GCB Bioenergy (2011) 3, 25–36, doi: 10.1111/j.1757-1707.2010.01079.x OPINION Genomic resources and transcriptome mining in Agave tequilana JUNE SIMPSON*,AI´ DA MARTI´ NEZ HERNA´ NDEZw ,MARI´ AJAZMI´ NABRAHAM JUA´ REZ*,SILVIADELGADOSANDOVAL*,ALFREDOSA´ NCHEZ VILLARREALw and CELSO CORTE´ SROMERO* *Department of Plant Genetic Engineering, Cinvestav Irapuato, Km. 9.6 Libramiento Norte Carretera Irapuato-Leo´n, Apdo, Postal 629, Zip Code 36821. Irapuato, Guanajuato, Me´xico, wColegio de Postgraduados Campus Campeche, Km 17.5 Carretera federal Haltunche´n-Edzna´, Zip code 24750. Sihochac, Champoto´n, Campeche, Mexico Abstract Different species of Agave are grown commercially in Mexico for the production of alcoholic beverages and fibers. These plants are well adapted to dry, arid conditions and can be cultivated on land which is unsuitable for staple crops such as corn or beans. A substantial amount of waste material in the form of discarded leaves or stem tissue is produced from commercial applications and an attractive alternative proposition is to employ this waste for bioenergy production. To date little basic research at the molecular- genetic level in agave has been carried out and more detailed and directed work in this area is necessary in order to fully develop agave species as bioenergy crops. The current genomic resources available for agave and the potential for transcriptome mining in relation to bioenergy applications are discussed. Keywords: A. tequilana, bioenergy, data mining, genomic resources, transcriptome Received 21 July 2010; revised version received 16 October 2010 and accepted 2 November 2010 annually with consequently less economic impact (Con- Introduction sejo Mexicano Regulador de la Calidad del Mezcal, Agave plants have been exploited in Mexico since 2009, http://www.comercam.org). For growers how- around 10 000–8 000 years BC (Garcı´a-Mendoza, 1992) ever, investment in agave plantations for tequila pro- and pre-Columbian cultures used them for fiber, food, duction can be risky, depending on both environmental construction and religious ceremonies. Agave plants conditions and demand. These problems are exacer- played such an important role in these societies that bated by the long life-cycle of the plant (5–8 years) they were awarded a specific deity, the goddess ‘Maya- and the restrictions which only allow Agave tequilana huel’ who is represented in different forms in several Weber cultivar ‘azul’ grown in specified regions of surviving codices (Lima, 1986). Today agave plants are Mexico to be harvested for tequila production. In recent still strong Mexican cultural icons, appearing in films, years some growers have been unable to find a market works of art and many forms of publicity. for materials from plantations initiated several years The best known modern agave products are of course before and many of these plantations can be found tequila and mezcal. Around 249 million liters of tequila untended and abandoned. are produced annually of which more than half are Until the latter part of the 20th century Mexico was exported, accounting for around $900 million dollars in also a major producer of agave fibers and textiles, income. Around 100 thousand hectares of agave are however this industry has declined from a total produc- grown for tequila production and the industry directly tion of 210 000 tons in 1916 to 32 000 tons in 2005 employs around 60 000 people (SAGARPA, 2009, (Eastmond & Robert, 2000; Caceres-Farfan et al., 2008), http://www.sagarpa.gob.mx; CRT:Consejo Regulador substantially reducing the potential sources of income del Tequila, 2009, http://www.crt.org.mx ). Production in the Yucata´n peninsula, the traditional centre of fiber of mezcal is much lower, around 680 thousand liters production in Mexico. These examples underline the need to seek alterna- Correspondence: June Simpson, e-mail: tives for the exploitation of agaves in Mexico and [email protected] several areas such as the use of agave sugars as dietary r 2010 Blackwell Publishing Ltd 25 26 J. SIMPSON et al. supplements and substitutes for sugar and fats (Lopez et al., 2003; Ortiz-Basurto et al., 2008; Urias-Silvas et al., 2008; Gomez et al., 2009; Ravenscroft et al., 2009; Leach & Sobolik, 2010) or for the production of paper (Idarraga et al., 1999) have been explored. Recently however, attention has focused on the potential of agave species as bioenergy crops (Borland et al., 2009; Somerville et al., 2010). Indeed, agaves offer many advantages for this goal: they are succulent, CAM plants which grow naturally in arid or semiarid conditions making them good candidates for the exploitation of marginal or uncultivated land and their exploitation for bioenergy production would not divert resources from staple food crop production as in the case of maize when used for bioenergy production, an extremely important consid- Fig. 1 Schematic representation of the taxonomy of the Agava- eration in Mexico. Among the advantages these plants ceae family. offer are: management in a similar manner to forest species with harvesting over several