Microsatellite development for the endangered Yangtze sturgeon (Acipenser dabryanus Duméril, 1869) using 454 sequencing

The Yangtze sturgeon (Acipenser dabryanus) is an endemic species in China. Using 454 sequencing, eight polymorphic tri‐, tetra‐, penta‐, and hexanucleotide microsatellite loci were isolated in this study. The raw sequence data from a one‐eighth run of 454 sequencings were 38.0 Mbp containing 94 222 reads/sequences. Of 80 microsatellite loci, only eight loci were polymorphic in a population of 30 individuals. The number of alleles per locus ranged from 4 to 14 (mean 7.62), and the observed heterozygosities varied between 0.46 and 0.88 (mean 0.74). Cross amplification was tested in congeneric species Acipenser sturio and Acipenser sinensis. These new microsatellite markers will be useful for further studies on genetic variation, parentage analysis, and conservation management for this critically endangered species.     

Twenty‐four novel microsatellites for the endangered Chinese sturgeon (Acipenser sinensis Gray, 1835)

The Chinese sturgeon (Acipenser sinensis) is an endemic and critically endangered species in China. In this study, a total of 24 polymorphic microsatellite loci were isolated and characterized using Illumina sequencing for A. sinensis. The number of alleles (Na) per locus ranged from 2 to 6 (mean 4.04), mean expected heterozygosities (He), Shannon‐Wiener Diversity Indices (SW) and evenness (E) per locus ranged from 0.235 to 0.786 (mean 0.62), from 0.396 to 1.608 (mean 1.13), and from 0.060 to 0.213 (mean 0.13), respectively. Exact tests revealed that nine loci showed significant (P < 0.01) deviation from Hardy‐Weinberg equilibrium (HWE). Cross amplification was tested in congeneric species A. schrenskii, A. baerii, A. dabryanus and Huso dauricus. The new microsatellite markers described herein will be useful for further studies on genetic variation, parentage analysis, and conservation management.     

Development and standardization of disomic microsatellite markers for lake sturgeon genetic studies

Lake sturgeon (Acipenser fulvescens) are of conservation concern in North America. To facilitate the recovery of this fish species, an understanding of their population genetic structure is necessary to develop and implement spatially and temporally appropriate management actions. Until recently, few genetic data using nuclear loci have been collected, primarily due to the paucity of suitable genetic markers because most microsatellite loci in lake sturgeon appeared to be tetrasomic. The authors identified nine microsatellite loci (from 254 examined) that were putative polymorphic disomic loci and tested their conformance to a disomic mode of inheritance using three lake sturgeon families. The objectives of the study were to: (i) confirm the disomic status of the nine loci through inheritance testing, and (ii) standardize the genetic markers among participating laboratories. At all nine loci, disomic inheritance were confirmed, and all nine loci segregated independently in the 26 of 36 loci pairs possible to test. One of the nine loci showed non‐Mendelian segregation, possibly due to meiotic drive and/or selection. Three progeny had peak patterns inconsistent with disomy at one or more loci. The nine loci when combined with four microsatellite loci previously confirmed in other studies as disomic in lake sturgeon now yield a suite of 13 microsatellite markers. These 13 markers have been standardized among four other laboratories to facilitate building an inter‐laboratory genetic database for lake sturgeon.     

From microsatellites to single nucleotide polymorphisms for the genetic monitoring of a critically endangered sturgeon

The use of genetic information is crucial in conservation programs for the establishment of breeding plans and for the evaluation of restocking success. Short tandem repeats (STRs) have been the most widely used molecular markers in such programs, but next‐generation sequencing approaches have prompted the transition to genome‐wide markers such as single nucleotide polymorphisms (SNPs). Until now, most sturgeon species have been monitored using STRs. The low diversity found in the critically endangered European sturgeon (Acipenser sturio), however, makes its future genetic monitoring challenging, and the current resolution needs to be increased. Here, we describe the discovery of a highly informative set of 79 SNPs using double‐digest restriction‐associated DNA (ddRAD) sequencing and its validation by genotyping using the MassARRAY system. Comparing with STRs, the SNP panel proved to be highly efficient and reproducible, allowing for more accurate parentage and kinship assignments' on 192 juveniles of known pedigree and 40 wild‐born adults. We explore the effectiveness of both markers to estimated relatedness and inbreeding, using simulated and empirical datasets. Interestingly, we found significant correlations between STRs and SNPs at individual heterozygosity and inbreeding that give support to a reasonable representation of whole genome diversity for both markers. These results are useful for the conservation program of A. sturio in building a comprehensive studbook, which will optimize conservation strategies. This approach also proves suitable for other case studies in which highly discriminatory genetic markers are needed to assess parentage and kinship.     

