REUNIONES Y CONGRESOS: resmenes y trabajos presentados

ISSN On line 1851-4987 Estacin Experimental Agropecuaria Manfredi

Ao: 2012/2 Characterization of sunflower inbred lines (Helianthus annuus L.) for high oleic acid content using SSR markers
Nancy G. Grandn1, Ma. Valeria Moreno1, Ma. Carolina Scorcione1, Jorge O. Gieco1, Daniel Alvarez2, Norma Paniego3, Ruth Heinz3
1

Laboratorio de Biotecnologa, INTA-EEA Manfredi. Ruta Nac. N 9. Km 636. (5988) Manfredi, Crdoba, Argentina. E-mail: nggrandon@manfredi.inta.gov.ar. 2Grupo Mejoramiento de Girasol, INTA-EEA Manfredi. Ruta Nac. N 9. Km. 636. (5988) Manfredi, Crdoba, Argentina. 3Instituto de Biotecnologia, CNIA-INTA Castelar. Los Reseros y Las Cabaas s/n. (1686) Hurlingham, Buenos Aires, Argentina.

ABSTRACT Sunflower seed oil contains a high proportion (about 90%) of unsaturated fatty acids: oleic (C18:1) and linoleic (C18:2) acids. They have been described as healthier, essential to human metabolism and potent hypocholesterolemic factors. High oleic acids levels can extend oxidative stability and life oils utility. Therefore, the increasing current market demand has been oriented for quality differentiated foods and focused sunflower breeding programs towards the development of improved cultivars with increased oleic acid content. The identification of QTL associated with this trait is a powerful molecular tool to facilitate sunflower breeding programs progress. The aim of this work was to generate a mapping population for the high oleic acid content from two contrasting inbred lines, identify polymorphic SSR markers between the two parental lines and characterize the population for oleic acid content. An F2 mapping population comprising 115 F2 individuals was developed from a cross between R285 (high oleic acid) and R023 (low oleic acid) inbred lines. Three hundred eighty six SSR markers which have been previously mapped in sunflower were used (275 HA and 111 ORS series). Genotyping was done by capillary electrophoresis and allele identification was performed using GeneMapper v. 4.0 software (Applied Biosystem). The mapping population of 115 F2 individuals was developed from a cross between R285 and R023 parental inbred lines. The experimental field design used was a randomized complete block assay with three replicates and the fatty acid composition was determined by gas chromatography. Eighty two polymorphic SSR markers (21.24%) between both parental inbred lines were identified. This first step allowed the selection of those polymorphic markers that were used to genotype the F2 mapping population. The phenotypic analysis revealed fatty acid content variation segregating within the mapping population (p<0.05). Three categories according to oleic acid content (low: 12.96 37.74%, intermediate: 41.35 58.7%, high: 63.88 87.91%) were identified. The generation of an F2 mapping population derived from contrasting inbred lines for oleic acid content, its phenotypic characterization and the identification of 82 polymorphic SSR markers between these parental lines, represent strategic tools to perform future QTL analysis and to generate molecular markers useful for marker assisted breeding.
1

