1427 a-e2 1178 ab 2467 a-c 1810 a 6.7 a-i 83 ab

Fla. 8972 957 e-r 501 g-o 2022 b-k 950 f-n 5.4 m-r 78 a-e

Fla. 8970 TYLCV, FCR 1043 d-q 837 b-j 2270 a-f 1624 a-c 6.5 a-k 78 a-e

Fla. 8942 TSWV, F3 781 j-r 620 d-n 1942 b-m 1415 a-f 6.7 a-h 71 a-j

Fla. 8973 TYLCV?, TSWV? 1396 a-g 757 c-l 2185 a-g 1170 c-j 5.9 i-p 76 a-f

Fla. 8971 TYLCV-tol, TSWV-tol 991 e-r 864 b-g 2017 b-l 1643 a-c 7.0 a-c 77 a-f

Fla. 8946B TYLCV 1090 c-o 732 c-m 1826 c-n 1103 e-m 6.0 h-o 73 a-g

Fla. 8976 TYLCV, FCR 617 o-r 568 e-n 1769 e-o 1472 a-e 7.2 ab 65 e-m

Soraya FCR 657 n-r 487 h-o 1643 f-p 1103 e-m 6.4 b-l 69 b-k

Florida 47 727 m-r 469 k-o 1370 l-p 777 i-o 5.5 l-r 55 k-n 1TYLCV = Tomato Yellow Leaf Curl Virus; FCR = Fusarium crown and root rot; F3 = Fusarium wilt race 3; N = nematode; tol =

S.F. Hutton and J. Li

[email protected]

Investigating the Relationship

Between the I-3 Locus and Increased

Bacterial Spot Sensitivity

Credit: Image courtesy of University of

California - Davis

Fusarium wilt of tomato

Fusarium oxysporum f. sp. lycopersici (Fol)

Three races

Host resistance

S. pimpinellifolium (I, I-2)

S. pennellii (I-3, I-7)

• Candidate loci: Chromosomes 1 3 7 9 10 11 12

• Stepwise ANOVA:

11 <.0001

7 (I-3) <.0001

3 .0098

12 .0529

8517 x 7946 F2

1 2 3 4 5 6 7 8

8517 x 7946

0.33 0.64 Average number OH9242 alleles:

1.10 0.93 Average number of chr 3 R alleles:

2.00 6.33

8517 Selective Genotyping

C2 10050 (chromosome 11, 54 cM)

I-3+ I-3-

8517 Selective Genotyping

I-3 SCAR (D. Maxwell)

Effect of a marker linked to fusarium wilt race 3 resistance on bacterial spot race T4 foliar disease severity in tomato across four populations and two seasons.

From Hutton et al. (2014) J. Amer. Soc. Hort. Sci. 139:282-289.

I-3 (UF_I-3 CAPS)

Population Season Genotypez Plant no. Mean DSIy Dis. (%)x

(Fla. 8233 x Fla. 7946) Fall 2009 +/+ 103 5.7 a 32

+/- 103 5.4 b 28

-/- 103 5.2 c 25

(Fla. 8517 x Fla. 7946) Fall 2008 +/+ 58 4.2 a 16

+/- 111 3.3 b 9

-/- 62 3.0 c 7

(Fla. 8517 x Fla. 7946) Fall 2009 +/+ 99 5.5 a 30

+/- 105 5.7 a 33

-/- 92 5.4 b 28

(Fla. 8326 x Fla. 7946) Fall 2008 +/+ 58 6.2 a 44

+/- 100 5.8 b 35

-/- 62 5.1 c 24

z +/+ = homozygous for fusarium wilt race 3 resistance or for the resistant allele at the chromosome 11 quantitative trait loci; +/- = heterozygous; -/- = homozygous-susceptible. y Disease severity index rated on the Horsfall-Barratt (Horsfall and Barratt, 1945) scale, where higher numbers indicated more disease. Different superscript letters represent statistically significant differences among genotypes in a population and year at P<0.05 based on Duncan’s multiple range test. X Percentage diseased tissue as converted from Horsfall-Barratt ratings.

