Uncovering Genetic Resistance to DNA Viruses
in Brassica Species

Adam K Hales, Laureana Lazarte, Alma Laney, Yu-Ya Liang
Report prepared by Adam K Hales

Introduction

  • A recently discovered putative DNA virus causes significant damage to Brassica species in Utah.
  • This virus is vectored by Brevicoryne brassicae, the cabbage aphid, and induces necrotic lesions and brittle leaves in infected plants.
  • The objective of this study was to investigate the genetic basis of resistance to this virus.

Methods

Plant Material

  • 200 Accessions were screened. This included 197 accessions from the USDA Germplasm System and 3 commercially available varieties.
  • Species diversity was of great importance to us in this study. To this extent, accessions from 10 different Brassica species were screened.
  • Additionally, it was important to us to screen accessions from across the world, as genetic information underlying virus disease resistance could be from the wild-type populations.

DNA Collection & Extraction

  • Leaf tissue was removed once plants had produced sufficient leaf tissue to obtain a sample.
  • After collection, leaf tissue was stored at -80C until DNA extraction was performed.
  • DNA extraction was done using a kit optimized for leaf tissue extraction.
  • DNA quality was evaluated using qubit prior to sequencing.
  • Sequencing results are currently being processed and will be used in a GWAS.

Experimental Design

  • This study was conducted in a controlled greenhouse environment using single pots to seperate accessions in a Randomized Complete Block Design for two growing seasons.
  • A drip system was designed and utilized to allow for uniform water application and water conservation.
  • Plants were grown for 45 days before inoculation. Plants were inoculated using a symptomatic cabbage aphid colony from the Laney Lab at Utah Valley University.
  • After inoculation, plants underwent an incubation period until plants began to show symptoms of infection which took roughly 14 days following inoculation.
  • To evaluate plants’ response to infection, a novel disease severity rating (DSR) was developed, as there were no previously reported evaluation metrics for this virus-host interaction.
  • The DSR used a scale from 1 to 10 to assess the plant’s condition following infection, where a score of ‘1’ indicated complete plant health, and ‘10’ represented complete plant death.

Results

DSR Progression

  • For growing season 1, average DSR began and ended higher than season 2.
  • In growing season 1, only two evaluations were conducted.
  • Three DSR evaluations were made for growing season 2.
  • Average DSR for all plants increased at an almost linear rate until final evalution.
  • Plant health declining with passing evaluations.

DSR Distribution Across Species

  • The average DSR for all species across the study can be seen below.
  • Note the low average DSR for gravinae, fruiticulosa, and oleraceae species.

Accession Performance

  • A list of accessions screened can be found below in ascending order of DSR
  • Note germplasm accession number, which is the official identification tag of that particular accession
Click to expand full list of accessions results