The results of this work will allow the advance in the genetic behavior dissection of high oleic acid trait, enabling the detection of QTL and linked markers, useful as a molecular tool for the sunflower breeding programs of INTA. Key words: oleic acid QTL SSR markers sunflower. INTRODUCTION Cultivated sunflower (Helianthus annuus L.) seed oil contains near 90% of unsaturated fatty acids: oleic (C18:1) and linoleic (C18:2) acids. They have been described as healthier, essential to human metabolism and potent hypocholesterolemic factors (Kris-Etherton and Yu, 1997). Moreover, high oleic acids levels can extend oxidative stability and oil life utility. The increasing current market demand has been oriented for quality differentiated foods and focused sunflower breeding programs towards the development of improved cultivars with increased oleic acid content. Sunflower seed oil composition and especially oleic acid content, is highly influenced by environmental factors as the temperature and the amount of moisture in the soil (Ljara et al., 1990; Baldini et al., 2002; Rondanini et al., 2003). In addition, high oleic acid genes show unstable expression for oleic acid content in different genetic backgrounds and therefore phenotypic selection for the high oleic acid trait could be difficult across different environments and seasons (Demurin and kori, 1996). DNA markers are not influenced by the environment and therefore selection based on markers linked to the high oleic acid trait will allow further advance in breeding for this character. Identifying molecular markers linked to the high oleic acid trait (HOA) that can be further used in marker-assisted selection (MAS) would greatly contribute in developing stable mid and high oleic acid breeding lines (Van der Merwe, 2010). Molecular markers are powerful tools to study genetic variation and relate them to phenotypic variation (Varshney et al., 2005). SSRs (Simple Sequence Repeats) show high reproducibility and genomic covering, co-dominance, neutrality and they are highly polymorphic (Spooner et al., 2005). Therefore, they have been extensively used to study genetic variability in different organisms. In plants SSRs are being used to assess genetic variability in germplasm collections for making appropriate choice of parents to generate breeding populations, mapping and tagging of genes or QTL (Quantitative Trait Loci) identification for agronomic and disease resistance traits, genome mapping, MAS of promising lines and marker assisted backcrossing (MAB) during breeding programs, gender identification, studying the population structure and taxonomic, as well as in the analysis of phylogenetic relationships (Kalia et al., 2011). Regarding HOA breeding, Fick (1984) found that the high oleic character was determined by a co-dominant gen called Ol, whereas Urie (1985) described this gen as dominant. A second modificator gen (Ml) of Ol was detected as necessary for the character expression (Miller et al., 1987). Later, three complementary genes Ol1, Ol2, Ol3 were described (Fernndez-Martnez et al., 1989). The identification of QTL associated with this trait is a powerful molecular tool to facilitate sunflower breeding programs progress. SSR mapping to study high oleic character in cultivated sunflower was used in some recent works (Ebrahimi et. al., 2008; Haddadi et. al., 2010). Furthermore, AFLP (Amplified Fragment Length Polymorphism) and RFLP (Restriction Fragment Length Polymorphism) mapping in this species were used. Different QTLs for oleic acid (OA) and stearic acid (SA) content were detected (Prez- Vich et. al., 2002). The sunflower Active Germplasm Bank of INTA Manfredi (AGB-IM) preserves circa 1200 accessions of different geographic origins, including East Germany, Argentina, Armenia, Australia, Bolivia, Brazil, Bulgaria, Canada, Chile, China, Spain, United States, France, Greece, Hungary, Israel, Italy, Morocco, Moldova, Poland, Romania, Russia, Syria, Turkey, Uruguay and ex-Yugoslavia. The collection encompasses diverse accession categories including open-pollinized populations, composites, cultivars, inbred lines, etc.
2

In the last six years, efforts to exploit germplasm bank genetic resources with genomics-driven plant breeding methods such as linkage and association mapping are being made to characterize the bank and to detect the genetic bases underlying agronomical trait. In this work, initial studies and recent advances in high oleic acid breeding, including the fatty acid phenotype and the molecular characterization of the parental lines of a mapping population underlying fatty acid composition traits, are presented. The aim of this work was to identify polymorphic SSR markers between two cultivated sunflower inbred lines with contrasting high oleic acid content, to generate an F2 mapping population derived from these lines and to assess its phenotypic characterization, enabling the future detection of QTL and linked markers useful for the sunflower breeding programs. MATERIALS AND METHODS An F2 mapping population was developed from a cross between R285 (high oleic acid) and R023 (low oleic acid) inbred lines. Table 1 shows the fatty acid profile of the parental lines. Field trails were conducted in randomized complete block design with three replicates. The fatty acid composition of F4 seeds was determined by gas chromatography (AOCS, 1998). Statistical analysis (ANOVA) was made with Infostat software (Di Rienzo et al., 2010). Table 1: Fatty acid composition of inbred parental lines
Parenta l C16: 0 C18: 0 C18: 1 C18: 2 C18: 3 C20: 0 C20: 1 22:0 C22: 1 C24: 0 O/L 27.7 9 IY 80.7 3.14 4.17 3.1 0.1 0 0.30 0.9 0.09 0.50 R285 87.2 0 130. 6.02 4.21 62.8 0.1 0 0.15 0.7 0.11 0.26 0.40 R023 25.4 65 C16:0: palmitic acid %, C18:0: stearic acid %, C18:1: oleic acid %, C18:2, linoleic acid %, C18:3: linolenic acid %, C20:0: arachidic acid %, C20:1: eicosanoic acid %, C22:0: behenic acid %, C22:1: erucic acid %, C24:0: lignoceric acid %, O/L: ratio oleic/linoleic, IY: iodine index.