Novel microsatellite markers for Atlantic sturgeon (Acipenser oxyrinchus) population delineation and broodstock management

Twenty‐one new disomic, polymorphic microsatellite DNA markers were isolated in Atlantic sturgeon, Acipenser oxyrinchus oxyrinchus. These markers yielded a total of 220 alleles in a survey of 16 fish; two to 21 alleles/locus were observed. Each locus segregated in a Mendelian fashion when tested in a family, and a set of 14 of the loci distinguished between siblings and half‐siblings. Average observed heterozygosity ranged from 18.8 to 100.0%, and no linkage disequilibrium was detected. These loci should detect sufficient genetic diversity to allow kinship analysis for broodstock management, gene marking for stocking assessment and life history studies, and delineation of fine‐scale population structure.     

Multiple molecular approaches yield no evidence for sex‐determining genes in lake sturgeon (Acipenser fulvescens)

Common DNA‐based sexing assays have been widely used for the conservation and management of mammals and birds. However, many fishes do not have genetic sex determination and in those that do, the plasticity of the genes involved means that species‐specific assays are normally required. Such DNA‐sexing markers would be especially valuable in lake sturgeon (Acipenser fulvescens) because of their sexual monomorphism, delayed sexual maturity, and conservation status. We tried to identify genetic differences between male and female lake sturgeon using several different molecular genetic methods, including randomly amplified polymorphic DNA, representational difference analyses, subtractive hybridization, and a candidate gene approach. Ultimately, a number of genes were identified but none was sex‐specific. Although the ultimate mechanism of sex determination is yet unknown, it is possible that sex determination is environmental in lake sturgeon, especially since recent studies have also failed to identify sex determination genes in other sturgeon species.     

The use of microsatellite loci for identification of sturgeon species (Acipenseridae) and hybrid forms

The genetic polymorphism of ten sturgeon species that inhabit the territory of the Russian Federation (Russian sturgeon, Siberian sturgeon, Amur sturgeon, Sakhalin sturgeon, Persian sturgeon, ship sturgeon, sterlet, stellate sturgeon, beluga, and kaluga) was examined at five microsatellite loci (Afug41, Afug51, An20, AoxD161, AoxD165) (in total, 3821 individuals). The examined loci were successfully amplified with the same primer set in all species tested and demonstrated a high level of variation. Alleles specific to different species have been identified, which allows them to be used to identify species of sturgeon and their food products. In addition, the possibility of identifying hybrid forms was demonstrated. The assignment test performed in the STRUCTURE software program showed a high probability of correctly assigning each individual to its species based on genotyping with five microsatellite loci examined (96–98%, on average). However, for Russian and Persian sturgeon, the rate of proper species assignment was considerably lower (75 and 84%, respectively).

     

Isolation and characterization of microsatellites in the Adriatic sturgeon (Acipenser naccarii)

We isolated and characterized seven polymorphic microsatellite loci from a partial genomic library of the Adriatic sturgeon (Acipenser naccarii) enriched for GATA repeats. Variability was tested in 20 specimens of this endangered species. Our data support the tetraploid condition for A. naccarii and will be useful for the investigation of the remnant genetic variability of this species as well as for genetic tagging studies.     

Microsatellite DNA variation in Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) and cross-species amplification in the Acipenseridae