Marketable

Genotypez

Fruit (no./plt)

Yield (kg/plt)

Fruit size (g)

Extra large fruity (%)

Fla. 7859 (I-3/I-3)

44.8 6.85 153 56

Fla. 7859 (I-3/+)

44.6 nsx

7.99 ns

z I-3/I-3 = Homozygous resistant to fusarium wilt race 3. I-3/+ = Heterozygous resistant to fusarium wilt race 3. y Size 5 x 6 and larger x ns and * indicate means in column are not significantly different or significantly different, respectively, by the F test at p < 0.05.

Marketable fruit number, yield per plant, fruit size, and marketable extra large fruit percentage for Fla. 7859 hybrids, isogenic except for heterozygous or homozygous Fusarium wilt race 3 resistance in spring 1999 at Bradenton, Florida.

Mean fruit size, fruit number and yield of I-3 haplotypes for two advanced,

fresh market tomato inbred lines1 in spring 2015 field trials at Balm, FL.

Fruit size Fruit no. Yield

Line Genotype (g per plant) (no. per plant) (kg per plant)

I-3/I-3 158 c 2 27.7 n.s. 4.4 n.s.

Fla. 7907B I-3/i/3 191 b 25.0 4.8

i-3/i-3 210 a 22.6 4.7

I-3/I-3 163 b 27.2 a 4.4 b

Fla. 8814 I-3/i/3 182 b 28.6 a 5.2 a

i-3/i-3 208 a 24.8 b 5.1 a

1 Design was a Randomized Complete Block Design with 3 blocks and 8-plant plots. 2 Mean separation by Duncan's multiple range test at P ≤ 0.05. For each inbred background, means within

columns followed by the same letter are not significantly different. ns = no significant difference.

Fusarium wilt race 3

Negative associations with I-3

Weaker vines

Bacterial spot sensitivity

Reduction in fruit size

Linkage drag or pleiotropic effects?

Weaker vines

Population

I-3/i-3 i-3/i-3 I-3 locus effect One alternative Two alternative

Locus Loci

Healthy Infected Healthy Infected X2

(p <0.05) present

(p<0.05) present

LA716 37 4 17 20 0.0005 yes 0.622 yes 0.000 no

LA750 32 5 14 24 0.0002 yes 0.105 yes 0.000 no

LA751 0 0 20 40 -- -- 0.010 no 0.000 no

LA1272 39 5 11 18 0.0001 yes 0.194 yes 0.000 no

LA1273 22 14 6 16 0.0995 no 0.793 yes 0.000 no

LA1275 14 19 20 26 0.9998 no 0.216 yes 0.000 no

LA1277 21 17 10 18 0.4801 no 0.622 yes 0.000 no

LA1282 35 14 16 13 0.5464 no 0.007 no 0.050 yes

LA1297 28 11 14 23 0.0313 yes 0.139 yes 0.000 no

LA1299 20 0 10 10 0.0040 yes 1.000 yes 0.010 no

Weaker vines

I-3 Map-based cloning Recombinant screening

Resistant parent: Fla. 7228

Susceptible parents: Fla. 7169, Fla. 7218 and 7217D

~2,800 F3 plants screened with three CAPS markers

Marker

Primer sequence (5’ – 3’) Marker

Digested fragment size (bp)

S. lycopersicum S. pennellii

Physical position (Mbp)

S. lycopersicum

I-3CAPS F: TGTGCGTCTCTTGTCAACCTTACC

R: TGCTGTCGAGAGAATCTGGTGAGT

(HincII) 221, 200 421 62.77

SLG-1 F: CGTTCATAATCTGCTAAGTTTCTA

R: GGTTTCTATTACTCCAGCATCCTC

(RsaI) 296,170 466 63.63

bB6 F: GCCCTACAGTCCTTTTTCCT

R: CGTAAGCCAAAAGGTGTGC

(AluI) 404 214,190 63.69

Marker development

Heinz 1706 vs. S. pennellii LA716 sequences compared

Additional SCAR markers developed

F2 (/)