DNA extraction was made using NucleoSpin Plant II kit (Machery Nagel, Germany) based in 0.03 g of liophylized material. Inbred lines genotyping was performed with 386 SSR markers. PCR mix containing 15 ng/l template DNA, 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs (Fermentas, Canada), 0.25 M of each primer, 0.5 U of Taq DNA polymerase (Life Technologies, Argentina) was amplified using AB GeneAmp system 9700 termocycler (Applied Biosystems, USA). Amplification conditions were a touchdown of 64C-52C (35 cycles) and final extension at 72C. Flourescent fragments were resolved using electrophoresis through an ABI 3130xl DNA analyzer (Applied Biosystems, USA). Fragment sizing was done using the ROX 500 internal-lane standard (Applied Biosystems; ROX, 6-carboxy-x-rhodamine). GeneMapper 4.0 software (Applied Biosystems, USA) was used to score SSR alleles. RESULTS AND DISCUSSION An F2 mapping population comprising 115 individuals was obtained from a cross between the contrasting inbred lines R285 and R023 for oleic acid content. Eighty two (21.24%) of 386 SSR markers analyzed (54 HA set, 28 ORS set) were polymorphic between both inbred lines and will be used in genotyping of F2 mapping population (Table 2). Remaining SSR markers were monomorphic (24.6%), null alleles (7.5%), others showed nonspecific amplification products with complex profile or multiallelic (21.5%) and 25.13% could not be amplified.

Table 2: Polymorphic SSR between R285 and R023 parental lines

Marker name
HA102 HA140 HA196 HA293 HA360 HA432 HA557 HA729 HA790 HA806 HA911 HA969 HA1108 HA1155 HA1848 HA1938 HA2057 HA2063 HA2077 HA2145 HA2178 HA2191 HA2193 HA2237 HA2272 HA2348 HA2448 HA2499 HA2500 HA2547 HA2605 HA2714 HA2946 HA3070 HA3272 HA3288 HA3298 HA3330 HA3348 HA3349 HA3373

Genbank accession number

BV727861 G67517 G67518 G67519 G67406 G67407 BV728012 BV727945 BV727948 G67410 BV727888 BV727890 BV727970 BV727896 BV728005 BV728039 BV728112 BV728113 BV727907 BV728363 BV728124 BV728131 BV727902 BV728143 BV728137 BV728037 BV728038 BV728042 BV728041 BV728047 BV728347 BV728147 BV728089 BV728164 BV728357 BV728101 BV728102 BV728245 BV728079 BV728219 BV728080

LG

Allele in R285 (bp)

160 145 176 121 236 170 126 125 145 186 180 110 185 96 242 229 117 180 112 188 156 204 137 122 252 286 212 148 137 206 88 223 135 106 162 193 134 148 203 259 183

Allele in R023 (bp)

154 158 179 113 223 165 128 128 152 192 178 122 144 90 260 226 121 171 116 223 154 206 127 132 228 280 214 138 141 142 75 227 116 111 164 218 131 146 185 265 181

Marker name
HA3582 HA3627 HA3632 HA3691 HA3700 HA3703 HA3847 HA3878 HA3886 HA4011 HA4057 HA4149 HA4239 ORS59 ORS229 ORS297 ORS316 ORS371 ORS420 ORS457 ORS510 ORS607 ORS613 ORS662 ORS687 ORS727 ORS799 ORS807 ORS844 ORS878 ORS887 ORS894 ORS899 ORS959 ORS1024 ORS1065 ORS1085 ORS1146 ORS1222 ORS1247 ORS1265

Genbank accession number

BV728254 BV728237 BV728239 BV728256 BV728302 BV728286 BV728314 BV728315 BV728360 BV728333 BV728355 BV728202 BV012516 BV012471 BV006634 BV005917 BV006649 BV005977 BV005997 BV006030 BV006704 BV006091 BV006121 BV006138 BV006163 BV006742 BV006217 BV006252 BV006281 BV006290 BV006297 BV006302 BV006356 BV006408 BV006445 BV006463 BV006511 BV006579 BV006602 BV006617

LG

Allele in R285 (bp)

122 190 206 396 172 213 140 199 186 214 208 188 112 185 172 226 179 251 136 228 248 276 230 230 166 189 140 268 305 191 243 251 322 246 227 273 277 344 440 339 228

