Development and Characterization of Microsatellite PCR Primers for Bonytail Chub

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Development and Characterization of Microsatellite PCR Primers for Bonytail Chub: OVERVIEW Bonytail chub (Gila elegans) is one of the four species of large, long-lived, endemic fishes that were once abundant and broadly distributed throughout the Colorado River basin (reviewed in Minckley et al. 2003). Bonytail chub is federally listed as endangered (U.S. Fish and Wildlife Service [USFWS] 1980) and is extremely rare in the wild (Minckley and Thorson 2007). Levels of genetic variability in the species are extremely low due to a historical bottleneck (Garrigan et al. 2002). The program relies entirely on hatcheries to produce fish for repatriation due to the scarcity of Bonytail chub. In 1981, 5 male and 6 female Bonytail chub captured in Lake Mohave were used to produce an F1 year class that became the initial broodstock (Hamman 1982); however, Hedrick et al. (2000) found that less than 8 individuals actually contributed. This finding has resulted in increased efforts to incorporate additional individuals from Lake Mohave into the broodstock; however, these efforts have not been successful (Minckley and Thorson 2007), hence Bonytail chub is genetically depaurperate. Demographic factors are important when considering the fate of imperiled taxa. Bonytail chub are iteroparous, long-lived species and have enormous reproductive capacity (mature females can likely produce thousands of eggs per spawn like other large chubs (e.g. Brouder et al. 2000). From a demographic perspective, this life history virtually guarantees a large number of surviving offspring when rearing conditions favor recruitment. The downside of this level of fecundity is that one or a few breeding pairs could produce the majority of offspring that survive each year, and hence pass on only a small subset of the total genetic diversity available in the spawning stock, which ultimately leads to increased inbreeding. Inbreeding can lead to reduced viability (Dowling et al.1996), so it is crucial to monitor how well progeny spawned and reared in off-channel habitats represent the genetic diversity of the initial spawning stock, and to ascertain the number of parents contributing to offspring that are ultimately repatriated to the river. The primary objective of this project is to test PCR primers that have been used previously in two other species of Gila (nigrescens and nigra) for their utility in Bonytail Chub as well as optimize existing primers for examination of individual reproductive success of chub in backwater habitats. RECIPIENT INVOLVEMENT To date, almost 20 microsatellite markers have been developed for Bonytail and other chubs but additional microsatellite markers (5-10 depending on levels of polymorphism) are necessary to facilitate the unambiguous identification of individuals (Keelor-Foster 2004). The recipient will test PCR primers that have been used previously in two other species of Gila (nigrescens and nigra) for their utility in Bonytail Chub as well as optimize existing primers for examination of individual reproductive success of chub in backwater habitats. Like Bonytail Chub, Gila nigrescens (Chihuahua chub) has experienced population bottlenecks in the past and has relatively low levels of genetic variability. Despite this, nine microsatellite loci that amplified reliably in this species (Osborne et al. 2012) have been successfully identified. The recipient will use standard molecular procedures to characterize the microsatellites, and will also test microsatellite markers on known parents and progeny produced at Dexter National Fish Hatchery and Technology Center to determine their reliability. RECLAMATION INVOLVEMENT No substantial involvement on the part of Reclamation is anticipated for the successful completion of the objectives to be funded by this award. It is anticipated that Reclamation's involvement will consist of standard federal stewardship responsibilities such as monitoring project performance, technical assistance at the request of the recipient, etc.