15 F3 recombinants

F3:4 haplotypes

+, / and --

Spring 2014 bacterial spot

evaluation

F3:5 homozygous

haplotypes

+ and --

Fall 2014 bacterial spot

evaluation

Fall 2014 Fol3 screening

F4:5 haplotypes

+, / and --

Spring 2015 bacterial

spot evaluation

I-3 Map-based cloning

Marker

Physical

Position

I-3 Recombinant Inbred Lines (RILs)2

19 23 18 26 7 21 11 14 17 24 12 15 8 9 10 I3/I3 i3/i3

59.70 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- + --

61.00 -- -- -- -- -- -- + + + + + + + + + + --

I-3 CAPS 62.77 -- -- -- -- -- -- + + + + + + + + + + --

7g501 63.18 -- -- -- -- + + + + + + + + + + + + --

7g595 63.27 -- -- -- -- + + + + + + + + + + + + --

7g665 63.34 -- -- -- -- + + + + + + + + + + + + --

7g728 63.41 -- -- -- -- + + + + + + + + + + + + --

7g916 63.59 -- -- + + + + + + + + + + + -- -- + --

7g923 63.60 -- -- + + + + + + + + + + -- -- -- + --

7g6362 63.62 + + + + + + + + + + -- -- -- -- -- + --

7g6363 63.63 + + + + + + + + + -- -- -- -- -- -- + --

SLG-1 63.63 + + + + + + + + + -- -- -- -- -- -- + --

7g957 63.63 + + + + + + + + -- -- -- -- -- -- -- + --

bB6 63.70 + + + + + + -- -- -- -- -- -- -- -- -- + --

64.00 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- + --

Fol3 Phenotype3 S S R R R R R R R R R R S S S R S

1 All fifteen I-3 recombinants derived from crosses between Fla.7228 (I-3/I-3) and susceptible breeding lines (i-3/i-3); The introgression size

was determined by molecular markers, where "+" indicates homozygous I-3/I-3 genotype; "--" indicates homozygous i-3/i-3 genotype. 2 Physical location is based on the S. lycopersicum genome assembly SL2.50. 3 Evaluation for fusarium wilt race 3 (Fol3); "R" indicates resistant; "S" indicates susceptible.