Allele in R023 (bp)

131 200 247 385 176 215 147 232 183 216 206 199 109 167 166 222 182 257 132 226 256 274 226 320 163 197 203 255 307 200 249 249 312 236 231 297 281 380 442 336 226

unknown 5 unknown 14 16 4 unknown unknown unknown unknown 8 unknown 10 12 7 unknown unknown 9 14 unknown unknown 16 16 unknown unknown unknown unknown unknown unknown unknown 8 14 unknown unknown 7 unknown unknown 13 unknown unknown unknown

16 5 unknown unknown 5 4 10 7 14 13 3 17 15 unknown 2 17 13 1 unknown 11 9 11 10 1 15 17 13 10 9 10 9 8 16 1 5 2 12 11 3 17 9

LG: linkage group according to Poormohammad Kiani et al. 2007. bp: base pairs

Fifty three out of 82 polymorphic SSR markers, have known position and localize on 16 sunflower linkage groups (LG) (Poormohammad Kiani et al., 2007) (Table 2). Twentyfive of them were also mapped by Ebrahimi et al. (2008) and Haddadi et al. (2010), on 14 LG. These authors reported that most important QTL for OA trait are located on LG 10 with several QTL controlling fatty acids content. Among polymorphic SSR markers detected in the present study, seven SSR mapping on LG 10 were identified.

The percentage of polymorphism (21.24%) was low comparing with previous works (Maringolo, 2007; Talia, 2008); however the polymorphic SSRs are considered informative for this study. Therefore, the incorporation of more SSR markers will contribute increase the probability of QTL detection for OA contains. Mean oleic acid content for the parental inbred lines and for all F4 families was determined as described in Materials and Methods. According to the OA content the F4 families analyzed in this work were categorized in three groups, low (12.96-37.74%), intermediate (41.35-58.70%) and high content (63.88-87.91%). Analyses of phenotypic variance (ANOVA) revealed variation for oleic acid content in the mapping population (p<0.05). The results of this work allowed the validation of 82 HA markers in cultivated sunflower inbred lines for high oleic traits. These have been used for QTL mapping for resistance to Sclerotinia head rot (Sclerotinia sclerotiorum (Lib.) De Bary) (Maringolo, 2007) and characterization of genomic regions involved in disease resistance (Talia, 2008). Currently they are being used to map QTL associated with tolerance to water stress. The identification of this polymorphic SSR marker set between these parental lines, along with the generation of the mapping population and its phenotypic characterization for OA content represent strategic tools to perform future QTL analysis and to generate molecular markers useful for marker assisted breeding. ACKNOWLEDGEMENTS Several grants from CONICET and INTA are gratefully acknowledged. REFERENCES Baldini, M., R. Giovanardi, S. Tahmasebi-Enferadi and G.P. Vannozzi. 2002. Effects of water regime on fatty acid accumulation and final fatty acid composition in the oil of standard and high oleic sunflower hybrids. Ital. J. Agron. 16(2):119-126. Demurin, Y. and D. kori. 1996. Unstable expression of Ol gene for high oleic acid content in sunflower seeds. In: Proc. 13th Int. Sunflower Association, Paris, France. p145150. Di Rienzo, J.A., F. Casanoves, M.G. Balzarini, L. Gonzalez, M. Tablada and C.W. Robledo, InfoStat version. 2010. Grupo InfoStat, FCA, Universidad Nacional de Crdoba, Argentina. URL http://www.infostat.com.ar Ebrahimi, A., P. Maury, M. Berger, S. Poormohammad Kiani, A. Nabipour, F. Shariati, P. Grieu and S. Sarrafi. 2008. QTL mapping of seed-quality traits in sunflower recombinant inbred lines under different water regimes. Genome 51:599-615. Fernndez-Martnez, J., A. Jimenez, J. Dominguez, J.M. Garcia, R. Grces and M. Mancha. 1989. Genetic analysis of the high oleic acid content in cultivated sunflower (Helianthus annuus L.). Euphytica 41:39-51. Fick, G. 1984. Inheritance of high oleic acid in the seed oil of sunflower. Proc. Sunf. Research Workshop. Bismark, N. Dacota, p9. Haddadi, P., B. Yazdi-samadi, N.B. Langlade, M.R. Naghavi, M. Berger, A. Kalantari, A. Calmon, P. Maury, P. Vincourt and A. Sarrafi. 2010. Genetic control of protein, oil and fatty acids content under partial drought stress and late sowing conditions in sunflower (Helianthus annuus). African Journal of Biotechnology Vol. 9 (40), p67686782. Kalia, R.K., M.K. Rai, S. Kalia, R. Singh, A.K. Dhawan. 2011. Microsatellite markers: an overview of the recent progress in plants. Euphytica 177:309334. Kris-Etherton, P.M. and S. Yu. 1997. Individual fatty acid effects on plasma lipids and lipoproteins: human studies. Am. J. Clin. Nutr. 65:1628-1644. Ljara, J.R., U. Diaz, and R.A. Quidiello. 1990. Definite influence of location and climatic conditions on the fatty acid conditions of sunflower oil. J. Amer. Oil Chem. Soc. 67:618-623.
5

