B. Que se haya registrado en su contabilidad por concepto y en forma acumulativa durante el ejercicio
5. Descripción del bien o mercancía enajenados
5.1 Transcriptome sequencing of the Adriatic sturgeon (original publication in Appendix I)
This work concerns the analysis of a 454-‐sequenced transcriptome obtained from brain and gonads cDNA libraries of two A. naccarii individuals (one male and one female full-‐sibs).
These are the first transcriptome data for sturgeon species.
I have contributed to the phase of preparation of samples for libraries.
The sequencing yielded 55,000 high quality Expressed Sequence Tags (ESTs) given by the assembling of 182,066 reads for the male and 167,776 for the female.
Besides its evolutionary value for the amount of new information obtained for a member of the Chondrosteans, this work focused on the identification of gene related to sex differentiation, which could be differentially expressed between the two sexes. The transcriptome was screened for 32 genes related to sex differentiation of which 5 appear to be specifically for male and 2 for female.
Moreover, the identification of a large amount of SNPs (21,791) and microsatellites EST-‐
linked (5,295) is an important step towards the establishment of a genome wide genetic markers panel that could be useful in the future in monitoring the effects of conservation activities in captivity and in the wild (releases).
5.2 Analysis of transposable elements in Acipenser naccarii (original publication in Appendix II)
During these years I also had the possibility to contribute to the discovery of Tana1, a new putatively active Tc1-‐like transposable element (Plasterk et al., 1999) found in the genome of sturgeons.
The complete sequence of Tana1 was first characterized in the 454-‐sequenced transcriptome of the Adriatic sturgeon (A. naccarii) by Dott. Michele Vidotto. The element found in the transcriptome was obtained by assembling many short reads but this cannot be a certain indication that the entire sequence is transcribed.
Therefore, the element was isolated and characterized from the cDNA of the same species (by PCR and cloning) to validate the bioinformatics data. Then, Tana1 was isolated from the genome of Adriatic sturgeon and we demonstrated its occurrence also in the genome of 12
additional sturgeon species including 3 genera of the Acipenseridae (Acipenser, Huso and Scaphirhynchus).
The integrity of the native form (with the entire sequence of the transposase), the presence of all expected functional domains (Shao and Tu, 2001; Yuan and Wessler, 2011) and its occurrence in the sturgeon transcriptome suggest a current or recent activity of Tana1. The results of this study were recently published (Pujolar et al., 2013; Appendix I).
Moreover, the discovery of DNA editing activity by ADAR (Adenosine Deaminase Acting on RNA) enzymes on the Tana1 inverted repeats opens new interesting perspectives for the next year to study the putative activity of this element and the role of post-‐transcriptional editing on the activity of transposable elements in vertebrates.
A NNEX A
Inheritance pattern of microsatellite loci in the polyploid Adriatic sturgeon (Acipenser naccarii)
Elisa Boscari, Federica Barbisan, Leonardo Congiu⁎
Department of Biology, University of Padova, Via Ugo Bassi 58/b, 35121 Padova, Italy
a b s t r a c t a r t i c l e i n f o
Article history:
Received 8 April 2011
Received in revised form 31 August 2011 Accepted 8 September 2011
The Adriatic sturgeon is a highly endangered tetraploid species whose conservation depends up ex-situ man-agement of the remnant genetic variability. Understanding whether the species follows a tetrasomic or a di-somic inheritance pattern is of primary importance to set up a parental allocation procedure and to establish a long-term breeding plan. Moreover, comprehending the inheritance modality can strongly contribute to understanding the origin of tetraploidy in this species. For this purpose, microsatellite inheritance patterns were analyzed in 7 complete families and at 7 loci for a total of 12 family/locus combinations. For each avail-able family, a preliminary selection of loci was performed, in order to avoid ambiguities due to allele dosage, null alleles or interference between parental contributions. Results allowed to unambiguously reject a strict disomic inheritance pattern and to suggest tetrasomy as the more likely model. Accordingly, parental chro-mosomes can be expected to pair in the gametes in all possible combinations, though a certain degree of pref-erential pairing could not be excluded for the limited statistical power reached. This study represents the first investigation of the inheritance pattern in the Adriatic sturgeon and provides relevant information for the correct management of its residual genetic diversity.
Published by Elsevier B.V.
1. Introduction
Sturgeons (infraclass Chondrostei, order Acipenseriformes) are a very ancient fish group, distributed in the Palearctic hemisphere and represented by about 25 species (Peng et al., 2007). The interna-tional Union for Conservation of Nature (IUCN, March 2010) recently identified sturgeons as the most endangered group of animals with 85% of the species being at risk of extinction according to the Red List of Threatened Species (http://www.iucnredlist.org). This vulner-ability is mostly due to the very high economic interest in these ani-mals, overexploited for the production of caviar, one of the most valuable products on the international food market (Ludwig, 2008).
