7 Breeding American Chestnuts for Blight Resistance Lisa M. Worthen, Keith E. Woeste, and Charles H. Michler Hardwood Tree Improvement and Regeneration Center U.S. Forest Service Northern Research Station Department of Forestry and Natural Resources Purdue University 715 West State Street West Lafayette, IN 47907 I. INTRODUCTION II. CHESTNUT GENETICS III. CHESTNUT BLIGHT IV. BLIGHT-RESISTANCE BREEDING A. Historical B. Seed Orchard Design and Forest Trials V. POPULATION GENETICS OF HYBRID CHESTNUT REINTRODUCTION A. Small Population Size B. Establishment, Colonization, and Expansion of Forest Tree Populations C. Monitoring Introduced Populations 1. Chloroplast Markers 2. Nuclear Markers 3. Morphological, Physiological, and Phenological Traits D. Interactions between Resistant and Native Chestnuts E. Linkage Disequilibrium F. Local Adaptation and Long-Term Fitness of Reintroduced Populations G. Lessons from Other Reintroduction Programs VI. CONCLUSION LITERATURE CITED Plant Breeding Reviews, Volume 33 Edited by Jules Janick Copyright Ó 2010 Wiley-Blackwell. 305 306 L. M. WORTHEN, K. E. WOESTE, AND C. H. MICHLER I. INTRODUCTION When Burnham, Rutter, and French (1986) published their proposal to use backcross breeding methods such as those used in annual crops to breed American chestnut trees that would be resistant to the chestnut blight disease, the prospect seemed the hopeful imaginings of dedicated few. In the 20 or so years since that publication, a nationwide effort involving thousands of volunteers, hundreds of backcross breeding experiments, many universities, and numerous government and private institutions has culminated in seed orchards that contain blight- resistant American chestnut hybrids. These hybrids will be the parents of seeds used for reforestation trials. While backcross breeding programs continue to incorporate new sources of resistance and new locally adapted American chestnut genotypes, chestnut breeders must prepare for the long-hoped-for return of American chestnuts to forests of the eastern United States and the appearance of American chestnuts in plantations. Breeders face unusual challenges when seeking to reintroduce popu- lations to their former habitats or to supplement wild populations that may no longer be viable. Conservation breeders may use traditional breeding methods, but unlike crop breeders, they must release the products of their program into environments over which landowners typically will exercise little control. Indeed, the goal is to return healthy individuals back into the wild—into environments marked by dynamic change and nonuniformity. In effect, conservation breeders are seeking to reverse the typical work flow of breeding. Crop breeders typically incorporate wild germplasm into cultivated or domesticated lines and seek to improve the predictability of yield by manipulating the genetics and culture of the crop. American chestnut breeders introgress genes from a cultivated species into a wild relative with the hope that their selections will thrive in unmanaged or minimally managed stands. Conservation breeding of forest trees also differs from traditional forest tree breeding in that forest geneticists select their releases for optimal performance in even-aged plantations established within discrete zones of adaptation, a relatively circumscribed environment compared to the goal of restoring a species to its entire former range. This chapter examines how outcomes of historical and current American chestnut breeding may affect reintroduced chestnut popula- tions. What levels of genetic diversity and family structure will charac- terize reintroduced populations? How will reintroduced populations survive, mate, and spread? What will be the fate of native American chestnut genes? Twenty years after Burnham, Rutter, and Frenchs 7. BREEDING AMERICAN CHESTNUTS FOR BLIGHT RESISTANCE 307 seminal publication in this same journal, an assessment of past breeding efforts and an examination of the genetic issues affecting future reintro- ductions is warranted. II. CHESTNUT GENETICS The genus Castanea is comprised of three sections and seven species (Johnson 1988). Four species—Castanea mollissima Blume (Chinese chestnut), C. seguinii Dode. (Seguin chestnut), C. crenata Sieb. and Zucc. (Japanese chestnut), and C. henryi Skan (Chinese chinqua- pin)—in eastern Asia. The European chestnut, C. sativa Mill., is native to western Asia and southern Europe, and C. dentata (Marsh.) Borkh. (American chestnut) and C. pumila Mill. (American chinquapin, often divided into subspecies Ozark and Alleghany chinquapin) occur in eastern North America. Genetic variation, population structure, and phylogenetic relation- ships within and among Castanea species have been analyzed using a variety of marker