Marker

Physical

Position

19 23 18 26 7 21 11 14 17 24 12 15 8 9 10 I3/I3 i3/i3

59.70 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- + --

61.00 -- -- -- -- -- -- + + + + + + + + + + --

I-3 CAPS 62.77 -- -- -- -- -- -- + + + + + + + + + + --

7g501 63.18 -- -- -- -- + + + + + + + + + + + + --

7g595 63.27 -- -- -- -- + + + + + + + + + + + + --

7g665 63.34 -- -- -- -- + + + + + + + + + + + + --

7g728 63.41 -- -- -- -- + + + + + + + + + + + + --

7g916 63.59 -- -- + + + + + + + + + + + -- -- + --

7g923 63.60 -- -- + + + + + + + + + + -- -- -- + --

7g6362 63.62 + + + + + + + + + + -- -- -- -- -- + --

7g6363 63.63 + + + + + + + + + -- -- -- -- -- -- + --

SLG-1 63.63 + + + + + + + + + -- -- -- -- -- -- + --

7g957 63.63 + + + + + + + + -- -- -- -- -- -- -- + --

bB6 63.70 + + + + + + -- -- -- -- -- -- -- -- -- + --

64.00 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- + --

Marker

Physical

Position

19 23 18 26 7 21 11 14 17 24 12 15 8 9 10 I3/I3 i3/i3

59.70 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- + --

61.00 -- -- -- -- -- -- + + + + + + + + + + --

I-3 CAPS 62.77 -- -- -- -- -- -- + + + + + + + + + + --

7g501 63.18 -- -- -- -- + + + + + + + + + + + + --

7g595 63.27 -- -- -- -- + + + + + + + + + + + + --

7g665 63.34 -- -- -- -- + + + + + + + + + + + + --

7g728 63.41 -- -- -- -- + + + + + + + + + + + + --

7g916 63.59 -- -- + + + + + + + + + + + -- -- + --

7g923 63.60 -- -- + + + + + + + + + + -- -- -- + --

7g6362 63.62 + + + + + + + + + + -- -- -- -- -- + --

7g6363 63.63 + + + + + + + + + -- -- -- -- -- -- + --

SLG-1 63.63 + + + + + + + + + -- -- -- -- -- -- + --

7g957 63.63 + + + + + + + + -- -- -- -- -- -- -- + --

bB6 63.70 + + + + + + -- -- -- -- -- -- -- -- -- + --

64.00 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- + --

Marker

Physical

Position

19 23 18 26 7 21 11 14 17 24 12 15 8 9 10 I3/I3 i3/i3

59.70 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- + --

61.00 -- -- -- -- -- -- + + + + + + + + + + --

I-3 CAPS 62.77 -- -- -- -- -- -- + + + + + + + + + + --

7g501 63.18 -- -- -- -- + + + + + + + + + + + + --

7g595 63.27 -- -- -- -- + + + + + + + + + + + + --

7g665 63.34 -- -- -- -- + + + + + + + + + + + + --

7g728 63.41 -- -- -- -- + + + + + + + + + + + + --

7g916 63.59 -- -- + + + + + + + + + + + -- -- + --

7g923 I-3 63.60 -- -- + + + + + + + + + + -- -- -- + --

7g6362 63.62 + + + + + + + + + + -- -- -- -- -- + --

7g6363 63.63 + + + + + + + + + -- -- -- -- -- -- + --

SLG-1 63.63 + + + + + + + + + -- -- -- -- -- -- + --

7g957 63.63 + + + + + + + + -- -- -- -- -- -- -- + --

bB6 63.70 + + + + + + -- -- -- -- -- -- -- -- -- + --

64.00 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- + --

I-3 Candidate genes

I-3 located within a <30 kb

Three candidate genes based on the International Tomato

Annotation Group (ITAG 2.4)

I-3 recently cloned (Catanzariti et al., 2015)

Synonyms 1Location(s):

Chromosome 7 Length (bp) Description Genomic Variations

Solyc07g055630 63593008..63595531 2,524

Protein kinase/ transmembrane

receptor protein serine/threonine

kinase

two 2 bp indels, one 6 bp

indel, and SNPs

Solyc07g055640 63597005..63603617 6,613 Receptor-like kinase,

Serine/threonine protein kinase

one 13 bp indel, one 7 bp

indel, one 6 bp indel, and

Solyc07g055650 63618089..63620976 2,888 Receptor-like kinase,

Serine/threonine protein kinase one 3 bp indel, and SNPs

Solyc07g055660 63621865..63622999 1,135 Unknown Protein one 8 bp indel, and SNPs

F2 (/)

15 F3 recombinants

F3:4 haplotypes

+, / and --

spot evaluation

F3:5 homozygous

haplotypes

+ and --

Fall 2014 bacterial spot

evaluation

Fall 2014 Fol3 screening

F4:5 haplotypes

+, / and --

spot evaluation

Experimental design

15 recombinants X 3 haplotypes each

RCBD with 6-plant plots and three blocks

Inoculated with X. perforans race T4

Rated on the 1-12 Horsfall and Barratt scale (1965)

Data analysis

Disease severities among I-3 haplotypes were compared within

families by ANOVA using the GLM procedure in SAS

Mean separation was conducted using Fisher's LSD0.05

Segregation patterns for markers spanning the I-3 introgression

DSI for I-3 introgression haplotypes 59.7

7g6362

7g6363

Spring 2014 Fall 2014 Spring 2015

+ / -- + -- + / --

R-14 -- seg seg seg seg seg seg seg seg seg seg seg seg -- -- -- 6.9 a 6.2 b 5.7 c 6.8 6.6 7.3 6.9 6.3