Overharvest and habitat alteration have led toa collapse of most commercial Atlantic sturgeon(Acipenser oxyrinchus oxyrinchus)fisheries while pushing the species to rarityor extirpation in most of its historical range. A biologically sound conservation program forthis species requires knowledge of its geneticdiversity and of the evolutionary relationshipsamong geographic populations. To address theseresearch needs, six microsatellite loci wereisolated from A. o. oxyrinchus. Pedigreeanalysis suggested that all are inherited in acodominant Mendelian pattern. The six lociwere tested in ten additional sturgeon speciesfrom three genera and three apparent ploidylevels (4n, 8n, 16n). Approximately 70% ofsuccessful locus-species amplifications werepolymorphic. Polysomy was observed most oftenin 8n and 16n species. Genetic diversity andpopulation structure of A. o. oxyrinchuswere assayed using three polymorphic Aoxmarkers and four markers developed from lakesturgeon (A. fulvescens). A. o.oxyrinchus were sampled from the AltamahaRiver, Georgia, USA north to the St. LawrenceRiver, Quebec, Canada. Gulf sturgeon, A.o. desotoi, were sampled from the SuwanneeRiver, Florida, USA, to assess differentiationbetween the subspecies. Seventy-seven alleleswere observed to segregate into uniquemultilocus genotypes for each of the 392individuals assayed. Mean diversity wasgreatest in the Chesapeake Bay (9.7 alleles perlocus) and Delaware River (7.4 alleles perlocus) collections, and lowest in the St.Lawrence River (4.6 alleles per locus). Meanheterozygosity across seven loci ranged from44.3% (St. Lawrence River) to 62.6% (Altamaha River). Significant allelic heterogeneity wasobserved in 82% of pairwise comparisons aswell as a global test (p < 0.0001) for A.o. oxyrinchus collections. Genetic distancesuggests the presence of at least sixsubpopulations in A. o. oxyrinchus: St.Lawrence River, St. John River, Hudson River,Delaware River, Albemarle Sound, and AltamahaRiver. Genetic and geographic distances werepositively correlated (r = 0.57, p < 0.03) amongA. o. oxyrinchus, suggesting isolation bydistance and philopatry. Hierarchical genediversity analysis indicated significantgenetic population structure at every level. Maximum likelihood assignment tests correctlyassigned individual fish to collection with ahigh rate of success (mean = 87.5%); this andother lines of evidence indicated that theChesapeake Bay collection represents a mixedpopulation of sub-adult sturgeon from northernand southern Atlantic coast populations. Population structure was correlated with thatsuggested by earlier mitochondrial (mt) DNAanalyses. Significant diversity was observedbetween two Canadian populations from whichonly a single mtDNA haplotype has beenreported.     

Comparative stable isotope analyses of green sturgeon (Acipenser medirostris) and white sturgeon (A. transmontanus) in the San Francisco estuary

Green sturgeon (Acipenser medirostris) and white sturgeon (A. transmontanus) are closely related, sympatric species that inhabit the San Francisco estuary. Green sturgeon have a more marine life history but both species spawn in the Sacramento River and reside for some duration in San Francisco Bay. These sturgeons are of conservation concern, yet little is known about their dietary competition when they overlap in space and time. To examine evidence of dietary differentiation, we collected whole blood and blood plasma from 26 green sturgeon and 35 white sturgeon in San Francisco Bay. Using carbon and nitrogen stable isotope analyses, we compared their relative trophic levels and foraging locations along the freshwater to marine gradient. Sampling blood plasma and whole blood allowed comparison of dietary integration over shorter and longer time scales, respectively. Plasma and whole blood δ¹³C values confirmed green sturgeon had more marine dietary sources than white sturgeon. Plasma δ¹⁵N values revealed white sturgeon fed at lower trophic levels than green sturgeon recently, however, whole blood δ¹⁵N values demonstrated the two species fed at the same trophic level over longer time scales. Larger individuals of both species had higher δ¹³C values than smaller individuals, reflecting more marine food sources in adulthood. Length did not affect δ¹⁵N values of either species. Isotope analyses supported the more marine life history of green than white sturgeon and potentially highlight a temporary trophic differentiation of diet between species during and preceding the overlapping life stage in San Francisco Bay.     

High‐throughput SNP‐genotyping analysis of the relationships among Ponto‐Caspian sturgeon species

Legally certified sturgeon fisheries require population protection and conservation methods, including DNA tests to identify the source of valuable sturgeon roe. However, the available genetic data are insufficient to distinguish between different sturgeon populations, and are even unable to distinguish between some species. We performed high‐throughput single‐nucleotide polymorphism (SNP)‐genotyping analysis on different populations of Russian (Acipenser gueldenstaedtii), Persian (A. persicus), and Siberian (A. baerii) sturgeon species from the Caspian Sea region (Volga and Ural Rivers), the Azov Sea, and two Siberian rivers. We found that Russian sturgeons from the Volga and Ural Rivers were essentially indistinguishable, but they differed from Russian sturgeons in the Azov Sea, and from Persian and Siberian sturgeons. We identified eight SNPs that were sufficient to distinguish these sturgeon populations with 80% confidence, and allowed the development of markers to distinguish sturgeon species. Finally, on the basis of our SNP data, we propose that the A. baerii‐like mitochondrial DNA found in some Russian sturgeons from the Caspian Sea arose via an introgression event during the Pleistocene glaciation.     

