1 Quantitative trait loci underlying host responses of soybean to Fusarium virguliforme toxins
2 that cause foliar sudden death syndrome.
3 4 5 Sivakumar Swaminathan1, Nilwala S. Abeysekara2, Min Liu3, Silvia R. Cianzio1 and Madan
6 K. Bhattacharyya1*
7 8 1
Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA
9 2
Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa 50011,
10 USA
11 3
12 of Agronomy, Shenyang Agricultural Univ., 120 Dongling Ave., Shenyang, Liaoning 110866,
13 China
Visiting Scholar, Department of Agronomy, Iowa State University; current address, Department
14 * Corresponding author; e-mail mbhattac@iastate.edu; Ph : 1-515-294-2505 ; Fax : 515-294-2299
15 16 17 18 19 20 21 1 Abstract
22 23 24 Sudden death syndrome (SDS) is a major soybean disease throughout most of the soybean
25 growing regions in the world including the United States. The disease is caused by the fungal
26 pathogen, Fusarium virguliforme (Fv). The fungus produces several toxins that are responsible
27 for development of interveinal leaf chlorosis and necrosis, which are typical foliar SDS
28 symptoms. Growing of resistant cultivars has been the most effective method in controlling the
29 disease. The objective of the present study was to identify quantitative trait loci (QTL) underlying
30 host responses of soybean to Fv toxins present in culture filtrates. To accomplish this objective,
31 two recombinant inbred line (RIL) populations, AX19286 (A95-684043 x LS94-3207) and
32 AX19287 (A95-684043 x LS98-0582), segregating for SDS resistance were evaluated for foliar
33 symptom development by applying two screening protocols, the stem cutting and the root feeding
34 assays. The AX19286 population revealed two major and seven minor QTL for SDS resistance.
35 In the AX19287 population, we identified five major QTL and three minor QTL. The two QTL
36 mapped to Chromosome 7 [molecular linkage groups (MLG) M] and Chromosome 20 (MLG I)
37 are most likely novel, and were detected through screening of the AX19287 population with stem
38 cutting and root feeding assays, respectively. This study established that Fv culture filtrates could
39 be employed in mapping QTL underlying foliar SDS resistance. The outcomes of the research
40 also suggest that multiple genetic mechanisms might be used by soybean to overcome the toxic
41 effects of the toxins secreted by the pathogen into culture filtrates.
42 43 44 2 45 Keywords: Fusarium virguliforme, soybean sudden death syndrome (SDS), quantitative trait loci
46 (QTL) mapping, cell-free Fv culture filtrates, toxins, recombinant inbred lines (RILs)
47 48 49 Key message
50 Soybean deploys multiple genetic mechanisms to confer tolerance to Fusarium virguliforme
51 toxins. This study revealed that F. virguliforme culture filtrates could be used in mapping QTL
52 underlying foliar SDS resistance.
53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 3 Introduction
68 69 70 Soybean [Glycine max (L.) Merr.] is one of the most important crops with high economic value
71 worldwide. Sudden death syndrome (SDS) is one of the most serious soybean diseases in both
72 North and South America. In North America, it is caused by the soil-borne fungus, Fusarium
73 virguliforme O’Donnell & T. Aoki (formerly F. solani (Mart.) Sacc. f. sp. glycines). In South
74 America, the disease is caused by four Fusarium spp., F. virguliforme, F. tucumaniae, F.
75 brasiliense and F. cuneirostrum (Aoki et al. 2003; 2005). The pathogen has never been isolated
76 from diseased foliar tissues. It is becoming evident that one or more fungal toxins secreted by the
77 pathogen in infected roots are responsible for the foliar SDS development (Pudake et al. 2013).
78 The pathogen secretes a large number of peptides and proteins to the culture filtrates including
79 five discovered in the xylem sap of the F. virguliforme (Fv)-infected soybean plants (Abeysekara
80 and Bhattacharyya 2014). Toxins secreted by the pathogen to the culture filtrates cause foliar
81 SDS-like symptoms in the susceptible soybean varieties (Brar et al. 2011; Ji et al. 2006; Jin et al.
82 1996; Li et al. 1999).
83 Fusaria have been reported to produce a variety of phytotoxins including the isomeric
84 compounds, marticin, and isomarticin (Kern 1972), anhydrofusarubin, fusarubin and javanicin
85 (Baker et al. 1981), fusaric acid (Kern 1972), lycomarasmin (Vesonder and Heeseltine 1983),
86 enniatin (Burmeister and Plattner 1987), fumonisin and moniliformin (Abbas et al. 1995), and
87 2,5-anhydro-Dglucitol (Tanaka et al. 1996). Two phytotoxins, monorden (Baker and Nemec
88 1994) and a 17-kDa proteinaceous toxin (Jin et al. 1996), were isolated from F. virguliforme
89 (formally known as F. solani f. sp. glycines) culture filtrates that cause necrosis in soybean
90 tissues. Recently a 13.5 kDa proteinaceous toxin, FvTox1, was isolated from the cell-free F.
4 91 virguliforme culture filtrates. It is a major causal agent of foliar SDS in soybean (Pudake et al.
92 2013).
93 First discovered in Arkansas in 1971, SDS has spread rapidly throughout most of the
94 soybean growing areas of the North Central United States (Leandro et al. 2012; Roy et al. 1997;
95 Rupe 1989) and now has become a major soybean disease. SDS management options are limited
96 and the use of SDS resistant cultivars has been the most effective method of managing the
97 disease. Unfortunately, SDS resistance is partial and several QTL must be incorporated into a
98 single cultivar to provide soybean with sufficient resistance against the pathogen.
99 Stephens et al. (1993) reported a single dominant gene, Rfs, responsible for SDS resistance in
100 the soybean cultivar ‘Ripley.’ However, investigation of segregating populations developed from
101 the cross ‘Essex’ x ‘Forrest’ revealed nine QTL for SDS resistance (Kassem et al. 2007; Kassem
102 et al. 2006). These QTL were distributed among Chromosome 13 (MLG F), 16 (MLG J), 6 (MLG
103 C2), 20 (MLG I), and 18 (MLG G). In the RIL from ‘Pyramid’ x ‘Douglas,’ three QTL for SDS
104 resistance were found in Chromosome 3 (MLG N), 6 (MLG C2), and 18 (MLG G) (Kassem et al.
105 2007; Njiti et al. 2002). In the ‘Hartwig’ x ‘Flyer RIL’ population, a QTL mapped to
106 Chromosome 18 (MLG G) was identified (Prabhu et al. 1999). Study of the RIL population from
107 ‘Ripley’ x ‘Spencer’ revealed three QTL for SDS resistance mapped to Chromosome 4 (MLG
108 C1), 17 (MLG D2), and 19 (MLG L) (de Farias-Neto et al. 2007). From study of an RIL
109 population developed from PI 567374 x ‘Omaha,’ two QTL for SDS resistance, mapped to
110 Chromosomes 17 (MLG D2) and 20 (MLG I), were discovered (de Farias Neto et al. 2007). 111 An investigation of the RILs developed from the cross ‘Hartwig’ x ‘Flyer’ revealed three
112 QTL mapped to Chromosome 6 (MLG C2), 17 (MLG D2) and 18 (MLG G) (Kazi et al. 2007;
113 Kazi et al. 2008). Study of a RIL population generated from PI 438489B x ‘Hamilton’ revealed
5 114 14 QTL for SDS resistance (Kassem et al. 2012). Seven of these QTL had been previously
115 identified on Chromosome 4 (MLG C1), Chromosome 6 (MLG C2), Chromosome 2 (MLG D1b),
116 Chromosome 18 (MLG G), Chromosome 19 (MLG L), Chromosome 3 (MLG N) and
117 Chromosome 10 (MLG O); the other seven QTL were novel and mapped to Chromosome 8
118 (MLG A2), Chromosome 11 (MLG B1), Chromosome 6 (MLG C2), Chromosome 1 (MLG D1a),
119 Chromosome 2 (MLG D1b) and Chromosome 10 (MLG O) (Kassem et al. 2012). A recent
120 genome-wide association mapping using more than 300 elite soybean cultivars resulted in the
121 identification of 20 loci underlying SDS resistance, of which 13 were novel loci (Wen et al.
122 2014). In summary, by 2014 more than 40 QTL for SDS resistance had been identified from nine
123 different recombinant inbred line populations.
124 Soybean breeders are constantly looking for new genetic sources to improve SDS
125 resistance in soybean cultivars. Although a large number of QTL for foliar SDS resistance have
126 been identified in response to toxins produced by the pathogen in infected diseased roots, no
127 mapping experiments have been conducted to identify genetic loci that underlie tolerance of
128 soybean just to F. virguliforme toxins involved in foliar SDS development. Here we have mapped
129 the QTL underlying tolerance of soybean to F. virguliforme toxins secreted to the culture filtrates.
