Published July 19, 2018 RESEARCH Genetic Diversity of a Germplasm Collection of Confectionery Sunflower Landraces from Spain B. Pérez-Vich, M. R. Aguirre, B. Guta, J. M. Fernández-Martínez, and L. Velasco* Instituto de Agricultura Sostenible (IAS-CSIC), Alameda del Obsipo ABSTRACT s/n, 14004 Córdoba, Spain. Received 14 Feb. 2018. Accepted 1 June Native to North America, non-oilseed, confec- 2018. *Corresponding author ([email protected]). Assigned to tionery sunflower (Helianthus annuus L.) has Associate Editor Stella Kantartzi. been traditionally cultivated in Spain since its Abbreviations: AMOVA, analysis of molecular variance; He, expected introduction from the New World in the 16th heterozygosity; IAS-CSIC, Instituto de Agricultura Sostenible–Consejo century. This created great genetic diversity Superior de Investigaciones Científicas; INIA, National Institute for in the form of local landraces, whose charac- Agricultural Research; PCoA, principal coordinates analysis; PCR, terization and conservation is of paramount polymerase chain reaction; PIC, polymorphic information content; importance. In this research, several seed and SSR, simple sequence repeat. plant traits, as well as flowering time, were evaluated in a collection of 192 landraces of confectionery sunflower from Spain. Evalu- unflower (Helianthus annuus L.) is currently a major crop ation was conducted in Córdoba, Spain, in Sat the world scale, with an annual production over 40 Tg of 2011, 2012, and 2013. The greatest variability grains (FAOSTAT, 2017). Around 90% of sunflower production is was observed for hundred-seed weight (4.21– dedicated to oil extraction, while the major part of the remaining 19.68 g), plant height (65.00–361.67 cm), head 10% corresponds to non-oilseed or confectionery sunflower. diameter (9.00–31.00 cm), and days to flow- The latter is mainly used for confection seeds, snack food, and ering (64.31–103.00 d). Genetic diversity in the collection was also evaluated with a set pet food (Fernández-Cuesta et al., 2012). Current oilseed and of 52 simple sequence repeat (SSR) markers, confectionery cultivars widely differ for achene characteristics. which produced 167 alleles, with an average Oilseed cultivars have small black seeds with low hull proportion of 3.2 alleles per locus (from 2 to 5). The SSR and high oil content, typically ?50%. Conversely, confectionery markers disclosed moderate variability in the cultivars have large seeds that are typically black or grey with germplasm collection, with average Nei’s white stripes, high hull proportion, and low oil content, typically expected heterozygosity of 0.29 (from 0.02 to <30% (Fernández-Martínez et al., 2009). 0.50). The analysis of the structure of the germ- Sunflower is native to North America, where it was grown by plasm collection revealed the existence of two Native Americans as far back as 3000 BC, mainly in the territory separated genetic pools, one of them widely corresponding to present-day Arizona and New Mexico (Putt, distributed throughout the country and another 1997). There is some discrepancy about the date when sunflower one tracing back to a reduced area in the north reached Europe and its origin. Putt (1997) stated that Spanish of Córdoba Province, where accessions with a high level of membership in this group are explorers introduced sunflower to Spain for the first time in 1510 still relatively common. Genetic diversity of from New Mexico. An alternative hypothesis is that Spanish this germplasm can be of use for widening the explorers found the sunflower plant in Peru in 1532, where it genetic base of cultivated sunflower. Published in Crop Sci. 58:1972–1981 (2018). doi: 10.2135/cropsci2018.02.0108 © Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1972 WWW.CROPS.ORG CROP SCIENCE, VOL. 58, SEPTEMBER–OCTOBER 2018 was worshipped as a sacred image of the sun god, and Table 1. Collection details of the 192 confectionery sunflower Spanish landraces used in the genetic diversity analysis. it was taken to Spain (Lehner and Lehner, 1960). The latter hypothesis is followed by the Dictionary of Spanish Accession Region Province Location Language of the Royal Spanish Academy, where sunflower NC020027 Castile–La Mancha Ciudad Real Daimiel is described as “native to Peru” (RAE, 2014). NC020055 Castile–La Mancha Ciudad Real Calzada de Calatrava NC020085 Castile–La Mancha Ciudad Real Valdepeñas After its introduction in the 16th century, sunflower NC020110 Castile–La Mancha Ciudad Real Villanueva de los Infantes has been traditionally grown in Spain as an ornamental NC020143 Castile–La Mancha Ciudad Real Terrinches plant in gardens and for the use of seeds as a snack. It NC020154 Castile–La