Study ID Species Average DSR Germplasm Accession Number
14 gravinae 2.466667 PI 662590
15 gravinae 2.533333 PI 662583
127 oleracea 3.133333 PI 343560
12 fruticulosa 3.266667 PI 597825
58 oleracea 3.400000 PI 662667
62 oleracea 3.400000 PI 143351
63 oleracea 3.466667 PI 143358
88 oleracea 3.466667 PI 687095
112 oleracea 3.533333 PI 507854
129 oleracea 3.533333 PI 343576
151 oleracea 3.533333 PI 169053
57 oleracea 3.533333 PI 662586
102 oleracea 3.600000 PI 662531
16 gravinae 3.615385 PI 597827
86 oleracea 3.666667 PI 188561
132 oleracea 3.733333 PI 343664
137 oleracea 3.733333 PI 296209
143 oleracea 3.733333 PI 229747
150 oleracea 3.733333 PI 165067
98 oleracea 3.733333 G 28880
115 oleracea 3.800000 PI 414936
91 oleracea 3.800000 PI 249534
124 oleracea 3.866667 PI 343529
133 oleracea 3.866667 PI 343666
73 oleracea 3.866667 PI 385953
146 oleracea 3.933333 PI 204680
152 oleracea 3.933333 PI 169050
55 oleracea 3.933333 PI 687129
72 oleracea 3.933333 PI 443022
121 oleracea 4.000000 PI 343489
153 oleracea 4.000000 PI 169044
19 rapa 4.000000 PI 662687
74 oleracea 4.000000 PI 385954
76 oleracea 4.000000 PI 372864
139 oleracea 4.066667 PI 291998
111 oleracea 4.071429 PI 688226
123 oleracea 4.076923 PI 343514
148 oleracea 4.111111 G 28893
11 fruticulosa 4.133333 PI 633082
56 oleracea 4.133333 PI 662840
60 oleracea 4.200000 PI 169054
83 oleracea 4.200000 PI 244830
122 oleracea 4.266667 PI 343494
134 oleracea 4.266667 PI 303629
79 oleracea 4.266667 PI 183214
107 oleracea 4.333333 PI 662641
128 oleracea 4.333333 PI 343565
149 oleracea 4.333333 G 30490
90 oleracea 4.333333 PI 687064
114 oleracea 4.400000 PI 508413
117 oleracea 4.400000 PI 372913
131 oleracea 4.400000 PI 343646
141 oleracea 4.400000 PI 280067
155 oleracea 4.400000 PI 171529
147 oleracea 4.461538 G 28894
108 oleracea 4.466667 PI 687074
110 oleracea 4.466667 PI 687048
144 oleracea 4.466667 PI 229470
71 oleracea 4.466667 PI 662707
69 oleracea 4.500000 PI 694037
100 oleracea 4.533333 PI 662712
116 oleracea 4.533333 PI 379108
65 oleracea 4.533333 PI 441511
75 oleracea 4.533333 PI 384428
61 oleracea 4.583333 PI 171533
120 oleracea 4.600000 PI 343488
154 oleracea 4.600000 PI 171536
66 oleracea 4.600000 PI 687080
68 oleracea 4.600000 PI 662538
93 oleracea 4.600000 PI 261775
99 oleracea 4.600000 PI 141576
78 oleracea 4.615385 PI 343480
130 oleracea 4.666667 PI 343630
101 oleracea 4.733333 PI 687047
103 oleracea 4.733333 PI 687082
135 oleracea 4.733333 PI 302985
138 oleracea 4.733333 PI 296132
64 oleracea 4.733333 PI 141572
84 oleracea 4.733333 PI 241611
136 oleracea 4.800000 PI 296331
77 oleracea 4.800000 PI 662518
80 oleracea 4.800000 PI 291567
85 oleracea 4.857143 PI 189028
109 oleracea 4.866667 PI 662705
81 oleracea 4.866667 PI 267721
67 oleracea 4.933333 PI 662561
70 oleracea 4.933333 PI 508410
113 oleracea 5.000000 PI 508411
119 oleracea 5.000000 PI 344065
82 oleracea 5.000000 PI 264331
105 oleracea 5.066667 PI 687041
87 oleracea 5.066667 PI 662560
118 oleracea 5.133333 PI 358284
94 oleracea 5.142857 G 30714