130 Two sets of RILs, AX19286 (A95-684043 x LS94-3207) and AX19287 (A95-684043 x LS98-
131 0582) were considered for this study. Progenitors of one SDS resistant parent (LS94-3207) were
132 previously used in mapping QTL for SDS resistance; whereas, the other one (LS98-0582) has
133 never been used in SDS resistance QTL mapping studies. We identified two putative novel QTL
134 for foliar SDS resistance from the AX19287 mapping population. In addition to identifying the
135 QTL, this study established that cell-free Fv culture filtrates could be used to identify QTL for
136 SDS resistance. It also laid a strong foundation for future studies to determine if any of the QTL
6 137 identified in this study carry the toxin-recognizing receptors for triggering resistance or
138 susceptibility to the pathogen.
139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 7 Materials and methods
160 161 Plant materials
162 163 164 Two hundred F7-derived RILs each from the AX19286 (A95-684043 x LS94-3207) and
165 AX19287 (A95-684043 x LS98-0582) populations were used in this study. The parent A95-
166 684043 (Cianzio et al. 2002) is an F4 plant selection from the cross of Jacques J285 x [Archer x
167 (Cordell x Asgrow A2234)] developed at the Iowa State University soybean research site located
168 at the Puerto Rico Agricultural Experiment Station, University of Puerto Rico. A95-684043 is a
169 maturity group (MG) III, high yielding line that carries soybean cyst nematode (SCN) resistance
170 introgressed from 'Peking,' PI 88788 and PI 90763. LS94-3207 is a soybean maturity group IV
171 (Reg. no. CV-467, PI 634335) line developed at Southern Illinois University, Carbondale, IL. It
172 carries resistance to multiple races of SCN and the SDS pathogen (Schmidt and Klein 2004).
173 LS94-3207 is a maturity group IV (Reg. no. CV-467, PI 634335) cultivar developed at Southern
174 Illinois University, Carbondale, IL. It carries resistance to multiple races of SCN and the SDS
175 pathogen (Schmidt and Klein 2004). LS94-3207 originated from an individual F5 plant selection
176 from the cross ‘Pharaoh’ x ‘Hartwig’. Pharaoh has the pedigree 'Forrest' (3) x V71-480. Hartwig
177 was derived from 'Forrest' x ‘PI 437654’. LS98-0582 is a MG IV soybean cultivar (Northrup
178 King S46-44 x Asgrow A4138) developed at Southern Illinois University (Heatherly and Hodges
179 1998). It has high levels of SCN and SDS resistance.
180 Stem cutting and root feeding assays
181 182 8 183 200 RILs from AX19286 and AX19287 populations were evaluated by feeding roots with cell-
184 free Fv culture filtrates. Stem cutting and root feeding assays were carried out with cell-free Fv
185 culture filtrates according to published protocols (Li et al. 1999; Mbofung et al. 2012). To prepare
186 the cell-free Fv culture filtrates, three isolates of the pathogen, Clinton 1B, Scott F2II 1a and Scott
187 B2 collected from Iowa (Cianzio et al. 2014), were grown separately on solid Bilay medium for
188 14 days. Mycelial plugs were then transferred to 100 ml liquid modified Septoria medium
189 (MSM) and incubated at room temperature in dark for 14 days (Song et al. 1993). Fungal culture
190 medium was then sequentially filtered through Whatman No.1 paper, 0.45 µm and 0.22 µm
191 Stericups (Millipore, Inc., Billerica, MA). The protein content of the fungal culture filtrates was
192 determined using the Bradford colorimetric assay (Bio-Rad, Inc., Hercules, CA). The liquid cell-
193 free Fv culture filtrates of Clinton 1B, Scott F2II 1a and Scott B2 isolates were mixed in equal
194 proportions. The protein estimation of liquid culture filtrates was done just before the assay.
195 Seeds of each RIL were planted in growth chambers under light at 23oC for 16 h and
196 maintained in the dark at 16°C for 8 h. The light intensity was 200 µmol photons m-2sec-1. Single
197 21-day old seedlings were cut below the cotyledons and placed in individual 50 mL plastic tubes
198 each containing 25 ml diluted cell-free Fv culture filtrates in sterile double distilled water
199 containing 225 µg of F. virguliforme excreted proteins (75 µg from each isolate) (Li et al. 1999).
200 Ten seedlings per RIL were assayed in each experiment. The experiment was repeated three more
201 times. The cut seedlings were kept in the growth chambers watered as needed.
202 The root feeding assay was adopted from the method developed in Leandro lab (Mbofung
203 et al. 2012). For the root feeding assay, the 21 day old seedlings were carefully pulled up from
204 the soil mixture (SB 300 Universal Professional Growing Mix, Sunshine; Sun Gro Horticulture,
205 Agawam, MA) without causing damage to the roots, which were then washed in tap water and
9 206 placed into 50 ml tubes containing 25 ml diluted cell-free Fv culture filtrates as described for stem
207 cutting assays.
208 In both stem and root assays, foliar SDS symptoms appeared 5-6 days following the
209 feeding of seedlings with the Fv culture filtrates. Disease scoring was done 14 days after the start
210 of the assays. The disease scoring scheme was: 0, no symptoms; 1, leaves showing slight
211 yellowing and/or chlorotic flecks or blotches (1-10 % of the foliage affected); 2, leaves with
212 obvious, interveinal chlorosis (11-20 % foliage affected) symptoms; 3, necrosis along the margin
213 of leaves (>2 cm wide in size; 21-40 % foliage affected); 4, necrosis along the entire margin of
214 the leaves and leaves curled with irregular shapes (41-75 % foliage affected); 5, interveinal
215 necrosis and most of the leaf areas necrotic (75-100 % foliage affected); 6, leaves starting to
216 defoliate. On the basis of foliar disease scores, the RILs were grouped as highly resistant with
217 scores <1.5, resistant with scores 1.50-2.00, moderately resistant with scores 2.01- 2.50,
218 susceptible with scores 2.51-3.00, and highly susceptible with scores >3.00 (Brar et al. 2011;
219 Hartman et al. 2004; Pudake et al. 2013).
220 Genotyping of RILs, genome map construction and QTL analysis
221 222 223 Genomic DNA was isolated by CTAB extraction (CIMMYT, 2005) and quantified by Nanodrop
224 ND-1000 (Thermo Fisher Scientific, Wilmington, DE, USA). DNA samples were diluted to a
225 final concentration of 100 ng DNA/µL. The DNA was subjected to Illumina Golden Gate
226 Genotyping assay at the Cregan lab (USDA ARS, Beltsville, MD). The 1,536 Universal Soy
227 Linkage Panel 1.0 was used for the Golden Gate assay (Fan et al. 2003; Hyten et al. 2008; Hyten
228 et al. 2010). Genetic linkage maps were constructed individually for the AX19286 and AX19287
10 229 populations using MAPMAKER V2.0. QTL for SDS resistance were mapped by conducting
230 composite interval mapping using the computer program QGene. A permutation test with 1,000
231 iterations was executed to calculate the critical LOD threshold values.
232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 11 252 Results 253 254 255 Foliar SDS scores in the stem cutting assays of the RILs suggest distinct patterns of
256 inheritance of SDS resistance from the two SDS resistant parents.
257 258 The highest disease severity was recorded for the susceptible control line Spencer (mean foliar
259 SDS score 4.4) followed by the susceptible parent of each of the two crosses, A95-684043 (mean
260 foliar SDS score, 3.7) (Fig. 1 and Fig. 2). The foliar SDS mean scores of the resistant parents
261 LS98-0582 and LS94-3207 were 2 and 1.6, respectively. We observed several transgressive
262 segregants (p <0.05) with foliar SDS scores exceeding either the susceptible or the resistant parent
263 (Fig. 1 and Fig. 2).
264 We observed that 12.5% of the RILs showed foliar SDS scores that were either similar to or
265 lower than the resistant parent in the AX19286 population; whereas in the AX19287 population,
266 only 5% of the RILs had foliar SDS scores either similar to or lower than the resistant parent
267 (Table 1). We also observed twice the number of susceptible RILs with foliar SDS scores >3.00
268 in the AX19287 population as compared to that in the AX19286 population (Table 1).
269 270 The foliar SDS scores in root feeding assays of the RILs suggest distinct patterns of
271 inheritance of SDS resistance from the two SDS resistant parents.