Maringolo, C.A. 2007. Regiones cromosmicas asociadas a resistencia a podredumbre hmeda del captulo de girasol (Sclerotinia sclerotiorum (Lib.) De Bary). MSc diss. Universidad Nacional de Mar del Plata, Mar del Plata, Buenos Aires, Argentina. Millar, J.F., D.C. Zimmerman and B.A. Vich. 1987. Genetic control of high oleic acid content in sunflower. Crop. Sci. 27:923-926. Oficial Methods and Recommended Practices of the AOCS. 1998. 5th Ed. American Oil Chemists Society, Champaign, Illinois, USA. Prez-Vich, B., J.M. Fernndez-Martnez, M. Grondona, S.J. Knapp and S.T. Berry. 2002. Stearoyl-ACP and oleoyl-PC desaturase genes consegregate with quantitative trait loci underlying high stearic and high oleic acid mutant phenotypes in sunaflower. Theor. Appl. Genet. 104:338-349. Poormohammad Kiani, S., P. Talia, P. Maury, P. Grieu, R. Heinz, A. Perrault, V. Nishinakamasu, E. Hopp, L. Gentzbittel, N. Paniego, A. Sarrafi. 2007. Genetic analysis of plant water status and osmotic adjustment in recombinant inbred lines of sunflower under two water treatments. Plant Science 172:773787. Rondanini, D., R. Savin and A.J. Hall. 2003. Dynamics of fruit growth and oil quality of sunflower (Helianthus annuus L.) exposed to brief intervals of high temperature during grain filling. Field Crops Res .83:79 90. Spooner, D., R. Van Treuren and M.C. De Vicente. 2005. Molecular Marker for Genebank Management. Technical Bulletin N10. IPGRI, Rome. p127 Talia, P.M. 2008. Desarrollo de un mapa gentico y fsico integrado para girasol cultivado y su aplicacin en la caracterizacin de regiones genmicas involucradas en la resistencia a enfermedades. PhD diss. Universidad de Buenos Aires, Buenos Aires, Argentina Urie, L. 1985. Inheritance of high oleic acid in sunflower. Crop. Sci. 25:986-989. Van der Merwe, R. 2010. Genotype by environment interaction for oil quality in high oleic acid sunflower lines. PhD diss. University of the Free State Bloemfontein, South Africa. Varshney R.K., A. Graner and M.E. Sorrells. 2005. Genic microsatellite markers in plants: features and applications. TRENDS in Biotechnology Vol.23 No.1. p48-55.

Trabajo presentado al 18th International Sunflower Conference: Program &

Abstracts. February 27 - March 1 - 2012. Mar del Plata, Argentina: ISA, ASAGIR, 2012, p. 217.

Abril de 2012 Para suscribirse al boletn enve un email a : biblioteca@manfredi.inta.gov.ar Para cancelar su suscripcin enve un email a biblioteca@manfredi.inta.gov.ar ISSN: 1851-4987 Este boletn es editado en la Estacin Experimental Agropecuaria Manfredi Direccin Postal. Ruta Nac. N 9 Km. 636 (5988) Manfredi, Provincia de Crdoba Repblica Argentina. Tel. Fax: 03572-493053/58/61 Responsables: Julieta del R. Zabala y Norma B. Reyna (c) Copyright 2001 INTA - Instituto Nacional de Tecnologa Agropecuaria Todos los derechos reservados.