Conservation and collection efforts for the endangered Alabama sturgeon (Scaphirhynchus suttkusi)

The Alabama sturgeon (Scaphirhynchus suttkusi) is the rarest and most endangered sturgeon species in North America. Over an 8‐year period, the Alabama Division of Wildlife and Freshwater Fisheries, U.S. Army Corps of Engineers, and U.S. Fish and Wildlife Service cumulatively expended 2447 man‐days in efforts to collect Alabama sturgeon broodstock in an attempt to initiate a conservation propagation program. Out of nearly 29 000 fishes collected between March 1997 and May 2005, only five were Alabama sturgeon. Attempts to spawn and propagate these sturgeons were unsuccessful, and all have since died in captivity. In context with past collection efforts and anecdotal accounts, these results indicate that the Alabama sturgeon is becoming increasingly rare with the passage of time. Although there is evidence that some level of recruitment continued to occur in the Alabama River during the past decade, the increasing rarity of Alabama sturgeon suggests that mortality rates are exceeding recruitment.     

Development of polymorphic microsatellite loci for the design of management and conservation strategies of the critically endangered Barrens topminnow (Fundulus julisia,Williams & Etnier, 1982)

The decline of the Barrens topminnow (Fundulus julisia), a small schooling fish endemic to the Barrens Plateau region of Middle Tennessee, is one of the most dramatic cases of species imperilment in eastern North America. Loss of undisturbed habitat, coupled with the introduction of the invasive Western Mosquitofish (Gambusia affinis) has led to the extirpation of 12 out of 14 natural populations since the 1980s. Identified is a set of 14 microsatellite loci to improve conservation and management strategies for the species. Four loci were cross‐amplified using primers designed for congeneric taxa and 10 loci were developed from whole genome Illumina sequencing. Initial surveys in two populations suggest significant structuring of genetic variation and differing levels of heterozygosity among populations. These markers will contribute valuable information to ongoing conservation and management efforts.     

Production of recombinant great sturgeon (Huso huso,Linnaeus, 1758) growth hormone (GH) by Pichia pastoris (Guillierm, 1956)

In this research, the encoding cDNA of growth hormone (GH) was cloned from the pituitary gland of great sturgeon Huso huso (three adults: two females and one male, 7–9 years old, 70–90 kg, reared in concrete ponds). In order to obtain the great sturgeon recombinant GH expression in Pichia pastoris, the mature encoding cDNA was first cloned in TA vector PTZ57R and then sequenced. After confirmation of the correct GH sequence, the GH coding sequence was subcloned into pHILS1 expression vector. The yeast Pichia pastoris GS115 strain was transformed with the expression plasmid. Results obtained from this study showed that great sturgeon GH recombinants were expressed upon induction with methanol and exported into the medium. The level of expression was examined using RNA analysis, SDS‐PAGE, and western blot analysis. RNA analysis of the recombinant strains showed a sharp, specific band in 800 bp. The specific band in transformants indicated the presence of GH RNA in the yeast. SDS‐PAGE and western blot analysis showed a specific 21 kDa band for the growth hormone. Culture conditions were optimized for pH = 6 and incubation time (after 24 h induction, peaking at 72 h) for maximal protein production. The results provide useful information for the future production of recombinant growth hormones in other sturgeon species.     

Isolation of microsatellite loci from the endemic and endangered Adriatic sturgeon (<Emphasis Type="Italic">Acipenser naccarii</Emphasis>)

With the aim of elaborating a breeding plan on a captive stock of the highly endangered Adriatic sturgeon (Acipenser naccarii), a total of 10 polymorphic microsatellite loci were isolated from an enriched library. The results of cross amplification of additional 8 loci previously isolated from A. oxyrinchus, A. fulvescens and Scaphyrinchus platorhynchus are also reported. Given the tetraploid condition of the species the genetic variability was estimated basing on the number of alleles per individuals and the average band sharing.     

Development of new microsatellite primers for green and white sturgeon

Sixty-eight primer sets for microsatellite loci were developed from microsatellite motif enriched genomic libraries of pooled DNA from the polyploid green and white sturgeon (Acipenser medirostris and A. transmontanus). Four individuals from each species were screened for polymorphism at these loci. Forty-eight loci amplified in both species, and some exhibited species-specific amplification for white or green sturgeon (8 and 12 loci, respectively). The number of alleles per locus ranged from one to 12. At least 68% of the green and 65% of the white sturgeon loci we developed are polysomic.     