272 273 The overall segregation patterns of the RILs of the two populations were comparable for scores
274 obtained through stem cutting assays of the lines (Figs. 3 and 4; Table 2). In both methods, host
12 275 responses to cell-free Fv culture filtrate preparations were recorded as foliar SDS scores. The
276 foliar SDS scores in the root feeding assays were however higher than the scores observed in the
277 stem cutting assays (Figs. 3 and 4). In the root feeding assays, the foliar SDS scores were 4.13,
278 2.13, and 2.10 for the soybean lines A95-684043, LS94-3207, and LS98-0582, respectively. The
279 foliar SDS score was also higher for the susceptible check cultivar Spencer, 5.2. Again, more
280 foliar SDS resistant transgressive segregants (p <0.05) were observed for the AX19286
281 population compared to the AX19287 population.
282 Identification of QTL underlying the foliar SDS resistance
283 284 285 Composite interval mapping was conducted to identify QTL for SDS resistance based on the
286 foliar SDS scores obtained by stem cutting and root feeding assays and the results are presented in
287 Table 4 and Fig. 5. In the stem cutting assays, two major QTL for foliar SDS resistance were
288 identified from the study of RILs of the AX19286 population. The two QTL were mapped to
289 Chromosome 2 (MLG D1b) and 13 (MLG F). The LOD score for the QTL mapped to
290 Chromosome 2 (MLG D1b) was 3.6. This QTL contributes 8.4% of the total variation and was
291 mapped to the BARC-041581-08046 - BARC-046084-10230 interval (Supplemental Fig. 1; Table
292 3).
293 030899-06963 interval (Supplemental Fig. 1). The QTL interval was 5 cM with a LOD score
294 value of 4.4. The QTL explained 16% of the total variation (R2 = 16 %; Table 3). Five minor QTL
295 with LOD scores of ≤ 3.6 were mapped to the Chromosomes 8 (MLG A2), 16 (MLG J) and 20
296 (MLG I) in this population (Table 3). These five QTL had been previously identified in studies, in
The QTL on Chromosome 13 (MLG F) is located in the BARC-065495-19507 - BARC-
13 297 which different segregating populations were evaluated through root-infection with the pathogen
298 (Table 4).
299 In the root feeding assays of the AX19286 population, two QTL were mapped to
300 Chromosome 5 (MLG A1) and 20 (MLG I) with LOD score values of 3.0 and 2.5, respectively.
301 The two QTL however explained only 7.0 and 6.3% of the total variation, respectively (Table 3).
302 Both of the QTL identified in root feeding assays were minor and had been previously identified
303 (de Farias-Neto et al. 2007; Kassem et al. 2007; Meksem et al. 1999; Yamanaka et al. 2006)
304 (Table 4). Interestingly on Chromosome 20 (MLG I), the QTL obtained in stem cutting and root
305 feeding assays both mapped to the same region of 50.0 – 65.0 cM marker interval (Tables 3 and
306 4; Fig. 5).
307 In the AX19287 population, two major foliar SDS QTL were identified by the stem cutting
308 assays and mapped to Chromosome 7 (MLG M) and 9 (MLG K). The QTL on Chromosome 7
309 (MLG M) was located in the 30 cM marker interval between BARC-028517-05936 and BARC-
310 065255-19294. The LOD score for this QTL was 6.6. This QTL explains 12.2% of the total
311 variation. The QTL on Chromosome 9 (MLG K) was mapped to an approximately 6 cM marker
312 interval between BARC-056323-14257 and BARC-010353-00615 with a LOD score value of 6.2.
313 It explains 13% of the total variation (Table 3; Supplemental Fig. 2). From analysis of the foliar
314 SDS scores of this population gathered through root feeding assays, a major QTL was mapped to
315 a 23 cM marker interval between BARC-052017-11314 and BARC-020713-04700 on
316 Chromosome 20 (LG I) with a LOD score value of 5.4. It explains 15% of the total phenotypic
317 variation (Table 3). Two additional QTL were mapped to Chromosomes 6 (MLG C2) and 13
318 (MLG F) from analyses of the foliar SDS scores obtained from the root feeding assays of the
319 RILs of this population. Each QTL contributes 12% of the phenotypic variation (Table 3). We
14 320 identified three minor QTL mapped to Chromosomes 6 (MLG C2), 8 (MLG A2) and 10 (MLG O)
321 from analyses of this population through root feeding assays (Table 3). 322 In between the AX19286 and AX19287 populations, we did not identify major common
323 QTL. However, two minor common QTL with low LOD scores were mapped to the 14.99 - 67.86
324 cM interval of Chromosome 8 (MLG A2) and to the 74.12-78.05 cM interval of the Chromosome
325 13 (MLG F) (Table 3 and 4; Fig. 5). 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 15 343 344 Discussion 345 346 347 Considering the fact that foliar SDS is easily scorable, most of the QTL for SDS resistance
348 were discovered based on foliar SDS. We therefore investigated if (i) stem cutting and (ii) root
349 feeding assays can be applied to identify QTL for SDS resistance. Our study revealed several new
350 findings: (i) Although the same culture filtrates was used in phenotyping the RILs of two
351 independent segregating populations, the two assays resulted in discovering different QTL for
352 foliar SDS resistance. This observation suggests a possible differential uptake of toxins through
353 cut stems and roots from the culture filtrates. This observation warrants further study to determine
354 if such differences do exist. (ii) Some of the QTL identified in this study had been previously
355 identified by scoring for SDS resistance following root inoculation with the pathogen. Our results
356 thus establish that cell-free Fv culture filtrates can be employed to screen soybean germplasm to
357 map QTL for foliar SDS resistance. (iii) We identified two novel QTL, which could be due to
358 either novel SDS resistance mechanisms in the new parents included in this mapping study or
359 identified due to the novel methodologies used in this study.
360 Fifteen of the 17 QTL identified in this research from the two populations with stem cutting
361 and root feeding assays had been reported earlier from investigation of other segregating materials
362 through inoculation of roots with F. virguliforme (Table 3 and 4; Fig. 5). From the study of the
363 AX19286 population, seven and two QTL were identified through stem cutting and root feeding
364 assays, respectively (Table 3 and 4; Fig. 5). The study of the AX19287 population led to
365 identification of two and six QTL through stem cutting and root feeding assays, respectively. The
16 366 two QTL mapped to Chromosome 20 (MLG I) and Chromosome 7 (MLG M) using the AX19287
367 population are most likely novel (Table 3 and 4; Fig. 5).
368 In our study we have identified, nine QTL from the AX19286 (A95-684043 x LS94-3207)
369 population. The SDS resistant parent of this population, LS94-3207 was developed from the cross
370 between ‘Forrest’ with ‘Hartwig’. Both ‘Hartwig’ and ‘Forrest’ have been previously used to map
371 SDS resistance QTL (Chang et al. 1996; Hnetkovsky et al. 1996; Kassem et al. 2006; Kassem et
372 al. 2007, Kazi et al. 2008; Meksem et al. 1999; Njiti et al. 1998; Yuan et al. 2012). Six of the nine
373 QTL identified from the AX19286 population of this study, mapped to Chromosomes 13 (MLG
374 F), 8 (MLG A2), 20 (MLG I starting at 63.33 cM), 20 (MLG I starting at 55.09 cM), 16 (MLG J),
375 and 20 (MLG I starting at 50.11), were previously identified either from ‘Forrest’ (Njiti et al.,
376 2002) or ‘Hartwig’ (Kazi et al., 2008) (Table 3 and 4; Fig. 5). The rest of the nine QTL identified
377 from the AX19286 population, mapped to Chromosomes 5 (MLG A1; Yamanaka et al. 2006), 8
378 (MLG A2; Kassem et al. 2012) and 2 (MLG D1b; Kassem et al. 2012), were previously identified
379 from SDS resistant parents, other than ‘Forrest’ and ‘Hartwig’ (Table 3 and 4; Fig. 5). From the
380 population AX19287 (A95-684043 x LS98-0582), eight QTL were identified in our mapping
381 study. LS98-0582 was developed from two lines that had never been used in SDS resistance QTL
382 mapping studies. However, except for the two putative novel QTL mapped to Chromosomes 7
383 (MLG M) and 20 (MLG I) the rest of the six QTL were identified in previous studies (Table 3 and
384 4; Fig. 5).
