QTL mapping of Sclerotinia basal stalk rot (BSR) resistance in sunflower using genotyping-bysequencing (GBS) approach Zahirul Talukder 1, Gerald Seiler 2, Qijian Song 3, Guojia Ma 4, Lili Qi 2 1 Department of Plant Sciences, North Dakota State University, Fargo, ND 2 USDA-ARS, Northern Crop Science Laboratory, Fargo, ND 3 USDA-ARS, Soybean Genomics and Improvement Lab, Beltsville, MD 4 Department of Plant Pathology, North Dakota State University, Fargo, ND
INTRODUCTION
GOAL OF THE PROJECT Overall objective To improve Sclerotinia disease resistance in the cultivated sunflower
Genotyping Plant Materials MATERIALS & METHODS Parents HA 441 (a maintainer line) and RHA 439 (a restorer line) Mapping population 106 F 7 -derived Recombinant Inbred Lines (RIL) developed through Single Seed Descent (SSD) method from the cross HA 441/RHA 439 Genotype-by-sequencing (GBS) technology was used for simultaneous discovery and genotyping of large numbers of single nucleotide polymorphism (SNP) markers GBS was conducted at the Genomic Diversity facility of Cornell University A total of 1,236 good quality polymorphic SNP markers were obtained from the GBS protocol for linkage mapping
Stalk rot evaluation MATERIALS & METHODS (CONT.) Environments 2012: Carrington, ND and Crookston, MN 2013: Crookston, MN 2014: Carrington, ND and Grandin, ND Field design Randomized complete block design (RCBD) Two replications each for 2012 and 2013 field trials Four replications each for 2014 field trials Field inoculation S. sclerotiorum mycelia grown on millet and 80 gm of inoculums deposited in furrows next to the rows of sunflower lines at V-6 growth stage Disease incidence (DI) scoring Percent of plants showing BSR lesion
QTL mapping Linkage Mapping MATERIALS & METHODS (CONT.) JoinMap 4.1 software was used for linkage analysis Maximum likelihood (ML) algorithm was used to determine marker order Final genetic map was constructed with 1,049 SNP markers placed on 17 linkage groups of sunflower genome QTL analysis was conducted separately for each environment and also using integrated data across environments Composite interval mapping (CIM) option of WinQTL Cartographer v2.5 was used for QTL mapping QTL analysis results were confirmed using PLABQTL v1.2
RESULTS Table 1. Analysis of variance (ANOVA) of BSR disease incidence (DI) for HA441/RHA 439 RIL population evaluated in five environments Component df Variance estimate Confidence limit (0.05) F/Z value lower upper Pr > F/Z Env 4 - - - 164.7 <.0001 Rep (Env) 9 σ 2 r = 13.19 5.42 66.09 1.71 0.0439 Genotype 107 σ 2 g = 161.81 119.21 232.30 5.92 <.0001 Genotype x Env 428 σ 2 ge = 61.15 39.35 107.90 3.95 <.0001 Error 963 σ 2 e = 345.73 315.86 380.07 Analysis was performed across all five environments using PROC MIXED of SAS version 9.3. All factors were treated as random effects except environment
Number of RILs 0 5 10 15 20 25 Carrington 2012 0 5 10 15 20 25 30 Crookston 2012 0 5 10 15 20 Crookston 2013 0 20 40 60 80 100 0 20 40 60 80 0 20 40 60 80 100 Disease incidence (%) Number of RILs 0 5 10 20 30 Carrington 2014 0 5 10 15 20 25 Grandin 2014 Legend RHA 439 HA 441 0 10 20 30 40 50 0 20 40 60 80 Disease incidence (%) Disease incidence (%) Fig. 1. Frequency distributions of BSR disease incidence (DI) in the HA441/RHA439 RIL population evaluated in five environments
0 20 40 60 80 100 0 40 80 Carrington 2012 0 20 40 60 ρ = 0.29 ** Crookston 2012 0 40 80 ρ = 0.55 *** ρ = 0.46 *** Crookston 2013 0 10 30 ρ = 0.53 *** ρ = 0.34 ** ρ = 0.46 *** Carrington 2014 0 20 60 ρ = 0.51 *** ρ = 0.48 *** ρ = 0.66 *** ρ = 0.57 *** Grandin 2014 0 20 60 0 20 40 60 80 100 0 20 40 60 0 20 40 60 80 100 0 10 20 30 40 0 20 40 60 80 Fig. 2. Spearman s rank correlation of BSR disease incidence (DI) among environments evaluated for HA 441/RHA 439 RIL population