R-24 -- seg seg seg seg seg seg seg seg seg -- -- -- -- -- -- 5.9 a 5.8 a 5.4 b 6.3 6.2 6.1 n.d. 6.3

R-15 -- seg seg seg seg seg seg seg seg -- -- -- -- -- -- -- 6.7 a 6.3 b 5.7 c 6.5 a 6.2 b 6.5 a 6.2 ab 5.8 b

R-8 -- seg seg seg seg seg seg seg -- -- -- -- -- -- -- -- 6.6 a 5.9 b 5.6 c 7.1 a 6.7 b 6.2 a 5.8 ab 5.4 b

R-9 -- seg seg seg seg seg seg -- -- -- -- -- -- -- -- -- 6.9 a 6.3 b 5.8 c 7.0 6.7 6.9 a 6.3 b 6.0 c

R-7 -- seg seg + + + + + + + + + + + + -- 6.7 6.6 6.6 6.8 a 6.2 b n.d. n.d. n.d.

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

R-12 -- + + + + + + + + seg seg seg seg seg seg -- 6.7 6.7 6.8 7.1 7.2 6.8 6.8 6.6

R-19 -- -- -- -- -- -- -- -- -- seg seg seg seg seg seg -- 5.17 5.2 5.1 6.1 5.9 n.d. 5.6 5.6

R-23 -- -- -- -- -- -- -- -- -- seg seg seg seg seg seg -- 6.0 a 5.7 b 5.6 b 6.1 5.9 5.7 n.d. 6.3

R-10 -- + + + + + + seg seg seg seg seg seg seg seg -- 6.6 6.7 6.7 6.7 6.8 6.8 7.0 6.8

R-18 -- -- -- -- -- -- -- seg seg seg seg seg seg seg seg -- 6.0 6.0 6.1 6.2 6.4 6.3 6.3 6.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7169 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- n/a n/a 5.3 d n/a 6.1 b n/a n/a 5.3 c

Fla.7218 -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- n/a n/a 6.1 c n/a 6.1 b n/a n/a 6 ab

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

Disease severities among I-3 recombinant haplotypes

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

Disease severities among I-3 recombinant haplotypes I-

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

7g6362

7g6363

+ / -- + -- + / --

R-21 -- seg seg + + + + + + + + + + + + -- 6.3 a 6.0 b 5.9 b 6.5 a 6.2 b 6.0 5.8 5.7