385 In this investigation we have shown for the first time, that screening of mapping
386 populations can be conducted using cell-free Fv culture filtrates containing toxins to map QTL for
387 SDS resistance. The fact that 15 of the 17 QTL identified in this study mapped to genomic
388 regions that have previously been reported to contain QTL for SDS resistance, as stated above,
17 389 establishes that the use of cell-free Fv culture filtrates can be used for mapping QTL for SDS
390 resistance and identifying novel SDS resistant germplasm. Application of cell-free Fv culture
391 filtrates in the evaluation of soybean genotypes for foliar SDS resistance is more reproducible
392 than the traditional assays based on root inoculation with the pathogen. In this new approach, we
393 can control the amounts of culture filtrates with a greater precision and more uniformly across the
394 genotypes. In the root inoculation method, much of the variations can be attributed to variation in
395 the amount and distribution of inocula in soil and other environmental conditions generally
396 encountered during the assays. The inconsistency of inoculum levels can greatly influence the
397 outcomes of the root infection assay as high inoculum levels can lead to breakdown of SDS
398 resistance (Gray and Achenbach 1996; Yamanaka et al. 2006).
399 The positions of the two putative QTL identified in this study, one on Chromosome 5
400 (MLG A1) from analysis of the AX19286 population via root feeding assay, and the other one on
401 Chromosome 9 (MLG K) from the AX19287 population through stem cutting assay match with
402 the map locations of two previously reported QTL for SDS resistance (Yamanaka et al. 2006). It
403 is worth noting that the segregating population used in the previous study was screened in the
404 greenhouse through root inoculation with F. tucumaniae, the common SDS pathogen in the South
405 America. 406 In the present investigation, alleles for SDS resistance among some of the segregating RILs
407 were most likely contributed by both susceptible and resistant parents. Similar observations were
408 also made during mapping QTL for SDS resistance using segregating populations generated from
409 crosses between ‘Pyramid’ and ‘Douglas’, ‘Essex’ and ‘Forrest’, and ‘Ripley’ and ‘Spencer’ (de
410 Farias-Neto et al. 2007; Iqbal et al. 2001; Njiti et al. 2002). In these studies, the SDS susceptible
411 parents were shown to carry QTL for SDS resistance; for examples, ‘Douglas’ was shown to
18 412 carry QTL on Chromosome 6 (MLG C2), ‘Essex’ to carry QTL on Chromosome 6 (MLG C2) and
413 Chromosome 20 (MLG I), and ‘Spencer’ to carry QTL on Chromosome 4 (MLG C1). In the
414 population developed from the cross between ‘Misuzudaizu’ and ‘Moshidou Gong 503’, the
415 susceptible parent ‘Misuzudaizu’ contributed beneficial genes for SDS resistance mapped to
416 Chromosome 5 (MLG A1) (Yamanaka et al. 2006). Presumably, the contribution of beneficial
417 alleles for SDS resistance from both parents could explain the generation of transgressive
418 segregants. We speculate that similar mechanism might be involved in generating the
419 transgressive segregants in this investigation (Fig. 1-4). Transgressive RILs with increased SDS
420 resistance as compared to their respective resistant parents are ideal for realizing genetic gain for
421 SDS resistance in soybean. Several RILs with enhanced tolerance to toxins of the cell-free Fv
422 culture filtrates identified in this study could be used as new germplasm sources for genetic
423 improvement of soybean for foliar SDS resistance.
424 425 426 427 428 429 430 431 432 433 434 19 435 Author contribution statement
436 M.K.B. and S.R.C. devised the strategy and planned the experiments; S.R.C. generated the
437 recombinant inbred lines; S.S. lead the screening experiments, interpreted results and wrote the
438 manuscript; M.L. conducted screening experiments; N.S.A. conducted quantitative trait loci
439 mapping and analyzed the results; M.K.B. critically reviewed the results and edited the
440 manuscript.
441 442 Conflict of interest
443 The authors declare that they do not have any conflict of interest.
444 445 Acknowledgements
446 This research was conducted with the financial support from the United Soybean Board. We
447 thank Peter Lundeen and Kyle Vandermolen for their technical assistance in conducting the
448 experiment. We thank Dan Fortin for statistical analysis and interpretation of the data. We thank
449 Jordan Baumbach for her assistance in running the Mapchart program and David Grant for kindly
450 reviewing the manuscript. We thank Perry Cregan for his help with the Illumina Golden Gate
451 Genotyping assay.
452 453 454 455 456 457 20 References
458 459 460 Abbas HK, Boyette CD, Hoagland RE (1995) Phytotoxicity of Fusarium, other fungal isolates,
461 and of the phytotoxins Fumonisin, fusaric acid, and Moniliformin to Jimson weed.
462 Phytoprotection 76:17-25
Abeysekara NS, Bhattacharyya MK (2014) Analyses of the xylem sap proteomes identified
463 candidate Fusarium virguliforme proteinacious toxins. PLoS One 9: e93667
464 465 Aoki T, O'Donnell K, Homma Y, Lattanzi A (2003) Sudden-death syndrome of soybean is caused
466 by two morphologically and phylogenetically distinct species within the Fusarium solani
467 species complex--F. virguliforme in North America and F. tucumaniae in South America.
468 Mycologia 95:660-684
469 Aoki T, O'Donnell K, Scandiani MM (2005) Sudden death syndrome of soybean in South
470 America is caused by four species of Fusarium: Fusarium brasiliense sp. nov., F.
471 cuneirostrum sp. nov., F. tucumaniae, and F. virguliforme. Mycoscience 46:162–183
472 Baker RA, Nemec S (1994) Soybean sudden death syndrome: Isolation and identification of a
473 new phytotoxin from cultures of the causal agent, Fusarium solani. Phytopathology
474 84:1144
Baker RA, Tatum JH, Nemec S (1981) Toxin production by Fusarium solani from fibrous roots
475 of blight-diseased citrus. Phytopathology 71:951-954
476 477 Brar H, Bhattacharyya M (2012) Expression of a single-chain variable-fragment antibody against
478 a Fusarium virguliforme toxin peptide enhances tolerance to sudden death syndrome in
479 transgenic soybean plants. Mol Plant-Microbe Interact 25:817-824
21 480 Brar HK, Swaminathan S, Bhattacharyya MK (2011) The Fusarium virguliforme toxin FvTox1
481 causes foliar sudden death syndrome-like symptoms in soybean. Mol Plant-Microbe
482 Interact 24:1179-1188
Burmeister HR, Plattner RD (1987) Enniatin production by Fusarium tricinctum and its effect on
483 germinating wheat seeds. Phytopathology 77:1483-1487
484 485 Chang SJC, Doubler TW, Kilo V, Suttner R, Klein J (1996) Two additional loci underlying
486 durable field resistance to soybean sudden death syndrome (SDS). Crop science 36:1684-
487 1688
488 Cianzio SR, Arelli P, Uphoff M, Mansur L, Schultz S, Ruff R (2002) Soybean germplasm line
489 A95-684043. ISURF Docket # 02975. Iowa State University, Ames, IA 50011-1010.
490 U.S.A.
491 Cianzio SR, Bhattacharyya MK, Swaminathan S, Westgate GM, Gebhart G, Rivera-Velez N,
492 Lundeen P, Van Der Molen K, Pruski TI (2014) Registration of AR10SDS soybean
493 germplasm partially resistant to sudden death syndrome and resistant to soybean cyst
494 nematode. J Plant Regist 8:200–210
495 de Farias-Neto AL, Hashmi R, Schmidt M, Carlson S, Hartman GL, Li S, Nelson RL, Diers BW
496 (2007) Mapping and confirmation of a new sudden death syndrome resistance QTL on
497 linkage group D2 from the soybean genotypes PI 567374 and ‘Ripley’. Mol Breeding
498 20:53-62
499 Fan JB, Oliphant A, Shen R, Kermani BG, Garcia F, Gunderson KL, Hansen M, Steemers F,
500 Butler SL, Deloukas P, Galver L, Hunt S, McBride C, Bibikova M, Rubano T, Chen J,
501 Wickham E, Doucet D, Chang W, Campbell D, Zhang B, Kruglyak S, Bentley D, Haas J,
22 502 Rigault P, Zhou L, Stuelpnagel J, Chee MS (2003) Highly parallel SNP genotyping. Cold
503 Spring Harb. Symp. Quant. Biol. 68:69–78
504 Gray LE, Achenbach LA (1996) Severity of foliar symptoms and root and crown rot of soybean
505 inoculated with various isolates and inoculum rates of Fusarium solani. Plant Dis
506 80:1197-1199
Hartman GL, Huang YH, Li S (2004) Phytotoxicity of Fusarium solani culture filtrates from
507 soybeans and other hosts assayed by stem cuttings. Australasian Plant Pathol 33:9-15
508 Hashmi RY (2004) Inheritance of resistance to soybean sudden death syndrome. SDS. in ‘Ripley’
509 x ‘Spencer’ F5 derived lines. PhD Dissertation, Plant Biology, SIUC, Carbondale, USA.