RESULTS (CONT..) Table 2. Significant Sclerotinia BSR resistance QTL identified in the HA 441/RHA 439 RIL sunflower population QTL LG Peak position (cm) Left Flanking markers Right R 2 1-LOD interval Qbsr-4.1 4 32.0 S4_147688288 S4_135190076 6.4 6.5 Qbsr-9.1 9 45.0 S9_153762438 S9_158145790 9.3 1.5 Qbsr-10.1 10 66.5 S10_288646223 S10_281294015 31.6 1.6 Qbsr-11.1 11 83.2 S14_148877201 S14_148877253 7.7 3.5 Qbsr-16.1 16 87.3 S16_157591485 S16_137964301 10.5 10.4 Qbsr-17.1 17 42.5 S17_228661362 S17_238998311 14.6 11.5
Qbsr-4.1 Qbsr-9.1 Qbsr-10.1. Qbsr-11.1 Qbsr-16.1 Qbsr-17.1 LG4 LG9 LG10 LG11 LG16 LG17 S4_31145092 0.000 S4_97655106 0.044 S4_97635847 0.491 S4_109632392 1.025 S4_88777357 1.037 S4_29937672 2.234 S18_285291457 S18_285291452 S18_285291455 S18_285291450 2.280 S4_29937645 2.288 S12_178870935 3.408 S4_45037620 5.470 S4_151599492 S4_56666249 S4_48439385 6.840 S18_51321740 7.357 S4_151599498 8.133 S4_58630500 9.510 S4_58662210 10.408 S4_58009804 10.568 S4_144516218 12.538 S14_121837369 S14_121841350 16.005 S4_101760955 17.231 S17_168121296 17.543 S4_79848575 18.082 S4_163953111 18.584 S4_107675211 18.957 S4_79910756 19.376 S18_36203479 19.636 S4_58726331 20.051 S4_84269148 S4_84269231 S4_84269171 20.541 S4_68619883 20.751 S4_68619845 21.085 S4_124807891 22.185 S4_140766705 22.859 S4_86922062 25.656 S4_132107538 25.924 S4_147688312 26.146 S4_129702941 26.468 S4_147690966 27.073 S4_132107549 28.097 S4_147688288 29.129 S4_135190076 33.143 S4_135190088 33.153 S1_26427234 S1_26427229 34.385 S4_155607259 S4_155607293 37.911 S9_644392 0.000 S9_1805927 1.055 S9_7477642 S9_7477651 4.223 S5_22293772 S5_22293715 11.686 S9_12972523 S9_15276575 S9_12972505 17.085 S9_25239528 25.069 S9_113434846 30.099 S9_50867496 S9_50782344 31.109 S9_81114421 31.599 S9_58823711 31.612 S9_46500495 S9_46500524 31.624 S9_76311935 33.576 S9_95276878 S9_95276886 33.854 S9_231703273 33.912 S9_89227722 34.002 S14_84055197 S9_89227691 S9_89227698 S9_89227717 S9_111269940 34.123 S9_129033402 S9_7368725 S9_129989533 S9_126831835 S9_129989525 S9_126831848 40.401 S9_126831858 S9_135225376 41.571 S9_142793246 S9_144600502 43.437 S9_153762386 44.953 S9_153762438 44.955 S9_158145790 46.586 S9_165008146 50.486 S9_170141410 52.292 S9_175124750 54.143 S9_174930226 S9_174930256 55.342 S9_174930209 S9_174930230 S9_174520670 S9_174930273 S9_174930275 55.832 S9_197928394 80.752 S9_198646722 S9_198646732 81.337 S9_189078469 82.036 S9_201855349 82.701 S18_340891152 82.709 S18_207227572 S9_205883017 S9_205882937 83.454 S9_213164953 S9_213164980 S9_213164982 S9_218465989 S9_218466008 85.501 S9_212940630 S9_212940659 85.603 S9_212510834 85.759 S9_214866446 85.890 S9_218466078 85.987 S9_227995116 S9_227995062 86.477 S9_225256464 S9_225256465 86.480 S9_234471958 89.909 S9_235042673 90.422 S9_236488316 90.436 S9_237560438 92.454 S9_242156583 99.027 S9_242156691 S9_242156592 99.562 S9_244813371 107.404 S9_244813342 S9_244813370 108.511 Figure 3. QTL in the linkage map S9_252173286 S9_252173312 S9_252173324 S9_252173366 137.383 S10_12611643 0.000 S10_13821302 S10_13821433 0.520 S10_13520561 1.569 S1_73676340 1.840 S10_13520542 2.085 S10_29486746 14.558 S10_29486735 S10_29486739 S10_29486740 14.563 S10_29486774 S10_29486792 S10_29486793 14.645 S10_24712547 15.704 S10_155896896 16.472 S10_33305037 16.564 S10_29043188 20.383 S17_209554231 25.165 S10_51995451 S10_51995465 31.759 S10_48854361 32.542 S10_48511591 32.556 S10_55219786 S10_55219794 33.074 S10_68260501 33.431 S10_214091914 34.735 S10_186448314 S10_203859523 36.520 S10_186181722 