systems (Villani et al, 1991; Huang et al. 1994, 1998; Lang and Huang 1999; Dane et al. 1999, 2003; Kubisiak and Roberds 2003; Tanaka et al. 2005; Lang et al. 2006, 2007; Wang et al. 2006; Han et al. 2007). Sequence-based phylogenetic analysis of the variable trnT-L-F region of Castanea cpDNA showed C. crenata the most basal species and placed the Chinese species in a monophyletic clade with the North American and European species as a sister group (Lang et al. 2006). In general, Castanea species show high levels of genetic diversity at neutral loci, little population structure, and partition most genetic diversity within populations—population genetic traits shared by many other long-lived, outcrossing forest trees (Petit and Hampe 2006). In North America, Castanea species were driven southward to one or two southern glacial refugia during the Wisconsin glacial maximum 18,000 to 20,000 years ago. C. dentata greatly expanded its range after the glacial retreat about 10,000 years ago, spreading northward along the Appala- chian ridge (Delcourt and Delcourt 1984; Huang et al. 1998; Delcourt 2002) and establishing itself as a dominant species in 800,000 km2 of eastern forest (Braun 1950). C. dentata, like all Castanea species, is diploid (2n ¼ 2x ¼ 24) and will easily hybridize with its congeners, although offspring from some com- binations suffer from low vigor or male sterility (Jaynes 1975). It is monoecious, primarily wind pollinated, and generally bears three heavy seeds per cupule. The American chestnut is an obligate outcrosser with a genetic self-incompatibility system that prevents self-fertilization 308 L. M. WORTHEN, K. E. WOESTE, AND C. H. MICHLER (McKay 1942). The self-incompatibility system appears to be gameto- phytic because chestnut has binucleate pollen grains, and multiple pollen tubes penetrate to the base of the style prior to fertilization (McKay 1942; Brewbaker 1957; Burnham et al. 1986). Chestnut flowers becomereceptiveina patterndescribedbyStout (1928) asduodichogamy: Staminate flowers borne on long, slender catkins mature first, followed by pistillate flowers, and, last, staminate flowers on bisexual catkins. Under favorable conditions, American chestnut trees reach reproductive matu- rity between 12 and 18 years of age and mast abundantly every year (Buttrick 1925; Weeks et al. 2006).Americanchestnutseedlingshave been shown to outgrow co-occurring hardwoods under high light conditions, but they can also survive for prolonged periods in shaded understories (Paillet and Rutter 1989; McCament and McCarthy 2005). Records indi- cate that in some locations, chestnut was a medium-size tree, but on favorable sites, American chestnut trees grew to 37 m tall and 1.5 m in diameter (Buttrick 1925; Illick 1928; Braun 1950). The largest recorded specimen was about 5.2 m in diameter (Detwiler 1915). The preblight population genetics of the American chestnut must be inferred from knowledge of postblight American chestnut populations and other forest trees with similar life history characteristics. Analyses using allozymes have shown that C. mollissima has the highest popu- lation mean genetic variability of all the members of the genus [total genetic variability (HT) ¼ 0.321, Hexp ¼ 0.311; Lang and Huang 1999] and C. dentata has the lowest mean variability at isozyme loci (HT ¼ 0.214, Hexp ¼ 0.167; Huang et al. 1998). Pigliucci et al. (1991) reported an ¼ intermediate mean expected heterozygosity for C. sativa (Hexp 0.24 À 0.27). Kubisiak and Roberds (2003) employed 6 microsatellite and 19 random amplified polymorphic DNA (RAPD) loci to examine 17 popula- tions of root crown collar sprouts of C. dentata. They used Neis (1978) gene diversity (h) and calculated a mean genetic diversity across RAPD loci (h ¼ 0.226) that, barring differences in marker systems, is similar to the diversity for American chestnut reported by Huang et al. (1998). Both reports are well within genetic diversity limits of similar forest tree species (Hamrick, Godt, and Sherman-Broyles 1992). Higher- than-average heterozygosities have been found in postblight American chestnut populations, leading some to speculate on the role of hetero- zygote advantage in the species remarkable growth (Stilwell et al. 2003). The spatial genetic structure characteristic of wind-pollinated, self- incompatible forest tree species is most often negligible or weakly present at only very close distances (Berg and Hamrick 1995; Streiff et al. 1998). Pierson et al. (2007) used minisatellite band sharing to calculate the differentiation between sampling plots in a large woodland 7. BREEDING AMERICAN CHESTNUTS FOR BLIGHT RESISTANCE 309 of naturalized American chestnut 600 km west of the species native range. They determined that no differentiation among sampling