97 oleracea 5.166667 PI 662581
104 oleracea 5.200000 PI 687055
95 oleracea 5.200000 PI 343674
1 carinata 5.333333 PI 613125
52 rapa 5.333333 PI 508417
191 napus 5.466667 Ames 26635
96 oleracea 5.466667 PI 662661
125 oleracea 5.500000 PI 343536
59 oleracea 5.500000 PI 320915
145 oleracea 5.533333 PI 212080
5 carinata 5.533333 PI 633080
140 oleracea 5.600000 PI 289697
4 carinata 5.600000 PI 596535
126 oleracea 5.666667 PI 343537
8 carinata 5.666667 PI 633076
187 napus 5.692308 PI 649127
24 rapa 5.733333 PI 662557
170 juncea 5.800000 PI 662777
22 rapa 5.857143 PI 662558
2 carinata 5.866667 PI 613128
142 oleracea 5.933333 PI 261642
193 napus 5.933333 PI 633122
106 oleracea 6.000000 PI 662792
190 napus 6.000000 PI 597352
195 napus 6.066667 PI 633121
6 carinata 6.076923 PI 597822
92 oleracea 6.111111 PI 209942
167 juncea 6.133333 Ames 31505
23 rapa 6.133333 PI 687052
3 carinata 6.133333 PI 596534
43 rapa 6.133333 PI 419027
162 juncea 6.200000 PI 662735
166 juncea 6.200000 Ames 12769
183 napus 6.200000 PI 537090
51 rapa 6.200000 PI 508420
53 rapa 6.200000 PI 508422
179 napus 6.266667 PI 619618
157 juncea 6.333333 PI 470082
192 napus 6.333333 PI 649129
30 rapa 6.333333 PI 633169
36 rapa 6.333333 PI 279857
7 carinata 6.357143 PI 633075
13 fruticulosa 6.400000 PI 649099
169 juncea 6.400000 PI 662769
182 napus 6.400000 PI 633123
185 napus 6.400000 PI 649126
21 rapa 6.400000 PI 508408
25 rapa 6.400000 PI 662556
42 rapa 6.400000 PI 419028
54 rapa 6.400000 PI 508430
35 rapa 6.466667 PI 234600
40 rapa 6.466667 PI 419074
44 rapa 6.466667 PI 418987
49 rapa 6.466667 PI 508425
163 juncea 6.500000 PI 649122
45 rapa 6.500000 PI 508429
177 juncea 6.533333 PI 633100
180 napus 6.533333 PI 610258
41 rapa 6.533333 PI 419069
47 rapa 6.533333 PI 508416
184 napus 6.538462 PI 470005
181 napus 6.571429 PI 605719
172 juncea 6.600000 PI 662736
173 juncea 6.600000 PI 633086
186 napus 6.600000 Ames 30186
37 rapa 6.600000 PI 418959
38 rapa 6.600000 PI 418958
48 rapa 6.600000 PI 508428
156 juncea 6.666667 PI 478332
161 juncea 6.666667 PI 633099
178 napus 6.666667 PI 634754
28 rapa 6.666667 PI 662545
50 rapa 6.666667 PI 508424
168 juncea 6.714286 PI 603011
160 juncea 6.866667 PI 662697
174 juncea 6.866667 PI 633112
176 juncea 6.866667 PI 633114
26 rapa 6.866667 PI 662553
31 rapa 6.866667 PI 597831
158 juncea 6.933333 PI 436559
17 maurorum 6.933333 PI 633117
175 juncea 6.933333 PI 649120
20 rapa 6.933333 PI 662688
32 rapa 6.933333 PI 687065
164 juncea 7.000000 PI 458928
27 rapa 7.000000 PI 662550
33 rapa 7.066667 G 30212
39 rapa 7.133333 PI 418957
171 juncea 7.200000 PI 633097
196 napus 7.307692 PI 597828
189 napus 7.333333 Ames 18510
29 rapa 7.333333 PI 633163
18 oxyrrhina 7.400000 PI 649157
165 juncea 7.533333 G 30990
34 rapa 7.666667 PI 633166
46 rapa 7.666667 PI 508427
194 napus 7.833333 PI 594321
10 deflexa 7.866667 PI 649096
197 napus 7.888889 PI 632742
188 napus 8.000000 PI 251236
9 deflexa 8.000000 PI 597823
159 juncea 9.500000 PI 432383
89 oleracea 9.500000 PI 263057

