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REUNIONES Y CONGRESOS: resúmenes y trabajos presentados

ISSN On line 1851-4987 Estación Experimental Agropecuaria Manfredi

Año: 2012/2

Characterization of sunflower inbred lines (Helianthus annuus L.)

for high oleic acid content using SSR markers

Nancy G. Grandón1, Ma. Valeria Moreno1, Ma. Carolina Scorcione1, Jorge O. Gieco1, Daniel Alvarez2, Norma Paniego3, Ruth Heinz3

1Laboratorio de Biotecnología, INTA-EEA Manfredi. Ruta Nac. Nº 9. Km 636. (5988) Manfredi, Córdoba, Argentina. E-mail: nggrandon@manfredi.inta.gov.ar. 2Grupo Mejoramiento de Girasol, INTA-EEA Manfredi. Ruta Nac. Nº 9. Km. 636. (5988) Manfredi, Córdoba, Argentina. 3Instituto de Biotecnologia, CNIA-INTA Castelar. Los Reseros y Las Cabañas 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 oil´s 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.

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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 (Lájara 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 Škorić, 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 (Fernández-Martínez 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 (Pérez- 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.

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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 phenotypiccharacterization, 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

Parental

C16:0

C18:0

C18:1

C18:2

C18:3

C20:0

C20:1 22:0 C22:

1 C24:

0 O/L IY

R285 3.14 4.17 87.2 3.1 0.1 0 0.30 0.9 0.09 0.50 27.79

80.70

R023 6.02 4.21 25.4 62.8 0.1 0 0.15 0.7 0.11 0.26 0.40 130.65

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/µl template DNA, 1X PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs (Fermentas, Canada), 0.25 µM 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 64ºC-52ºC (35 cycles) and final extension at 72ºC. 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.

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Table 2: Polymorphic SSR between R285 and R023 parental lines Marker name

Genbank accession number

LG Allele in R285 (bp)

Allele in R023 (bp)

Marker name

Genbank accession number

LG Allele in R285 (bp)

Allele in R023 (bp)

HA102 BV727861 unknown 160 154 HA3582 BV728254 16 122 131

HA140 G67517 5 145 158 HA3627 BV728237 5 190 200

HA196 G67518 unknown 176 179 HA3632 BV728239 unknown 206 247

HA293 G67519 14 121 113 HA3691 BV728256 unknown 396 385

HA360 G67406 16 236 223 HA3700 BV728302 5 172 176

HA432 G67407 4 170 165 HA3703 - 4 213 215

HA557 BV728012 unknown 126 128 HA3847 BV728286 10 140 147

HA729 BV727945 unknown 125 128 HA3878 BV728314 7 199 232

HA790 BV727948 unknown 145 152 HA3886 BV728315 14 186 183

HA806 G67410 unknown 186 192 HA4011 BV728360 13 214 216

HA911 BV727888 8 180 178 HA4057 BV728333 3 208 206

HA969 BV727890 unknown 110 122 HA4149 BV728355 17 188 199

HA1108 BV727970 10 185 144 HA4239 BV728202 15 112 109

HA1155 BV727896 12 96 90 ORS59 BV012516 unknown 185 167

HA1848 BV728005 7 242 260 ORS229 BV012471 2 172 166

HA1938 BV728039 unknown 229 226 ORS297 BV006634 17 226 222

HA2057 BV728112 unknown 117 121 ORS316 BV005917 13 179 182

HA2063 BV728113 9 180 171 ORS371 BV006649 1 251 257

HA2077 BV727907 14 112 116 ORS420 BV005977 unknown 136 132

HA2145 BV728363 unknown 188 223 ORS457 BV005997 11 228 226

HA2178 BV728124 unknown 156 154 ORS510 BV006030 9 248 256

HA2191 BV728131 16 204 206 ORS607 BV006704 11 276 274

HA2193 BV727902 16 137 127 ORS613 BV006091 10 230 226

HA2237 BV728143 unknown 122 132 ORS662 BV006121 1 230 320

HA2272 BV728137 unknown 252 228 ORS687 BV006138 15 166 163

HA2348 BV728037 unknown 286 280 ORS727 BV006163 17 189 197

HA2448 BV728038 unknown 212 214 ORS799 BV006742 13 140 203

HA2499 BV728042 unknown 148 138 ORS807 BV006217 10 268 255

HA2500 BV728041 unknown 137 141 ORS844 BV006252 9 305 307

HA2547 BV728047 unknown 206 142 ORS878 BV006281 10 191 200

HA2605 BV728347 8 88 75 ORS887 BV006290 9 243 249

HA2714 BV728147 14 223 227 ORS894 BV006297 8 251 249

HA2946 BV728089 unknown 135 116 ORS899 BV006302 16 322 312

HA3070 BV728164 unknown 106 111 ORS959 BV006356 1 246 236

HA3272 BV728357 7 162 164 ORS1024 BV006408 5 227 231

HA3288 BV728101 unknown 193 218 ORS1065 BV006445 2 273 297

HA3298 BV728102 unknown 134 131 ORS1085 BV006463 12 277 281

HA3330 BV728245 13 148 146 ORS1146 BV006511 11 344 380

HA3348 BV728079 unknown 203 185 ORS1222 BV006579 3 440 442

HA3349 BV728219 unknown 259 265 ORS1247 BV006602 17 339 336

HA3373 BV728080 unknown 183 181 ORS1265 BV006617 9 228 226

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). Twenty-five 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.

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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

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Maringolo, C.A. 2007. Regiones cromosómicas asociadas a resistencia a podredumbre

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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

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Trabajo presentado al 18th International Sunflower Conference: Program & Abstracts. February 27 - March 1 - 2012. Mar del Plata, Argentina: ISA, ASAGIR, 2012, p. 217.

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Abril de 2012

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