years, production of large amounts of biomass, methodology for ethanol mezcal (Agave angustifolia) and tequila (Agave tequilana) production from sugars well developed and low-main- are found within the Rigidae group and are underlined. tenance in terms of water and agrochemicals. An added More recently, molecular-genetic techniques have advantage is the possibility to also use the bagasse been employed to study the taxonomy, diversity and produced from the plants harvested for the production evolution of the Agavaceae (Colunga-GarciaMarin et al., of alcoholic beverages and fibers for bioethanol produc- 1999; Eguiarte et al., 1999; Martinez-Palacios et al., 1999; tion, effectively exploiting what would otherwise be a Piven et al., 2001; Navarro-Quezada et al., 2003; Gil-Vega waste product. A recent report (Hernandez-Salas et al., et al., 2006; Good-Avila et al., 2006; Bousios et al., 2007; 2009) describes the feasibility of exploiting not only the Gil-Vega 2007; Abraham-Juarez et al., 2009; Parker et al., sugar component of agave bagasse but also the cellu- 2010). Concerns have been raised over the very narrow losic component. germplasm pool exploited for Agave plants used in Although the current and potential economic impor- commercial applications, since plants are asexually tance of agaves is irrefutable, perhaps surprisingly, propagated and for tequila production by law only a relatively little basic research has been carried out on single genotype (A. tequilana Weber var. azul) can be these species, especially at the genetic and molecular grown (http://www.crt.org.mx). This leads to vulner- level. This is in contrast to comparable species such as ability in the crop under adverse environmental condi- pineapple where specific genes have been characterized tions or attack by pests and pathogens. Indeed the (Antony et al., 2008), transcriptome data generated combination of increased disease incidence and the (Moyle et al., 2005a) and a dedicated website developed international ‘boom’ in tequila sales led to a severe (Moyle et al., 2005b). shortage A. tequilana plants towards the end of the 1990s (Bowen & Valenzuela-Zapata, 2008; Bowen & Valenzuela-Zapata, 2009). However several molecular Available resources for genetic studies in Agave studies have shown that a greater diversity exists with- Although many details of agave taxonomy remain to be in A. tequilana than was first thought (Gil-Vega et al., defined, several excellent works are available, including 2006; Bousios et al., 2007; Abraham-Juarez et al., 2009) the classical morphological descriptions of Gentry and this diversity could be exploited if the terms of the (Gentry, 1982), Dahlgren (Dahlgren et al., 1985) and controlled designation of origin (Bowen & Valenzuela- Garcı´a-Mendoza (Garcı´a-Mendoza, 1992; Garcı´a-Mendoza, Zapata, 2008) were widened. This situation is less 2000; Garcia-Mendoza & Chiang 2003; Garcia-Mendoza, critical for mezcal and fiber production since many 2010). A simplified overview of the classification of the different cultivars and even different species may be Agavaceae family taken from these descriptions is used. shown in Fig. 1. Within the family the genus Agave is No formal reports are available of genetic or breeding economically the most important and for this genus studies although intra and interspecific crosses have alone, 200 species and 247 taxa have been been successful albeit at low efficiencies (Escobar- described (Garcı´a-Mendoza 2000). The main species Guzma´n et al., 2008) and hybridization has been exploited economically for fiber (Agave fourcroydes), observed in the field (Valenzuela, 1997). r 2010 Blackwell Publishing Ltd, GCB Bioenergy, 3, 25–36 GENOME AND TRANSCRIPTOME MINING IN A. TEQUILANA 27 Table 1 Ploidy levels for Agave species of the subgenus (a) Littaea and (b) Agave Ploidy level Species (a) DIPLOID, 2 Â , 60 chromosomes A. celsii, A. filifera, A. nizandensis, A. pendula, A. schotti, A. striata, A. stricta, A. toumeyana, A. univittata, A. victoriae-reginae, A. xalapensis, A. xylocantha TRIPLOID, 3 Â , 90 chromosomes A. warelliana, A. ornithobroma TETRAPLOID, 4 Â , 120 chromosomes A. kerchovei, A. lechugilla, A. victoriae-reginae HEXAPLOID, 6 Â , 180 chromosomes A. gilbeyi, A. giesbrechtii ANEUPLOID, No. chromosomes in parenthesis A. lechugilla (55 110), A. stricta (50), A. warelliana (100) (b) DIPLOID, 2 Â , 60 chromosomes A. amaniensis, A .americana, A. angustifolia, A. asperrima, A. avellanidens, A. brevispina, A. colorata, A. deserti, A. fourcroydes, A. gigantensis, A. palmeri, A.parryi, A. potatorum, A. rodocantha, A. shawii, A. tequilana TRIPLOID, 3 Â , 90 chromosomes A. angustifolia, A. cantala, A.lurida, A. vexans TETRAPLOID, 4 Â , 120 chromosomes A. americana, A. angustifolia, A. ferox, A. goldmaniana, A. lurida, A. parryi, A. salmiana A. tequilana, A. utahuensis PENTAPLOID, 5 Â , 150 chromosomes A. fourcroydes, A. mapisaga, A. sisalana, A. tequilana HEXAPLOID, 6 Â , 180 chromosomes A. americana, A. asperrima, A. angustifolia, A. decipiens, A. atrovirens, A. vivipara, A. wightii OCTAPLOID, 8 Â , 240 chromosomes A.