Model and Output

  • We fitted a linear mixed-effects rgression model to assess the effects of origin and species on DSR, while accounting for the random effects of block, growing season, and replicate.
  • For this model, block is nested within season as block randomization differed between the two growing seasons.
Click to expand full LMER model results


  DSR
Predictors Estimates CI p
(Intercept) 5.54 3.02 – 8.05 <0.001
Origin [Afghanistan] 1.65 0.60 – 2.70 0.002
Origin [Algeria] 1.63 0.62 – 2.63 0.001
Origin [Armenia] 0.97 -0.08 – 2.02 0.072
Origin [Australia] 1.17 0.32 – 2.02 0.007
Origin [Bangladesh] 2.01 -0.11 – 4.13 0.063
Origin [Brazil] 0.65 -0.40 – 1.70 0.226
Origin [Bulgaria] -0.75 -1.80 – 0.30 0.163
Origin [Canada] 0.84 0.03 – 1.65 0.043
Origin [China] 0.79 0.03 – 1.54 0.042
Origin [Colombia] -0.22 -1.27 – 0.84 0.687
Origin [Cuba] 0.84 -0.06 – 1.73 0.067
Origin [Czech Republic] -0.35 -1.40 – 0.70 0.515
Origin [Denmark] 0.12 -0.94 – 1.17 0.828
Origin [Egypt] 0.81 -0.05 – 1.68 0.065
Origin [Ethiopia] 1.25 0.20 – 2.30 0.020
Origin [France] 0.80 -0.18 – 1.78 0.109
Origin [Germany] 0.96 0.06 – 1.86 0.036
Origin [Hungary] 0.52 -0.46 – 1.50 0.295
Origin [India] 0.96 0.11 – 1.82 0.027
Origin [Iran] 0.10 -0.72 – 0.93 0.806
Origin [Israel] 0.18 -0.87 – 1.24 0.732
Origin [Italy] 0.35 -0.47 – 1.16 0.407
Origin [Japan] 0.84 0.02 – 1.65 0.044
Origin [Kazakhstan] 0.43 -0.44 – 1.30 0.335
Origin [Kenya] 0.13 -0.75 – 1.01 0.778
Origin [Mongolia] 0.85 -0.22 – 1.91 0.121
Origin [Nepal] 1.17 0.27 – 2.07 0.010
Origin [Netherlands] 1.13 0.29 – 1.98 0.009
Origin [North Korea] 1.07 0.04 – 2.09 0.042
Origin [North Macedonia] 0.65 -0.40 – 1.70 0.226
Origin [Norway] -0.05 -0.97 – 0.87 0.916
Origin [Pakistan] 1.02 0.08 – 1.96 0.033
Origin [Poland] 1.07 0.21 – 1.94 0.015
Origin [Portugal] 0.25 -0.80 – 1.30 0.641
Origin [Puerto Rico] 1.10 0.03 – 2.17 0.044
Origin [Romania] -0.06 -1.15 – 1.04 0.920
Origin [Russia] 1.32 0.45 – 2.19 0.003
Origin [Scotland] 0.52 -0.54 – 1.57 0.336
Origin [Serbia] 0.48 -0.44 – 1.40 0.303
Origin [South Africa] 0.41 -0.47 – 1.28 0.361
Origin [South Korea] 0.72 -0.05 – 1.49 0.066
Origin [Spain] 0.30 -0.53 – 1.14 0.477
Origin [Sweden] 0.81 -0.08 – 1.69 0.075
Origin [Taiwan] 0.11 -0.73 – 0.95 0.804
Origin [Tanzania] 1.05 -0.10 – 2.19 0.074
Origin [Tunisia] 1.05 -0.02 – 2.11 0.055
Origin [Turkey] 0.23 -0.56 – 1.03 0.566
Origin [Ukraine] -0.02 -1.07 – 1.04 0.976
Origin [United Kingdom] 0.54 -0.27 – 1.36 0.191
Origin [United States] 0.71 -0.06 – 1.49 0.072
Origin [Uzbekistan] 0.12 -0.94 – 1.17 0.828
Origin [Yemen] 0.72 -0.34 – 1.77 0.182
Origin [Zambia] 0.15 -0.98 – 1.27 0.801
Species [deflexa] 2.38 1.69 – 3.07 <0.001
Species [fruticulosa] -2.14 -2.96 – -1.32 <0.001
Species [gravinae] -4.10 -5.00 – -3.20 <0.001
Species [juncea] 0.41 -0.06 – 0.88 0.086
Species [maurorum] -0.08 -1.15 – 0.99 0.879
Species [napus] 0.17 -0.32 – 0.65 0.499
Species [oleracea] -1.51 -1.93 – -1.08 <0.001
Species [oxyrrhina] 1.76 0.65 – 2.87 0.002
Species [rapa] 0.34 -0.10 – 0.78 0.134
Random Effects
σ2 2.05
Ï„00 Block:Season 0.08
Ï„00 Rep 2.66
Ï„00 Season 1.78
ICC 0.69
N Block 3
N Season 2
N Rep 5
Observations 2803
Marginal R2 / Conditional R2 0.168 / 0.740
  • When accounting for random effects, this model explained about 74 percent of the dataset.