S10_180598505 S10_194821521 S10_194821559 S10_178090551 S10_84090104 36.521 S10_95977932 S10_113548928 S10_186342515 36.618 S10_150591982 36.722 S10_180974546 36.775 S2_89833593 36.858 S10_183532127 36.931 S10_195528451 37.006 S10_151061965 37.754 S10_77103494 38.441 S10_63981519 S10_63981551 38.619 S10_136184591 38.627 S10_35482253 38.648 S10_170702517 38.675 S10_164762225 38.726 S10_161453490 S10_161453520 39.942 S10_161453440 S10_161453447 S10_161453455 S10_161453473 39.985 S10_69814996 40.624 S10_123613821 41.075 S10_225357703 S10_225357728 S10_225357741 S10_225357750 41.793 S10_138424052 S10_138424072 41.916 S10_80335890 41.947 S10_122221562 41.954 S10_91859457 41.974 S10_138424055 41.976 S10_84185265 42.263 S10_77515376 42.483 S10_115818130 42.827 S10_89143058 42.832 S10_77103506 42.837 S10_147954110 42.847 S10_111598076 S10_115551094 42.929 S10_243379609 42.968 S10_194833129 43.006 S9_12197049 43.048 S10_171159013 S10_171159030 43.720 S10_180598406 44.406 S10_136480584 44.613 S10_113948286 S10_137966058 44.893 S10_118648111 S10_118648112 S10_118648133 S10_142588798 S10_202082122 45.124 S10_107467487 45.391 S10_170702489 45.670 S10_95977973 45.843 S10_197109971 45.857 S10_141461186 S10_141461189 S10_163756997 S10_163757016 S10_163757037 S10_217499333 S10_180400602 S10_147753272 S10_165227510 45.867 S10_113923671 S10_101942670 S10_156620325 S10_171158979 45.901 S10_190784209 47.742 S10_78806330 48.031 S10_78698491 48.444 S10_53127452 48.883 S10_116492546 48.919 S10_77819300 S10_77819306 S10_77819333 S10_77819334 49.880 S10_142891776 49.960 S10_197451375 50.057 S10_200952383 S10_184248469 51.390 S10_75151456 51.400 S10_194123689 51.449 S10_199645225 54.286 S8_41092026 54.687 S8_41092038 54.789 S10_213363776 54.859 S8_41092068 54.904 S10_211718712 54.995 S10_229736312 55.031 S10_242593555 56.152 S10_250138221 57.019 S10_150172489 57.909 S10_248236249 58.792 S10_259165916 60.135 S10_247683641 S10_247683660 60.149 S10_253471019 60.708 S10_260572826 61.980 S10_260572720 62.009 S10_260572805 62.011 S10_260572750 62.100 S10_266039361 63.736 S10_266039402 S10_266039405 S10_266039410 63.784 S10_273692455 S10_273692469 64.989 S10_271770211 65.055 S10_273508550 66.004 S10_288646223 S10_288646244 S10_288646251 66.498 S10_281294015 67.495 S10_68632751 S10_68632796 68.496 S10_121117479 S10_282979897 S10_282979911 68.986 S10_287274458 68.996 S10_287274466 69.919 S10_68632742 S10_68632744 69.934 S10_68632740 S10_68632741 69.968 S10_282979830 70.418 S10_287942242 70.819 S10_287788432 70.822 S14_114847790 S14_114847770 73.246 S10_291449460 S10_291449466 S10_291427082 S18_31459139 74.030 S11_1708014 S11_1708019 0.000 S11_1856627 S11_1856587 S11_1856625 S11_1856533 S11_1856586 1.513 S11_5419963 4.481 S11_8068067 S11_8068077 S11_8068006 6.201 S11_45311886 46.315 S11_45311878 46.879 S11_64516821 51.863 S11_52815125 S11_52815111 51.866 S11_64238570 52.158 S11_179313107 52.353 S11_64516748 52.360 S11_6897848 S11_6897850 54.399 S11_86052300 57.400 S15_73005964 S15_73005878 58.553 S11_54636693 59.443 S11_54636663 59.467 S11_129907818 60.798 S11_110888992 S11_110889049 62.052 S11_129907820 S11_119217815 S11_119217799 S11_129907840 S11_107011050 S11_106414735 63.046 S11_106414729 S11_107011078 S11_170010902 S15_62034877 S11_170688942 75.589 S11_170010905 75.610 S18_251450812 78.713 S18_228859350 80.555 S11_175992023 80.761 S14_148877201 83.216 S14_148877253 83.359 S11_185809691 S11_185809716 84.356 S12_103520906 92.553 S12_103520860 92.590 S11_45838127 94.186 S18_127310864 S18_127310857 94.213 S11_198240398 S11_198240352 107.685 S11_196771216 117.483 S11_48512806 S11_48512784 118.616 S11_48512700 119.120 S16_1632953 0.000 S14_3858175 10.005 