R-11 -- + + + + + + + + + + + + seg seg -- 6.4 6.3 6.4 6.8 6.7 7.4 6.8 7.2

R-17 -- + + + + + + + + + + + seg seg seg -- 6.9 7.0 7.0 6.9 6.7 7.6 7.6 7.4

Fla.7228 -- + + + + + + + + + + + + + + --

7.3 a n/a n/a 6.7 a n/a 6.7 a n/a n/a

Fla.7217D -- -- -- -- -- -- -- -- -- -- -- -- -- -- -- --

n/a n/a 6.4 b n/a 6.2 b n/a n/a 5.7 bc

Market

Type Generation Source

Markers

7g5862

7g5951

7g6096

7g6102

Fla.7776 round inbred UF/IFAS LL LL LL LL LL LL LL LL LL LL LL LL LL

LA7163 wild wild TGRC PP PP PP PP PP PP PP PP PP PP PP PP PP

Fla.7228 round inbred UF/IFAS LL PP PP PP PP PP PP PP PP PP PP PP PP

Fla.7907B round inbred UF/IFAS LL PP PP PP PP PP PP PP PP PP PP PP PP

NC123S round inbred NCSU LL PP PP PP PP PP PP PP PP PP PP PP PP

NC0256 round hybrid NCSU LL LP LP LP LP LP LP LP LP LP LP LP LP

Tasti-Lee® round hybrid Bejo LL LP LP LP LP LP LP LP LP LP LP LP LP

Fla.7547 round inbred UF/IFAS LL LL PP PP PP PP PP PP PP PP PP PP PP

I3-R1 round inbred UF/IFAS LL LL PP PP PP PP PP PP PP PP PP PP PP

Dixie Red round hybrid Seminis LL LL LP LP LP LP LP LP LP LP LP LP LP

FTM3309 round hybrid Sakata LL LL LP LP LP LP LP LP LP LP LP LP LP

SolarFire round hybrid HMClause LL LL LP LP LP LP LP LP LP LP LP LP LP

Sebring round hybrid Syngenta LL LL LP LP LP LP LP LP LP LP LP LP LP

Soraya round hybrid Syngenta LL LL LP LP LP LP LP LP LP LP LP LP LP

SevenTYIII plum hybrid Syngenta LL LL LP LP LP LP LP LP LP LP LP LP LP

Caddo round hybrid Enza LL LL LP LP LP LP LP LP LP LP LP LP LP

Tomato3365 round hybrid Hazera LL LL LP LP LP LP LP LP LP LP LP LP LP

Pawnee round hybrid Enza LL LL LL LL LP LP LP LP LP LP LP LP LP

Skyway 687 round hybrid Enza LL LL LL LL LP LP LP LP LP LP LP LP LP

Relevance to modern I-3 germplasm

Market

Markers

7g5862

7g5951

7g6096

7g6102

Market

Markers

7g5862

7g5951

7g6096

7g6102

Market

Markers

7g5862

7g5951

7g6096

7g6102

Summary

Compelling evidence that the association between I-3

and increased sensitivity to bacterial spot is due to

linkage drag

Negative alleles have likely been maintained in much

modern I-3 germplasm

Ongoing research to develop a minimal I-3 introgression

that is potentially free of negative effects

TBD: whether the effect of I-3 toward reduced fruit size

is likewise due to linkage drag

Funding

Florida Tomato Committee

USDA SCRI

Collaborators and Personnel

GE Vallad, GCREC Veg Pathology

JW Scott

UF Tomato Breeding lab

Funding

Florida Tomato Committee

USDA SCRI

Collaborators and Personnel

GE Vallad , GCREC Veg Pathology

JW Scott

UF Tomato Breeding lab

Marker

Primer sequence (5’ – 3’) Marker

(Restriction

enzyme)

Digested fragment size (bp)

S. lycopersicum S.

pennellii

Physical position (Mb)2

S. lycopersicum S. pennellii

7g5862 F: AGATGGTGGGATGTTGGC

R: ATCCAACTCTGTGCTTGGAC SCAR 69 82 58.62 69.18

7g5951 F: CTTCAGTATCTGCATAAATGAAAC

R: CCAAATGGAATACCCCGT SCAR 63 89 59.51 70.89

7g6096 F: GAGATATGTGCTTATCAATATTTGAAT

R: GAGATAAAAACATGATAAAACCACATA SCAR 80 72 60.92 71.89

7g6102 F: ATTGAAGTTTTAATTAATGATTTTGT

R: TACACCAAACAATTAACTCAAC SCAR 82 75 61.02 71.94

I-3CAPS F: TGTGCGTCTCTTGTCAACCTTACC

R: TGCTGTCGAGAGAATCTGGTGAGT

(HincII) 221, 200 421 62.77 74.06

7g501 F: ACTCTGTCCACCAAAGCTCAA

R: TGATTTTTCAATTTTCAGGCTTC SCAR 133 150

7g595 F: CATTTAGTCAGACGGCTAATGA

R: CTTGAGTTTGGTGTTTAAATTGGA SCAR 150 164 63.27 72.78

7g665 F: TCACCTTTACGCGAGTTTCA

R: AGCGCACCTTGGTACTGAAT SCAR 137 160 63.34 72.71

7g728 F: CACAACTGCCAAACATGACC

R: TAACCAAATTTATTGGCTTTTGGA SCAR 132 117 63.40 72.64

7g9161 F: GGATGTAGTGGTCAAAGGTATGG

R: ATCGACTCCTTCTCCCATTTC SCAR 153 143 63.59 74.62

7g923 F: ATTGGTTAAGTGAGACGTGTG

R: TAATGAATGCTGTGGCTAGGA SCAR 125 112 63.60 74.63

7g6362 F: GACTCACATCTGTCTACTGAATC

R: CTATTTACATATTAATTTTGGAATGAC SCAR 92 84 63.62 74.64

3 - Materials and Methods

Plant materials

42 S. pennellii accessions from the Tomato

Genetic Resource Center (TGRC)