510 511 Heatherly LG, Hodges HF (1998) Soybean production in the Midsouth. CRC Press, 416 pages
512 Hnetkovsky N, Chang SJC, Doubler TW, Gibson PT, Lightfoot DA (1996) Genetic mapping of
513 loci underlying field resistance to soybean sudden death syndrome (SDS). Crop science
514 36:393-400
515 Hyten D, Choi I-Y, Song Q, Specht J, Carter T, Shoemaker R, Hwang E-Y, Matukumalli L,
516 Cregan P (2010) A high density integrated genetic linkage map of soybean and the
517 development of a 1,536 universal soy linkage panel for quantitative trait locus mapping.
518 Crop Sci 50:960-968
519 Hyten D, Song Q, Choi I-Y, Yoon M-S, Specht J, Matukumalli L, Nelson R, Shoemaker R,
520 Young N, Cregan P (2008) High-throughput genotyping with the GoldenGate assay in the
521 complex genome of soybean. Theor Appl Genet 116:945-952
522 Iqbal MJ, Meksem K, Njiti VN, Kassem M, Lightfoot DA (2001) Microsatellite markers identify
523 three additional quantitative trait loci for resistance to soybean sudden-death syndrome
524 (SDS) in Essex by Forrest RILs. Theor Appl Genet 102:187-192
23 525 Ji J, Scott MP, Bhattacharyya MK (2006) Light is essential for degradation of ribulose-1,5-
526 bisphosphate carboxylase-oxygenase large subunit during sudden death syndrome
527 development in soybean. Plant Biol 8:597-605
528 Jin H, Hartman GL, Nickell CD, Widholm JM (1996) Characterization and purification of a
529 phytotoxin produced by Fusarium solani, the causal agent of soybean sudden death
530 syndrome. Phytopathology 86:277-282
531 Kassem MA, Meksem K, Wood AJ, Lightfoot DA (2007) Loci Underlying SDS and SCN
532 resistance mapped in the 'Essex' by 'Forrest' soybean recombinant inbred lines. Rev Biol
533 Biotech 6:2-10
534 Kassem MA, Ramos L, Leandro LF, Mbofung GYC, Hyten DL, Kantartzi SK, Grier IV RL, Njiti
535 VN, Cianzio S, Meksem K ( 2012) The ‘PI 438489B’ by ‘Hamilton’ SNP-based genetic
536 linkage map of soybean [Glycine max (L.) Merr.] identified quantitative trait loci that
537 underlie seedling SDS resistance. Journal of Plant Genome Sciences 1:18-30
538 Kassem MA, Shultz J, Meksem K, Cho Y, Wood AJ, Iqbal MJ, Lightfoot DA (2006) An updated
539 'Essex' by 'Forrest' linkage map and first composite interval map of QTL underlying six
540 soybean traits. Theor Appl Genet 113:1015-1026
Kazi S, Njiti VN, Doubler TW, Yuan J, Iqbal JM, Cianzio S, Lightfoot DA (2007) Registration of
541 the Flyer × Hartwig recombinant inbred line mapping population. J Plant Reg 1:175-178
542 543 Kazi S, Shultz J, Afzal J, Johnson J, Njiti VN, Lightfoot DA (2008) Separate loci underlie
544 resistance to root infection and leaf scorch during soybean sudden death syndrome. Theor
545 Appl Genet 116:967-977
Kern H (1972) Phytotoxins produced by Fusaria. In: Wood RKS, Ballio A, Graniti A (eds)
546 Phytotoxins in plant diseases. Academic Press, New York, pp 35–48
547 24 Leandro LF, Tatalovic N, Luckew, A (2012) Soybean sudden death syndrome—Advances in
548 knowledge and disease management. CAB Rev 7:1-14
549 550 Li S, Hartman GL, Widholm JM (1999) Viability staining of soybean suspension-cultured cells
551 and a seedling stem cutting assay to evaluate phytotoxicity of Fusarium solani f. sp.
552 glycines culture filtrates. Plant Cell Rep 18:375-380
553 Mbofung GYC, Harrington TC, Steimel JT, Navi SS, Yang XB, Leandro LF (2012) Genetic
554 structure and variation in aggressiveness in Fusarium virguliforme in the Midwest United
555 States. Can J Plant Pathol 34:83-97
556 Meksem K, Chancharoenchai K, Njiti VN, Chang SJC, Doubler TW, Nijti N, Cregan PE, Gray
557 LE, Gibson PT, Lightfoot DA (1999) Clustering among loci underlying soybean resistance
558 to Fusarium solani, SDS and SCN in near-isogenic lines. Theor Appl Genet 99:1131-1142
559 Njiti VN, Meksem K, Iqbal MJ, Johnson JE, Kassem M, Zobrist KF, Kilo VY, Lightfoot DA
560 (2002) Common loci underlie field resistance to soybean sudden death syndrome in
561 Forrest, Pyramid, Essex, and Douglas. Theor Appl Genet 104:294-300
562 Prabhu RR, Njiti VN, Bell Johnson B, Johnson JE, Schmidt ME (1999) Selecting soybean
563 cultivars for dual resistance to soybean cyst nematode and sudden death syndrome using
564 two DNA markers. Crop Sci 39:982-987
565 Pudake R, Swaminathan S, Sahu B, Leandro L, Bhattacharyya MK (2013) Investigation of the
566 Fusarium virguliforme fvtox1 mutants revealed that the FvTox1 toxin is involved in foliar
567 sudden death syndrome development in soybean. Current Genetics 59:107-117
Roy KW, Hershman DE, Rupe JC, Abney TS (1997) Sudden death syndrome of soybean. Plant
568 Dis 81:1100-1111
569 25 Rupe JC (1989) Frequency and pathogenicity of Fusarium solani recovered from soybeans with
570 sudden death syndrome. Plant Dis 73:581-584
571 572 Schmidt ME, Klein JH (2004) Registration of 'LS94-3207' soybean. Crop Sci 44:1482-1483
573 Song HS, Lim SM, Clark JM Jr. (1993) Purification and partial characterization of a host specific
pathotoxin from culture filtrate of Septoria glycines. Phytopathology 83:659-661
574 Stephens PA, Nickell CD, Kolb FL (1993) Genetic-analysis of resistance to Fusarium solani in
575 soybean Crop Sci 33:929-930
576 577 Tanaka T, Hatano K, Watanabe M, Abbas HK (1996) Isolation, purification and identification of
578 2,5-anhydro-D-glucitol as a phytotoxin from Fusarium solani. Journal of Natural Toxins
579 5:317-329
580 Vesonder RF, Heeseltine CW (1983) Metabolites of Fusarium. In Nelson PE, Toussoun TA,
581 Marasas WFO (eds). Fusarium: diseases, biology, and taxonomy. Pennsylvania State
582 University Press, University Park, pp 350–364
583 Wen Z, Tan R, Yuan J, Bales C, Du W, Zhang S, Chilvers MI, Schmidt C, Song Q, Cregan PB,
584 Wang D (2014) Genome-wide association mapping of quantitative resistance to sudden
585 death syndrome in soybean. BMC Genomics 15:809
586 Yamanaka N, Fuentes F, Gilli J, Watanabe S, Harada K, Ban T, Abdelnoor R, Nepomuceno A,
587 Homma Y (2006) Identification of quantitative trait loci for resistance against soybean
588 sudden death syndrome caused by Fusarium tucumaniae. Pesquisa Agropecuária
589 Brasileira 41:1385-1391
590 Yuan J, Rabia Bashir R, Salas G, Sharma H, Srour A, Lightfoot DA (2012) New approaches to
591 selecting resistance or tolerance to SDS and Fusarium root rot. Journal of Plant Genome
592 Sciences 1: 10-17
26 Table 1. Distribution of foliar SDS scores of two segregating populations gathered by stem-cut assays.
595 596 Population
AX19286
AX19287
597 598 599 600 601 602 603 604 605 <1.50*
(HR)
3
1
Percentage of RILs ¶
1.51 –
2.01 – 2.50* 2.51 – 3.00* > 3.00*
2.00* (R)
(MR)
(S)
(HS)
20
24
33
20
5
22
30
42
Mean (±
St.Dev)
2.50 (±0.71)
2.89 (±0.45)
Range
1.21-4.75
1.44-4.67
*= Foliar SDS score; HR = highly resistant; R = resistant; MR = moderately resistant;
S = susceptible; HS = highly susceptible
¶ 200 RILs from each population were categorized according to the mean disease score.
Table 2. Distribution of foliar SDS scores of two segregating populations gathered by root-feeding assays.