S18_275204098 34.307 S18_288887960 42.062 S16_145718327 42.161 S16_49811030 44.243 S16_50191075 S16_50191076 45.167 S16_84315726 46.708 S16_50191015 46.818 S16_48467973 S16_48467980 S16_48467986 47.760 S16_50904940 S16_50904957 S16_50904960 S16_50904964 52.469 S16_93128012 S16_93128063 52.743 S18_147148283 54.055 S18_147148196 55.119 S14_84248089 55.126 S16_66999252 S16_66999259 S16_66999276 55.616 S18_345897555 56.134 S16_21476474 58.342 S16_65576805 S16_65576818 S16_65576823 S16_65576845 58.872 S18_339776844 59.447 S11_25658764 61.296 S11_25658807 61.883 S10_67640756 62.726 S18_312262489 63.506 S2_19750370 63.518 S18_167573265 63.525 S1_87168254 S1_87168269 64.234 S18_101318310 64.549 S1_102731875 64.892 S16_164544798 65.164 S10_193762907 65.372 S10_194225104 65.507 S16_82607789 65.554 S18_46696184 66.588 S3_112915821 66.630 S16_199279958 S16_199279959 66.701 S16_130591100 67.990 S16_169259204 S16_169259206 S16_169259209 S16_169259226 S14_138593035 68.634 S16_95780947 70.148 S16_209384536 70.213 S16_128088513 71.065 S9_76686846 71.092 S16_101987202 71.135 S16_128088499 71.367 S18_357681143 S18_357681171 71.622 S18_18087856 73.425 S18_18087987 73.451 S16_108372619 75.102 S16_111827476 75.107 S16_114881297 75.731 S16_113502097 76.388 S16_45544415 78.816 S18_173956673 S16_157591485 S18_173956574 S18_173956588 87.268 S16_137964301 87.876 S16_160048607 92.074 S16_167725831 97.704 S16_190747132 100.369 S11_189238486 100.896 S16_7297182 103.068 S16_192348104 S16_192348103 S16_192348130 S16_192348141 103.569 S16_192273156 S16_192273171 103.581 S16_195546279 105.151 S16_195546238 105.680 S16_218376259 S16_218376260 S16_218376286 S16_218376150 S16_218376183 138.031 S17_19817784 S17_19817789 S17_19817831 0.000 S17_68137388 S17_68081670 21.288 S17_130634469 21.290 S17_228661362 42.485 S6_7451648 43.483 S17_238998311 43.504 S17_150544445 S17_238998317 S4_55631481 S17_238998336 S17_230083994 43.526 S18_307160683 50.332 S18_307160690 50.871 S17_257739126 60.325 S17_260425253 68.911 S18_269660990 S18_269660954 98.461 Legend Environment specific QTL Carrington 2012 Carrington 2014 Crookston 2013 Crookston 2012 Grandin 2014 QTL detected in all environments QTL detected in combined analysis
70 60 50 40 30 20 10 0 Cargill 270 Croplan 305 HA 441 RHA 439 RIL22 RIL25 RIL61 RIL87 RIL93 RIL102 RIL03 RIL10 RIL58 RIL96 Disease Incidence (%) Susceptible check Resistant check Parents Most resistant RILs Most susceptible RILs Fig. 4. Mean BSR disease incidence (DI) of the most resistant and most susceptible RILs in seven environments during 2012-2015
70 60 50 40 30 20 10 0 Cargill 270 Croplan 305 HA 441 RHA 439 RIL22 RIL25 RIL61 RIL87 RIL93 RIL102 RIL03 RIL10 RIL58 Disease Incidence (%) RIL96 BSR QTL Susceptible check Resistant check Parents Most resistant RILs Most susceptible RILs Qbsr-10.1 a A A A A A A A a a a a Qbsr-17.1 B b B B B B B B b b b b Fig. 5. QTL allele distribution in the parents and in the RILs
Conversion of flanking SNPs into AS-PCR primers SNP marker: S10_288646223 SNP marker: S10_281294015 Fig. 6. PCR gel picture of two SNP markers linked to the BSR resistance QTL amplified using allele-specific primers designed for length polymorphism
FUTURE PLAN Conduct 2 nd year field evaluation of HA 89/H. argophyllus advanced backcross (AB)-QTL population for BSR resistance Conduct 1 st year field evaluation of HA 89/H. praecox AB-QTL population for BSR resistance Genotyping and linkage map construction of AB- QTL population derived from the cross of HA 89/H. argophyllus
ACKNOWLEDGEMENT Prof. Kevin McPhee Michelle Gilley Angelia Hogness Christopher Misar Megan Ramsett Dr. Tom Gulya National Sclerotinia Initiative for financial support