Inter-specific F1s development ( x ‘Suncoast’)

F1BC1s development (F1 x ‘Suncoast’)

Control lines

Plant materials

Seeds were sown in spent coal media in a growth room

Seedlings were inoculated 7-9 days after sowing

Inoculation

Inoculum:

Fol races 1, 2 and 3, at concentration of ~107 spores /ml

Root dip and transplanted to seedling trays

S. pennellii accession (~32 plants) Fol 1, 2 and 3

F1 family (~32 plants) Fol 3

F1BC1 family (~80 plants) Fol 3

Disease evaluation

Healthy plants : no vascular browning

Susceptible plants : dead or with vascular browning

Genetic analysis for Fol3 resistance

I-3 genotyping

F1BC1 plants individually genotyped with one of three I-3 associated markers (< 1 cM), I-3CAPS, 7g728, and 7g923.

F1BC1 was either I-3/i-3 or i-3/i-3

Genetic analysis for novel resistance to Fol3

I-3 genotyping

F1BC1 plants individually genotyped with one of

three I-3 associated markers (< 1 cM), I-3CAPS,

7g728, and 7g923.

Chi-Square contingency test for I-3 locus effect (among I-

3/i-3 plants)

If number of resistant plants is significantly more than

expected in I-3/i-3 plants

Chi-Square contingency test for novel resistance to Fol3

(among i-3/i-3 plants or together with I-3/i-3 if no I-3 effect)

One novel resistance allele (expected 1R:1S)

Two novel resistance alleles (expected 3R:1S)

Phylogenetic analysis by genotyping by sequencing (GBS)

Two plants per accession

84 barcoded multiplexed library

Illumina HiSeq 2500

Raw data was analyzed by GBS pipeline 3.0

3 - Results

3-1. Evaluation on resistance Fol1 for S. pennellii accessions

3-2. Evaluation on resistance Fol2 for S. pennellii accession

3-3. Evaluation on resistance Fol3 for S. pennellii accession

3-4. Evaluation on resistance Fol3 for F1s (‘Suncoast’ x accessions)

3-5. Evaluation on resistance Fol3 for F1BC1s (‘Suncoast’ x F1s)