Population
AX19286
AX19287
606 607 608 609 610 611 612 613 614 615 <1.50*
(HR)
1
0
Percentage of RILs ¶
1.51 –
2.01 – 2.50* 2.51 – 3.00* > 3.00*
2.00* (R)
(MR)
(S)
(HS)
2
9
13
75
2
7
16
75
Mean (±
St.Dev)
3.49 (±0.66)
3.46 (±0.54)
*= Foliar SDS score; HR = highly resistant; R = resistant; MR = moderately resistant;
S = susceptible; HS = highly susceptible
¶ 200 RILs from each population were categorized according to the mean disease score.
616 617 28 Range
1.33 -5.42
1.56- 5.06
618 Table 3. Locations of QTL associated with SDS resistance determined by stem-cut and root-feeding assays.
Population
Assay
QTL
a
AX19286
Stem cut
1**
Root feeding
AX19287
Stem cut
Root feeding
b
d 2
Parent
contribution
4.4
16
A95-684043
14.99- 51.86
3.4
8.4
A95-684043
Sat_162 - GMENOD2B
51.86- 58.43
2.3
5.8
A95-684043
Barc-041581-08046 - BARC-046084-10230
Sat_139- Sat_069
93.34- 102.59
3.6
8.4
LS94-3207
20/ I
BARC-038869-07364 - BARC-059937-16229
Satt650- Sct_189
63.33-113.76
2.4
5.9
LS94-3207
6
20/ I
BARC-029803-06418 - BARC-041445-07985
Sat_268-Sat_104
55.09-65.62
2.5
6.2
LS94-3207
7
16/ J
BARC-016775-02320 - BARC-014745-01638
Sat_339-Satt280
27.99- 38.70
2.1
5.2
LS94-3207
8
05/A1
BARC-059081-15595 - BARC-065229-19273
Sat_171 – Sat_267
57.79 – 78.44
3.0
7.0
LS94-3207
9
20/ I
BARC-020245-04514 - BARC-038869-07364
Satt270- Satt650
50.11- 63.33
2.5
6.3
LS94-3207
10**
09/ K
Barc-056323-14257 - Barc-010353-00615
Satt167-Satt617
45.74- 50.93
6.2
13
LS98-0582
11**
07/ M
Barc-028517-05936 - Barc-065255-19294
Satt336-Sat_121
103.98-133.83
6.6
12.2
LS98-0582
12
08/ A2
BARC-042199-08209-BARC-044217-08646
Sct_067-AW132402
14.99 - 67.86
3.6
10.2
LS98-0582
13**
20/I
Barc-052017-11314 - Barc-057793-14926
Satt562-Satt127
22.84- 35.34
5.4
15
LS98-0582
14
06/ C2
Barc-010457-00640 - Barc-025767-05060
Satt307-Satt202
121.26- 126.23
3.2
8.6
A95-684043
15**
06/C2
Barc-021735-04194 - Barc-062515-17881
Satt376-Satt307
97.83-121.26
6.3
12
A95-684043
16
10/O
Barc-017045-02182 - Barc-060901-16948
Sat_221-Satt466
51.00-53.66
2.0
5
A95-684043
17**
13/ F
Barc-010501-00676 - Barc-042515-08280
Sct_033- Satt334
74.12-78.05
6.0
12
LS98-0582
Marker/interval
Flanking SSR markers
13/ F
BARC-065495-19507 - BARC-030899-06963
Satt334-Sat_317
72.97-78.05
2
08/ A2
Barc-031701-07215 - Barc-016685-03321
Sct_067- Sat_162
3
08/ A2
Barc-016685-03321 - BARC-038631-07266
4**
02/ D1b
5
Ch/MLG
Position (cM)
c
LOD
b
R (%)
619 620 a
c
621 assays. Threshold LOD cut off value in AX19287 was 3.8 and 3.7, respectively for stem cutting and root feeding assays (p= 0.05); per cent
Ch, Chromosome/ MLG, molecular linkage group; position of QTL based on the composite interval mapping (www.soybase.org); Logarithm
of odd ratios (LOD) at the QTL peak. Threshold LOD cut off value in AX19286 was 3.6 and 3.7, respectively for stem cutting and root feeding
d
29 622 623 contribution of particular QTL for the phenotypic variation in a particular population. **, Major QTL found in this study, which was selected based
on the threshold LOD cut off value (p= 0.05). Other QTL were treated as minor.
624 625 Table 4. QTL for SDS resistance reported prior to 2015 and identified in this investigation.
626 627 628 629 630 631 632 633 634 635 636 637 638 639 640 641 642 643 LG
Essex x
Forrest
A1
A1
A2
A2
A2
B1
B1
B2
C1
C2
C2
C2
D1a
D1b
D1b
D1b
D2
E
E
F
F
F
G
G
G
H
I
I
J
K
K
L
L
M
M
M
N
N
N
O
O
References 1-7
Pyramid x
Douglas
Hartwig x
Flyer
Ripley x
Spencer
PI 567354 x
Omaha
PI 438489B x
Hamilton
Asgrow X
Cordell
Misuzudaizu
x Moshidou
Gong 503
Elite soybean
cultivars
A95-684043 X
LS94-3207
3, 8
3, 9
3, 10 ,11
10
12
13
30 Minsoy X
Noir1
3
14
15
A95-684043 x
LS98-0582
a
Map
position (cM)
53.36-71.38
81.51-88.58
14.99 – 33.95
34.00 – 55.00
105.18
58.00 – 84.19
82.88- 102.55
25.9
65.08 – 90.11
56.50
82.23 – 107.58
121.26 – 150.0
75.25
3.79
74.79- 98.75
102.59- 112.62
85.15- 92.12
32.09
45.04
16.08 – 33.18
42.00 – 85.00
87.01
0.00 – 10.00
19.00 – 52.00
95.10 – 110.50
8.50
22.84- 36.40
46.22 – 99.83
11.74 - 42.50
44.99 – 61.66
104.79
36.04
56.13 – 92.66
67.6
95.44
103.98-133.83
45.13-53.25
92.55 - 102.05
136
55
130
644 a
The approximate map position is calculated based on the marker information available for the QTL/QTL peak from the literature and
it is based on the composite genetic map at www.soybase.org. Black boxes indicate the confirmed QTL from published literature.
645 646 647 648 , stem cutting assay;
, root feeding assay.
References: 1- Kassem et al., 2006; 2- Kassem et al., 2007, 3- Meksem et al., 1999; 4- Yuan et al., 2012; 5- Hnetkovsky et al.,1996; 6Njiti et al., 1998; 7- Chang et al., 1996; 8- Njiti et al., 2002; 9- Kazi et al., 2008; 10- de Farias Neto et al., 2007; 11- Hashmi, 2004;
12- Kassem et al., 2012; 13- Njiti and Lightfoot 2006; 14- Yamanaka et al. 2006; 15- Wen et al., 2014
649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 31 Figure Legends
665 666 667 Fig. 1 Frequency distribution of host responses to cell-free Fv culture filtrates evaluated
668 by stem cutting assay. Frequency distribution of foliar SDS scores of the F7 RILs of the
669 AX19286 population developed by crossing A95-684043 with LS94-3207 is presented.
670 Arrows indicate the parents and the most susceptible variety, ‘Spencer’. The values are
671 means of three biological replications. The disease symptoms were determined according
672 to Pudake et al. (2013).
673 674 Fig. 2 Frequency distribution of host responses to cell-free Fv culture filtrates evaluated
675 by stem cutting assay. Frequency distribution of foliar SDS scores of the F7 RILs of the
676 AX19287 population developed by crossing A95-684043 with LS98-0582 is presented.
677 Arrows indicate the parents and the most susceptible variety, ‘Spencer’. The values are
678 means of three biological replications. The disease symptoms were determined according
679 to Pudake et al. (2013).
680 681 Fig. 3 Frequency distribution of host responses to cell-free Fv culture filtrates evaluated
682 by root feeding assay. Frequency distribution of foliar SDS scores of the F7 RILs of the
683 AX19286 population developed by crossing A95-684043 with LS94-3207 is presented.
684 Arrows indicate the parents and the most susceptible variety, ‘Spencer’. The values are
685 means of three biological replications. The disease symptoms were determined according
686 to Pudake et al. (2013).
687 32 688 Fig. 4 Frequency distribution of host responses to cell-free Fv culture filtrates evaluated
689 by root feeding assay. Frequency distribution of foliar SDS scores of the F7 RILs of the
690 AX19287 population developed by crossing A95-684043 with LS98-0582 is presented.
691 Arrows indicate the parents and the most susceptible variety, ‘Spencer’. The values are
692 means of three biological replications. The disease symptoms were determined according
693 to Pudake et al. (2013).