Below is a table of the significant predictors of DSR.

Significant Fixed Effects on DSR (p < 0.05)
term estimate std.error statistic df p.value
Origin:Afghanistan 1.650 0.537 3.072 2731.972 0.002
Origin:Algeria 1.628 0.512 3.180 2731.985 0.001
Origin:Australia 1.169 0.433 2.703 2731.977 0.007
Origin:Canada 0.840 0.415 2.026 2731.981 0.043
Origin:China 0.785 0.386 2.034 2731.972 0.042
Origin:Ethiopia 1.250 0.537 2.328 2731.972 0.020
Origin:Germany 0.958 0.457 2.096 2731.971 0.036
Origin:India 0.964 0.435 2.218 2731.974 0.027
Origin:Japan 0.836 0.414 2.019 2731.972 0.044
Origin:Nepal 1.170 0.457 2.561 2731.971 0.010
Origin:Netherlands 1.134 0.431 2.629 2731.973 0.009
Origin:North Korea 1.067 0.523 2.038 2731.971 0.042
Origin:Pakistan 1.021 0.479 2.133 2731.988 0.033
Origin:Poland 1.074 0.440 2.439 2731.986 0.015
Origin:Puerto Rico 1.099 0.546 2.012 2731.990 0.044
Origin:Russia 1.322 0.444 2.974 2731.992 0.003
Species:deflexa 2.377 0.353 6.740 2731.980 0.000
Species:fruticulosa −2.141 0.417 −5.131 2731.978 0.000
Species:gravinae −4.100 0.457 −8.979 2732.001 0.000
Species:oleracea −1.505 0.214 −7.019 2732.013 0.000
Species:oxyrrhina 1.762 0.566 3.115 2731.977 0.002

The effect of origin on DSR is shown below. Note that the asteriks indicate the level of significance.

  • *** indicates a p-value of <0.001
  • ** indicates a p-value of ≤ 0.01.
  • * indicates a p-value of ≤ 0.05.
  • No asteriks indicate no statistical significance.

The effect of species on DSR is shown below. Note that the asteriks indicate the level of significance.

  • *** indicates a p-value of <0.001
  • ** indicates a p-value of ≤ 0.01.
  • * indicates a p-value of ≤ 0.05.
  • No asteriks indicate no statistical significance.

Discussion

Effect of Origin on DSR

  • Accessions with origins of Afghanistan, Algeria, and Russia seem to be more susceptible to this given virus.
  • For all other origins, data is not statistically significant.

Effect of Species on DSR

  • Species that are negatively correlated with DSR could exhibit resistance against the virus, and could posess disease resistant alleles that would be valuable in breeding practices.
    • These species include gravinae, fruiticulosa, and oleraceae. Each of which were roughly 1-3 DSR points lower than the other species.
  • Species that are positively correlated with DSR could be exhibit susceptibility to this putative DNA virus.
    • These species include deflexa and oxyrrhia.

Acknowledgements

  • UVU Scholarly Activities Committee
  • UVU Undergraduate Research Scholarly and Creative Activities Program
  • UVU Plant Breeding and Genetics Lab – Dr. Yu-Ya Liang
  • Dr. Alma Laney and Lab