Identification of microsatellite loci in lake sturgeon,Acipenser fulvescens,and their variability in green sturgeon,A. medirostris

From (CATC)_n, (GATA)_n, (AAAC)_n, and (CA)_n–enriched libraries for the lake sturgeon Acipenser fulvescens, 254 primer pairs were developed. These primer pairs resulted in the identification of 128 microsatellite loci in either A. fulvescens or A. medirostris. Polymorphic loci were identified in both sturgeon species for 48 of the primer pairs and 14 of the primer pairs amplified polymorphic loci only in A. medirostris. Most of the identified loci appear to be tetrasomic (79.1% in A. fulvescens and 64.5% in A. medirostris). These results offer estimates of the degree of diploidization in each of these species.     

Genetic discrimination of middle Mississippi River <Emphasis Type="Italic">Scaphirhynchus</Emphasis> sturgeon into pallid,shovelnose, and putative hybrids with multiple microsatellite loci

The pallid sturgeon (Scaphirhynchus albus), which is protected under the US endangered species act, and shovelnose sturgeon (S. platorhynchus), which is legally harvested in some locations, are sympatric throughout the range of pallid sturgeon. There is considerable morphological overlap between the species making discrimination problematic. The inability to reliably differentiate between species across all life stages has hampered pallid sturgeon recovery efforts. Furthermore, the two species are believed to hybridize. This study used allele frequency data at multiple microsatellite loci to perform Bayesian and likelihood-based assignment testing and morphological measures and meristics to discriminate pallid, shovelnose, and putative hybrid sturgeons from the middle Mississippi River. Bayesian model-based clustering of the genetic data indicated that two natural genetic units occur in the region. These units correspond to morphologically identified pallid and shovelnose sturgeon. Some individuals were morphologically intermediate and many of these failed to strongly assign genetically as either pallid or shovelnose sturgeon, suggesting they may be hybrids. These data indicate that pallid sturgeon and shovelnose sturgeon are genetically distinct in the middle Mississippi River (F _ST = 0.036, P < 0.0001) and suggest that hybridization between pallid sturgeon and shovelnose sturgeon has occurred in this region with genetic distance estimates indicating the greatest distance is between pallid and shovelnose sturgeon, while hybrid sturgeon are intermediate but closer to shovelnose. This study demonstrates that assignment testing with multiple microsatellite markers can be successful at discriminating pallid sturgeon and shovelnose sturgeon, providing a valuable resource for pallid sturgeon recovery and conservation.

Improved genetic identification of acipenseriform embryos with application to the endangered pallid sturgeon Scaphirhynchus albus

We produced pallid sturgeon Scaphirhynchus albus embryos at five pre-hatch developmental stages and isolated and quantified genomic DNA from four of the stages using four commercial DNA isolation kits. Genomic DNA prepared using the kit that produced the largest yields and concentrations were used for microsatellite DNA analyses of 10–20 embryos at each of the five developmental stages. We attempted to genotype the hatchery-produced embryos at 19 microsatellite loci and confirmed reliable genotyping by comparing the microsatellite genotypes to those of known parents. Embryos at stages 5 and 8 did not produce reliable genotyping while those at stages 14, 24 and 33 did. We used the same DNA isolation method on 262 wild-caught acipenseriform embryos collected from the lower Yellowstone River. A total of 200 of the wild embryos were successfully identified to stages 8 to 34 and the rest could not be staged. Using a combination of single nucleotide polymorphism and microsatellite markers, 249 of the wild-caught embryos were genetically identified as paddlefish Polyodon spathula, five were identified as shovelnose sturgeon Scaphirhynchus platorynchus and eight failed to amplify. None were identified as pallid sturgeon. This study demonstrates that early-stage wild-spawned acipenseriform embryos can be genetically identified less than 24 h post-spawn. This methodology will be useful for recovery efforts for endangered pallid sturgeon and can be applied to other acipenseriform species.     

Development and cross‐species amplification of 18 microsatellite markers in the Sumatran tiger (Panthera tigris sumatrae)

Eighteen polymorphic microsatellite loci from the highly endangered Sumatran tiger (Panthera tigris sumatrae) were isolated and characterized. Upon polymerase chain reaction amplification, 16 of these markers produced a single, sharp band in all three tiger and 10 non‐tiger felid species examined. Of the two remaining loci, 6HDZ057 and 6HDZ635 failed to amplify genomic DNA from puma (Felis concolor) and cheetah (Acinonyx jubatus), respectively. The amplification of these markers across four genera is an indication of their usefulness for population genetics studies and conservation work in a wide range of felid species.