694 695 Fig 5 The composite genetic map of the previously identified SDS resistance QTL (black
696 rectangles) and the QTL for SDS resistance identified in this study (gray rectangles). The
697 maps were constructed using Mapchart 2.2, marker information from SoyBase
698 (http://www.soybase.org), and data from published papers (see Table 4 for details).
699 700 701 702 703 704 705 706 707 708 709 710 33 Figure 1
Click here to download Figure Figure 1.ppt
Fig. 1
35
30
25
LS94-3207
Number of lines
20
15
A95-684043
10
5
Spencer
0
Foliar SDS score
Figure 2
Click here to download Figure Figure 2.ppt
Number of lines
Fig. 2
A95-684043
LS98-0582
Spencer
Foliar SDS score
Figure 3
Click here to download Figure Figure 3.ppt
Number of lines
Fig. 3
A95-684043
LS94-3207
Spencer
Foliar SDS score
Figure 4
Click here to download Figure Figure 4.ppt
Number of lines
Fig. 4
A95-684043
LS98-0582
Spencer
Foliar SDS score
Figure 5
Click here to download Figure Figure 5.ppt
Fig. 5
Fig. 5 - Continued
Fig. 5 - Continued
Fig. 5 - Continued
Fig. 5 - Continued
Fig. 5 - Continued
Supplemental Figures
Map Scal e is 10.0 cM per cm
Kos ambi Mappi ng Function
Seg ment Break Dist >= 999.9 cM
Seg ment Break Fra c >= 50 .0 %
Lo g-Likeli hood : -5 94.9 3
Iterati ons : 3
Lo nges t Seg cM : 147 .006
Lo op Tolera nce : 0 .010
In ner Tolera nce : 0.010
LG D1b
Dist
cM
2.5
0.7
16 .1
1.7
5.4
1.3
0.0
0.0
1.8
2.2
1.8
4.4
2.1
1.8
2.2
0.7
1.8
2.0
19 .6
16 .9
0.9
0.0
0.9
0.0
0.3
0.3
1.6
0.0
0.6
0.9
6.0
9.8
0.6
0.6
0.3
2.2
2.0
1.7
7.0
3.2
0.6
11 .5
3.9
0.6
5.6
0.4
Marker
Name
Barc-013 955-0 1283
Barc-041 773-0 8087
Barc-041 475-0 8016
Barc-020 103-0 4462
Barc-054 295-1 2453
Barc-018 187-0 2537
Barc-063 263-1 8286
Barc-065 787-1 9749
Barc-016 063-0 2049
Barc-016 573-0 2145
Barc-056 237-1 4178
Barc-051 039-1 0958
Barc-063 205-1 8270
Barc-037 223-0 6748
Barc-050 325-0 9554
Barc-019 149-0 3315
Barc-053 459-1 1856
Barc-057 545-1 4810
Barc-030 665-0 6919
Barc-031 301-0 7041
Barc-042 403-0 8252
Barc-014 233-0 3138
Barc-061 653-1 7307
Barc-057 357-1 4727
Barc-056 333-1 4265
Barc-010 849-0 0771
Barc-060 135-1 6407
Barc-063 685-1 8434
Barc-053 163-1 1724
Barc-060 541-1 6718
Barc-030 195-0 6829
Barc-055 839-1 3759
Barc-032 025-0 7239
Barc-041 581-0 8046
Barc-048 437-1 0593
Barc-031 263-0 7027
Barc-030 479-0 6875
Barc-021 647-0 4164
Barc-046 084-1 0230
Barc-017 895-0 2427
Barc-028 373-0 5856
Barc-054 149-1 2354
Barc-044 747-0 8795
Barc-059 321-1 5931
Barc-042 881-0 8448
Barc-019 805-0 4379
Barc-906 743-0 1012
0.0
Barc-051 405-1 1095
Data 0.0
Fil e: p op1 F.txt Barc-059 591-1 5929
0.3
Barc-046
112-1 0273
0.0 e is 10.0 cM per
Barc-060
Map Scal
cm 261-1 6509
1.4
Barc-050 009-1
787-0 7616
9886
81
.7
Barc-062
Kos ambi
Mappi
ng Function
Data
Fil
e:
p
op1
F.txt
0.0
Barc-043 173-0 8548
Seg ment
Dist
999.9
Map
Scal eBreak
is 10.0
cM>=
per
cm cM
0.0
Barc-019 977-0 3735
Seg ambi
ment Mappi
Break ng
FraFunction
c >= 50 .0 %
Kos
0.0 Break
Barc-016
943-0 2391
Seg
ment
>=
Lo g-Likeli
hood :Dist
-4 83.4
4999.9 cM
0.0
Barc-018
551-0 9507
2971
Seg
ment
Fra c >Barc-065
= 50 .0 %495-1
Iterati
ons Break
:3
Lo
g-Likeli
hood
:
-4
83.4
4
0.6
Barc-038
405-1 0071
Lo nges t Seg cM : 296 .341
Iterati0.0
ons : 3
Barc-023 293-0 5321
Lo op Tolera nce : 0 .010
Lo nges
cM : 296 .341
0.0t Segnce
Barc-024 749-0 5639
In ner
Lo
op Tolera
Tolera nce ::0.010
0 .010
0.9
Barc-024 765-0 5652
Dist
In ner
Tolera nce : 0.010Marker
0.3
Barc-016 891-0 2361
cM
Name
Dist
Marker
0.0
Barc-059
591-1 5929
cM
Name
0.0
Barc-060 261-1 6509
Barc-051
81 .7
Barc-062 955-1
009-1 1307
7616
Barc-051 955-1 1307
5.7
5.7
Barc-043 267-0 8567
79
.9
Barc-043 267-0 8567
8.4
8.4
0.7
Barc-014 411-0
411-0 1355
1355
0.7
Barc-014
1.6
Barc-049
723-0
9133
1.6
Barc-049
Barc-065 723-0
495-1 9133
9507
0.0
Barc-051 405-1 1095
0.3
Barc-046 112-1 0273
1.4
Barc-050
1.4
Barc-050 787-0
787-0 9886
9886
0.0
Barc-043 173-0 8548
0.0
Barc-043 173-0 8548
0.0
Barc-019 977-0 3735
0.0
Barc-019 977-0 3735
0.0
Barc-016 943-0 2391
0.0
Barc-016 551-0
943-0 2971
2391
0.0
Barc-018
Barc-050
235-0 9520
0.0
Barc-018
0.6
Barc-038 551-0
405-1 2971
0071
0.6
Barc-038 293-0
405-1 5321
0071
0.0
Barc-023
0.0
Barc-024
Barc-023 749-0
293-0 5639
5321
0.9
Barc-024
0.0
Barc-024 765-0
749-0 5652
5639
79
.9
0.3
Barc-016
0.9
Barc-024 891-0
765-0 2361
5652
0.0
Barc-059 591-1 5929
0.3
Barc-016 891-0 2361
0.0
Barc-060 261-1 6509
0.0
Barc-059
591-1
5929
81 .7
Barc-062 009-1 7616
0.0
Barc-060 261-1 6509
63
.8
81 .7
Barc-062 009-1 7616
Click here to download Figure Supplemental Figures.ppt
LG F
Barc-065 495-1 9507
Barc-065 495-1 9507
Barc-050 235-0 9520
2.6
1.2
0.7
0.7
79 .9
1.5
12 .3
63
.8
3.6
79
.9
8.3
0.7
0.6
0.0
0.0
5.5
0.7
2.6
0.3
1.2
6.2
0.7
2.5
0.7
3.6
1.5
63 .8
12 .3
3.6
8.3
0.7
0.6
63
.8
0.0
0.0
2.6
1.2
5.5
0.7
0.7
0.7
0.3
1.5
6.2
12 .3
2.5
3.6
3.6
8.3
2.6
Barc-030 899-0 6963
Barc-063 863-1 8477
Barc-042 515-0 8280
Barc-017 917-0 2456
Barc-013 633-0 1184
Barc-055 499-1 3329
Barc-024 045-0 4714
Barc-047 893-1 0417
Barc-032 717-0 9021
Barc-044 875-0 8829
Barc-031 567-0 7110
Barc-055 229-1 3122
Barc-045 235-0 9520
8913
Barc-050
Barc-055 613-1 3490
Barc-030 887-0
899-0 6033
6963
Barc-028
Barc-063 561-0
863-1 6521
8477
Barc-025
Barc-042 144-1
515-0 0286
8280
Barc-046
Barc-050
Barc-017 235-0
917-0 9520
2456
Barc-061
571-1
7276
Barc-013
633-0
Barc-038 355-1 1184
0050
Barc-055 499-1 3329
Barc-024 045-0 4714
Barc-047 893-1 0417
Barc-032 717-0 9021
Barc-044 875-0 8829
Barc-031 567-0 7110
Barc-055 899-0
229-1 6963
3122
Barc-030
Barc-045 863-1
235-0 8477
8913
Barc-063
Barc-042
Barc-055 515-0
613-1 8280
3490
Barc-017
Barc-028 917-0
887-0 2456
6033
Barc-013 633-0 1184
Barc-025 561-0 6521
Barc-055 499-1 3329
Barc-046
144-1
Barc-024 045-0 0286
4714
Barc-061 893-1
571-1 0417
7276
Barc-047
Barc-030
899-0
6963
Barc-032
Barc-038 717-0
355-1 9021
0050
Barc-063
863-1
8477
Supplemental Fig. 1. Composite interval mapping of major quantitative trait loci (QTL) associated with soybean sudden
death syndrome (SDS) for the AX19286 (A95-684043 X LS94-3207) recombinant inbred population. Genetic maps of the
chromosomes with the positions of the marker loci and the centimorgan (cM) distances between loci are shown on the left.
