Development and inheritance of molecular markers in the kelp bass Paralabrax clathratus

Abstract

Keywords Mendelian Microsatellite Mitochondrial Paralabrax SerranidaeWhile molecular tools have transformed fisheries biology,their development and implementation can necessitate theuse of delicate equipment, be expensive, and requireextensive optimization [1]. Counting only consumables, wehave calculated that in-house sequencing of 500–700 baseson a Beckman CEQ 8000 (Beckman Coulter, Fullerton,CA, USA) costs *$4–5 per sample under ideal conditions.It is a process not amenable to isolated or rudimentary labconditions and is not easily performed as a quick-returnassay. Loci scored as dye-labeled fragments on an auto-mated sequencer are also expensive [2]; they requiredevelopment of a microsatellite library and cost commer-cially *US$12,500 [3]. Confirmation of Mendelianinheritance in the marker set is yet another cost burden andrepresents an important but infrequently performed pre-requisite for statistical testing. A recent survey found 1 in15 microsatellite loci, as expressed in their data scores, hadnon-Mendelian patterns [4].The reporting of methodologies and inheritance studiesthat are cost effective and have minimal equipmentrequirements can therefore be invaluable to a new researchprogram or a monitoring effort that requires fast turn-around times. A set of markers will be available for thespecies in question and frequently its congeners; thestrategies for developing the markers, such as our inheri-tance methodology below, can have even widerapplication. Based on this rationale, this short paper pre-sents genetic methods developed for the kelp bass,Paralabrax clathratus, that confirm the robustness ofmarkers and allow for economical and expeditious assays.Kelp bass are a temperate serranid found abundantly fromPoint Conception, CA, to just south of Punta Eugenia,Mexico [5], and are an important component of the$300 million California sport fishing industry [6, 7]. P.clathratus occurs in current and proposed southern Cali-fornia marine reserves and has been the subject of adiversity of research [8–11].We document a restriction fragment length polymor-phism (RFLP) assay of the mitochondrial control regionthat can assess a substantial amount of the sequencediversity with only a thermal cycler and an agarose gel box.We also present a series of controlled crosses to confirmMendelian inheritance for previously published microsat-ellite markers [12], a task generally difficult to performwith many marine fish [13].Development of RFLPsSequences for the mitochondrial control region of 105 kelpbass from ten locations (Genbank accession numbersDQ192295–DQ192399) were used. Sequences werealigned in Sequencher 4.5 (Gene Codes, Ann Arbor, MI,USA) and surveyed with the program’s restriction enzymelibrary. Pairwise U