Mancha Ciudad Real Alhambra has also been used as a border plant in small vegetable NC020165 Castile–La Mancha Ciudad Real Alhambra gardens (Pardo-Pascual, 1942). This has resulted in the NC020355 Castile–La Mancha Ciudad Real Almodovar del Campo development of numerous local landraces whose conser- NC020418 Castile–La Mancha Ciudad Real Membrilla vation is of paramount importance. However, the NC020569 Castile–La Mancha Guadalajara Siguenza introduction of improved cultivars in the second half of NC024377 Castile–La Mancha Albacete Balazote the 20th century, together with a gradual replacement of NC024407 Castile–La Mancha Albacete Povedilla sunflower by maize Zea( mays L.) as a border plant in small NC024429 Castile–La Mancha Albacete Villapalacios vegetable gardens, has led to a drastic genetic erosion of NC024454 Castile–La Mancha Albacete Bienservida confectionery sunflower germplasm in Spain. In the mid- NC024619 Castile–La Mancha Cuenca Zafra de Zancara NC024640 Castile–La Mancha Cuenca Pozorrubio 1980s, the then-called National Institute for Agricultural NC026188 Castile–La Mancha Toledo Ocaña Research (INIA) started the collection of local land- NC026190 Castile–La Mancha Toledo Ocaña races of confectionery sunflower, with the collaboration NC052903 Castile and León León La Pola de Gordon of J.M. Fernández-Martínez (Instituto de Agricultura NC056389 Murcia Murcia Lorca Sostenible–Consejo Superior de Investigaciones Cientí- NC069019 Castile and León Zamora Fuentesauco ficas [IAS-CSIC], Córdoba, Spain). Collection efforts NC069151 Castile and León Salamanca Cantalpino were interrupted until the 2000s, when new collection NC069457 Castile and León Zamora Justel expeditions were conducted by L. Velasco (IAS-CSIC, NC074507 Castile and León Soria Burgo de Osma-Ciudad Córdoba, Spain). This resulted in a germplasm collection of de Osma 196 accessions of confectionery sunflower that is currently NC074589 Castile and León Burgos Melgar de Fernamental NC076152 Castile and León Palencia Calzada de los Molinos maintained at the Centre of Plant Genetic Resources of NC077705 Extremadura Badajoz Fregenal de la Sierra INIA. Part of the collection was previously evaluated for NC079052 Andalusia Huelva Encinasola seed quality traits, which revealed large genetic variation NC083389 Andalusia Jaén Castillo de Locubin for some of the traits (Velasco et al., 2014). The objec- NC083633 Andalusia Cordoba Villanueva de Cordoba tive of the present research was to evaluate the germplasm NC086191 Andalusia Cadiz Villamartin collection for several seed and plant traits and to analyze NC087931 Andalusia Jaén Pozo Alcon genetic diversity using microsatellite (simple sequence NC087939 Andalusia Jaén Santiago-Pontones repeat [SSR]) molecular markers. NC094593 Andalusia Córdoba Belmez NC094594 Andalusia Córdoba Encinas Reales MATERIALS AND METHODS NC094595 Andalusia Córdoba Encinas Reales NC094597 Andalusia Córdoba Hinojosa del Duque Germplasm Collection NC094598 Andalusia Córdoba Espiel The study was based on 187 accessions of the INIA germplasm NC094599 Andalusia Córdoba Espiel collection of confectionary sunflower (http://wwwx.inia.es/ NC094600 Andalusia Córdoba Espiel coleccionescrf/PasaporteCRF.asp) plus five additional accessions NC094601 Andalusia Córdoba Alcaracejos collected by the authors. The accessions were collected in the NC094602 Andalusia Córdoba Córdoba following Spanish regions: Andalusia (129 accessions), Castile– NC094603 Andalusia Córdoba Benameji La Mancha (40), Extremadura (15), Castile and León (7), and NC094604 Andalusia Córdoba Hinojosa del Duque Murcia (1). The complete list of accessions used in this research NC094605 Andalusia Córdoba Espiel and their collection locations is given in Table 1. An overview of NC094606 Andalusia Córdoba Benameji the distribution of collection sites is shown in Fig. 1. NC094607 Andalusia Córdoba Hinojosa del Duque NC094608 Andalusia Córdoba El Viso Plant Cultivation and Tissue Collection NC094609 Andalusia Córdoba Hinojosa del Duque The accessions were grown in the field at the experimental NC094611 Andalusia Córdoba Villanueva del Duque farm of the Institute for Sustainable Agriculture in 2011, 2012, NC094612 Andalusia Córdoba Espiel and 2013. Most of the accessions were characterized by a high NC094613 Andalusia Córdoba El Viso level of self-incompatibility and high plant stature, >4 m in NC094614 Andalusia Córdoba Encinas Reales some cases. Both characteristics hindered to a large extent seed NC094615 Andalusia Córdoba Villanueva de Córdoba multiplication. All the plants were bagged and sib-mated using NC094616 Andalusia Córdoba Pozoblanco CROP SCIENCE, VOL. 58, SEPTEMBER–OCTOBER 2018 WWW.CROPS.ORG