1.2
Barc-042 515-0 8280
(
QTL associated with SDS resistance)
0.7
0.7
0.6
0.0
0.7
0.0
1.5
5.5
12
.3
0.7
3.6
0.3
6.2
8.3
2.5
0.7
3.6
0.6
0.0
0.0
5.5
Barc-044 875-0 8829
Barc-031 567-0 7110
Barc-017 917-0 2456
Barc-055 229-1 3122
Barc-013
Barc-045 633-0
235-0 1184
8913
Barc-055
Barc-055 499-1
613-1 3329
3490
Barc-024
Barc-028 045-0
887-0 4714
6033
Barc-025 893-1
561-0 0417
6521
Barc-047
Barc-046 717-0
144-1 9021
0286
Barc-032
Barc-061
Barc-044 571-1
875-0 7276
8829
Barc-038 355-1 0050
Barc-031 567-0 7110
Barc-055 229-1 3122
Barc-045 235-0 8913
In ner Tolera nce : 0.010
Dist
cM
Data Fil e: Po p2 C2.txt
Map Scal e is 10.0 cM per cm
Kos ambi Mappi ng Function
Seg ment Break Dist >= 999.9 cM
Seg ment Break Fra c >= 50 .0 %
Lo g-Likeli hood : -2 92.7 1
Iterati ons : 3
Lo nges t Seg cM : 121 .573
Lo op Tolera nce : 0 .010
In ner Tolera nce : 0.010
LG C2
Dist
cM
Marker
Name
Data Fil e: Po p2 F.txt
Map Scal e is 10.0 cM per cm
Kos ambi Mappi ng Function
Seg ment Break Dist >= 999.9 cM
Seg ment Break Fra c > = 50 .0 %
Lo g-Likeli hood : -3 05.8 1
Iterati
ons : 3
11 2.4
Lo nges t Seg cM : 178 .738
Lo op Tolera nce : 0 .010
In ner Tolera nce : 0.010
LG F
Dist
cM
Barc-056 069-1 4029
9.0
1.2
0.0
4.8
10 .4
Barc-064 413-1 8929
Barc-024 137-0 4780
Barc-042 045-0 8161
Barc-016 957-0 2165
Barc-062 461-1 7811
3.3
11 .3
Barc-048 543-1 0663
10 .9
2.4
Barc-021 735-0 4194
Barc-044 133-0 8626
LG I
Dist
cM
Barc-900 926-0 0961
0.0
10 .3
7.9
0.4
3.8
39 .6
Marker
Name
Data Fil e: Po p2 I.txt
Map Scal e is 10.0 cM per cm
Kos ambi Mappi ng Function
Seg ment Break Dist >= 999.9 cM
Seg ment Break Fra c > = 50 .0 %
Lo g-Likeli hood : -9 4.40
Iterati ons : 3
Lo nges t Seg cM : 26.443
Lo op Tolera nce : 0 .010
In ner Tolera nce : 0.010
0.9
2.5
5.2
2.1
1.5
110.0
2.4
3.2
13 .9
0.0
0.0
Barc-051 955-1 1307
0.3
Barc-041 237-0 7944
1.2
0.9
Barc-050 235-0 9520
16 .5
0.0
Barc-024 569-0 4982
1.6
Barc-025 897-0 5144
6.0
Barc-038 413-1 0074
0.0
Barc-041 671-0 8065
Barc-030 853-0 6954
Barc-013 633-0 1184
Barc-010 501-0 0676
Barc-030 899-0 6963
Barc-017 917-0 2456
Barc-042 515-0 8280
Barc-055 499-1 3329
Barc-047 893-1 0417
25 .0
0.0
1.9
15 .3
1.2
Barc-055 939-1 3876
Barc-010 457-0 0640
Barc-062 515-1 7881
Barc-010 777-0 0746
Barc-025 767-0 5060
Barc-050 235-0 9520
7.9
0.4
3.8
11 .3
0.9
2.5
5.2
2.1
1.5
0.0
3.2
13 .9
Barc-024 569-0 4982
Barc-025 897-0 5144
Barc-038 413-1 0074
Barc-041 671-0 8065
Barc-030 853-0 6954
Barc-013 633-0 1184
Barc-010 501-0 0676
Barc-030 899-0 6963
Barc-017 917-0 2456
Barc-042 515-0 8280
Barc-055 499-1 3329
Barc-047 893-1 0417
Marker
Name
Data Fil e: Po p2 k.txt
Map Scal e is 10.0 cM per cm
Kos ambi Mappi ng Function
Seg ment Break Dist >= 999.9 cM
Seg ment Break Fra c >= 50 .0 %
Lo g-Likeli hood : -3 29.9 8
Iterati ons : 3
Lo nges t Seg cM : 87.689
Lo op Tolera nce : 0 .010
In ner Tolera nce : 0.010
LG K
Dist
cM
Barc-057 041-1 4548
Barc-057 087-1 4579
Barc-057 033-1 4543
17 .7
Barc-021 887-0 4232
Barc-027 552-0 6609
Barc-042 619-0 8314
Barc-052 017-1 1314
16 .3
Barc-046 570-1 2662
Barc-038 157-0 9983
Barc-057 793-1 49261.0
Barc-020 713-0 47001.5
Barc-027 790-0 66722.8
0.0
1.8
0.3
0.0
0.9
0.6
0.3
0.3
0.6
1.2
3.2
6.2
6.7
4.5
0.4
13 .1
3.1
5.3
Marker
Name
Barc-059 521-1 5876
Barc-028 040-0 6720
Marker
Name
Barc054 3471 2492
Data 1.6
Fil e: p op2-chro7 .txt
Barc041
Map Scal e is 10.0 cM per
cm 6670 8063
71 .0
.2 Mappi ng Function
Kos 14
ambi
Seg ment Break Dist >= 999.9 cM
0.0 Break Fra c >=
Barc039
Seg ment
50 .0 %1950 7465
Barc042
8150 8424
Lo g-Likeli hood : -1 53.1
2
Iterati ons : 3
Lo nges t Seg cM : 141 .621
Lo op Tolera nce : 0 .010
In ner Tolera nce : 0.010
Barc028 5170 5936
Dist
Marker
LG M
cM
71 .0
1.6
14 .2
54 .5
0.0
Name
Barc054 3471 2492
Barc041 6670 8063
Barc039 1950 7465
Barc042 8150 8424
Barc028 5170 5936
Barc-014 659-0 1609
Barc-026 035-0 5236
Barc-021 491-0 4133
Barc-028 249-0 5804
Barc-026 143-0 5278
Barc-057 243-1 4642
Barc-051 647-1 1184
Barc-048 623-1 0678
Barc-058 901-1 5494
Barc-061 977-1 7602
Barc-023 509-0 5422
Barc-042 823-0 8429
Barc-055 529-1 3356
Barc-056 323-1 4257
Barc-050 815-0 9887
Barc-061 897-1 7586
Barc-010 353-0 0615
Barc-014 813-0 1678
Barc-017 625-0 2635
Barc-044 609-0 8737
Barc-060 805-1 6910
Barc-038 909-0 7393
0.3
Satt250
Barc065 2551 9294
71 .0
54 .5
0.3
Satt250
Barc028 2551
5170 9294
5936
Barc065
54 .5
0.3
Satt250
Barc065 2551 9294
Supplemental Fig. 2. Composite interval mapping of major quantitative trait loci (QTL) associated with soybean sudden
death syndrome (SDS) for the AX19287 (A95-684043 X LS98-0582) recombinant inbred population. Genetic maps of the
chromosomes with the positions of the marker loci and the centimorgan (cM) distances between loci are shown on the left.
(
QTL associated with SDS resistance)