inocula

3-6. Genetic analysis for resistance to Fol3

Population

I-3/i-3 i-3/i-3 I-3 locus effect I-3 alternative I-3 alternative

One locus Two loci

(p <0.05) present

(p<0.05) present

LA716 37 4 17 20 0.0005 yes 0.622 yes 0.000 no

LA750 32 5 14 24 0.0002 yes 0.105 yes 0.000 no

LA751 0 0 20 40 -- -- 0.010 no 0.000 no

LA1272 39 5 11 18 0.0001 yes 0.194 yes 0.000 no

LA1273 22 14 6 16 0.0995 no 0.793 yes 0.000 no

LA1275 14 19 20 26 0.9998 no 0.216 yes 0.000 no

LA1277 21 17 10 18 0.4801 no 0.622 yes 0.000 no

LA1282 35 14 16 13 0.5464 no 0.007 no 0.050 yes

LA1297 28 11 14 23 0.0313 yes 0.139 yes 0.000 no

LA1299 20 0 10 10 0.0040 yes 1.000 yes 0.010 no

LA1302 15 0 11 4 0.2022 no 0.000 no 0.140 yes

LA1303 15 11 11 28 0.1298 no 0.107 yes 0.000 no

LA1340 23 13 16 23 0.2702 no 0.729 yes 0.000 no

LA1356 30 17 12 16 0.3717 no 0.299 yes 0.000 no

LA1367 11 3 40 26 0.6564 no 0.014 no 0.020 no

LA1376 20 11 19 17 0.8149 no 0.179 yes 0.002 no

LA1515 22 5 27 26 0.0710 no 0.891 yes 0.000 no

LA1522 27 9 16 28 0.0078 yes 0.070 yes 0.000 no

LA1649 38 9 12 18 0.0038 yes 0.273 yes 0.000 no

LA1656 22 11 18 10 0.9980 no 0.015 no 0.089 yes

3-6. Continued

Population

I-3/i-3 i-3/i-3 I-3 locus effect2 I-3 alternative I-3 alternative

One locus Two loci

(p <0.05) present

LA1674 34 14 19 13 0.7705 no 0.004 no 0.071 yes

LA1693 13 8 16 21 0.6006 no 1.000 yes 0.000 no

LA1724 11 15 20 19 0.9181 no 0.710 yes 0.000 no

LA1732 9 10 12 18 0.9677 no 0.317 yes 0.000 no

LA1733 10 16 11 26 0.9136 no 0.008 no 0.000 no

LA1734 7 6 11 16 0.8944 no 0.527 yes 0.000 no

LA1735 9 14 9 43 0.2443 no 0.000 no 0.000 no

LA1809 10 17 10 32 0.7062 no 0.000 no 0.000 no

LA1911 7 6 6 22 0.2300 no 0.019 no 0.000 no

LA1912 3 34 2 29 0.9954 no 0.000 no 0.000 no

LA1920 20 17 8 34 0.0145 yes 0.000 no 0.000 no

LA1926 12 6 1 16 0.0031 yes 0.000 no 0.000 no

LA1940 15 20 11 35 0.3514 no 0.001 no 0.000 no

LA1941 20 19 21 20 1.0000 no 0.823 yes 0.000 no

LA1942 25 9 21 20 0.2724 no 0.050 yes 0.006 no

LA1943 23 16 23 15 0.9993 no 0.087 yes 0.002 no

LA1946 10 10 8 33 0.1113 no 0.000 no 0.000 no

LA2580 4 11 20 33 0.8902 no 0.015 no 0.000 no

LA2657 13 17 9 32 0.2953 no 0.001 no 0.000 no

LA2963 7 29 6 33 0.9751 no 0.000 no 0.000 no

26 accessions potentially to have novel resistance

gene(s) to Fol3

14 accessions have only one novel resistance gene

8 accessions have one novel resistance gene and I-3

4 accessions have two novel resistance genes

How to choose germplasm for a specific project?

How are these accession related to another?

Genotyping by sequencing for all accessions

111,684,014 reads with good barcodes

17,184,756 sequence tags

340,962 tags were aligned to the unique positions in tomato genome

99,516 mapped SNPs were generated

18,449 SNPs filtered containing 0% missing were used for phylogenetic analysis by TASSEL 5.0

3-7. Isolation by distance test for S. pennellii accessions

A total 861 pairs of comparisons in genetic distance and distance of origins among all 42 Solanum

pennellii accessions were included in the regression analysis to prove isolation by distance. A significant

correlation (p < 0.0001, slope = 0.000028, r2 = 0.3167 and adjusted r2 = 0.3159) was determined

between genetic distance and distance of origins

0 200 400 600 800 1000 1200 1400 1600 1800

Genetic distance = 0.20033 + 0.00002848 x Physical distance

Distance (km)

Phylogeny of all Solanum pennellii accessions was analyzed under “Neighbor Joining” clustering method,

and an archaeopteryx tree was created by GUI 5.0 (http://www.maizegenetics.net/). Accessions have one

dark dot have one novel resistance gene to Fol; two dark dots have two novel resistance genes.

3-8. Phylogenetic relations and novel Fol3 resistance

Phylogeny of all Solanum pennellii accessions was analyzed under “Neighbor Joining” clustering method,

and an archaeopteryx tree was created by GUI 5.0 (http://www.maizegenetics.net/). Accessions have one

dark dot have one novel resistance gene to Fol; two dark dots have two novel resistance genes.

investigating the relationship between the i-3 locus and ...tgc.ifas.ufl.edu/2016/7. investigating...

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