Since 1998, international trade in all species of sturgeons has been regulated under CITES (http://www.cites.org) owing to concerns over the impact of unsustainable harvesting and illegal trade (Ludwig, 2008; Pikitch et al., 2005). Moreover, the dramatic decline in natural sturgeon populations in recent years prompted conservation efforts for most sturgeon species by means of restocking with animals pro-duced by controlled reproduction (Congiu et al., 2011).
The correct management of highly imperiled populations by means of ex-situ conservation programs must be supported by adequate ge-netic investigations (Doukakis et al., 2010; Ludwig, 2006), which in sturgeons deal with a very complex genome and different levels of
ploidy (Fontana et al., 2008; Ludwig et al., 2001). Two main groups of species can be identified based on their number of chromosomes, which is about 120 or 240, respectively. The level of ploidy to be as-cribed to these chromosome numbers is still being debated. Some au-thors consider species of the two groups to be diploid and tetraploid, respectively (Fontana et al., 2007), while others attribute the tetraploid or octoploid conditions to these same groups (Birstein and Vasil'ev, 1987). The high number of chromosomes was likely reached through multiple polyploidization events starting from a 60-chromosome com-mon ancestor (Fontana et al., 2008). After the first duplication event, the resulting tetraploid genome (4n=120) underwent a functional diploi-dization (2n=120). For this reason, species presenting about 240 chro-mosomes, considered to be octoploid by some authors, can be considered as functionally tetraploid, originating from a second event of chromosome doubling that likely took place more than once in Aci-penseridae (Fontana et al., 2008) and possibly due to different mecha-nisms. Accordingly, the different tetraploid species (2n=240) distributed in all the northern hemisphere might be either auto- or allo-tetraploids (Fontana et al., 2008).
Depending on the origin of tetraploidy, different modalities of chro-mosome segregation into gametes are expected, namely tetrasomic and disomic (Stift et al., 2008). The tetrasomic pattern would be followed when each chromosome has four homologous copies (A1A2A3A4) and each copy may pair randomly with any other homolog during meiosis.
This is usually observed in autotetraploids (derived from an intra-specific genome duplication). The six possible combinations all can seg-regate into gametes (A1A2, A1A3, A1A4, A2A3, A2A4, A3A4) (Stift et al., Aquaculture 321 (2011) 223–229
⁎Corresponding author. Tel.: +39 49 8276218; fax: 39 49 8276209.
E-mail address:[email protected](L. Congiu).
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2008). On the contrary, a disomic inheritance pattern would be ob-served in the presence of four homeolog (i.e., not properly homolog) chromosomes (A1A2B1B2) that cannot randomly pair. This generally oc-curs in allotetraploids in which tetraploidy is reached through hybridi-zation. Each chromosome pairs only with its homolog in meiosis and only four different allele combinations can segregate into gametes (A1B1, A1B2, A2B1, A2B2) (Stift et al., 2008). Besides the pure tetrasomic and disomic modalities, different degrees of preferential pairing be-tween chromosomes could lead to intermediate inheritance patterns in which all the possible chromosome combinations are expected with different frequencies (Stift et al., 2008).
Apart from providing information on the origin of their karyotype, un-derstanding the inheritance pattern in tetraploid sturgeon species repre-sents a necessary step toward the correct management of the residual genetic variability and the establishment of a breeding program for both conservation and aquaculture purposes (Rodzen et al., 2004). This is the case for the Adriatic sturgeon (Acipenser naccarii), endemic to the North Adriatic Sea and its tributaries. Since May 2010, it has been listed by IUCN as critically endangered and possibly extinct in the wild, on the basis of an estimated population decline greater than 80% (possibly 100) over the past three generations. Moreover, no evidence of natural spawning has been reported in the last 15 years. The few animals occa-sionally captured in the wild are probably of aquaculture origin and were released during the several restocking activities that have taken place since 1992 (Congiu et al., 2011; Ludwig et al., 2003). All Adriatic sturgeons released in the past were obtained by artificial reproduction starting from a limited broodstock of 50 animals of wild origin (hereinaf-ter F0) stocked in an aquaculture plant located in Orzinuovi (Brescia, Italy) since the early 1970s. Besides the F0stock, additional few stocks of F1 an-imals, produced by artificial generation, were retained in captivity for up to 15 years and are now available as breeders. Currently, the conservation of the Adriatic sturgeon is totally dependent on the correct management of these captive breeders through the establishment of a long-term breed-ing plan aimed at preservbreed-ing the genetic diversity available. Unfortunate-ly, no information is available about the degree of relatedness existing among these future F1breeders and the establishment of a reliable paren-tal allocation procedure is indispensable for a long-term breeding plan (Congiu et al., 2011).
However, the establishment of a parental allocation procedure on a tetraploid species cannot neglect the modality of inheritance. The geno-types expected in the progeny of a given parent vary under different transmission models. Moreover, the knowledge of the inheritance pat-tern is basic for the development of virtual offspring simulation proce-dures that can be very useful in assessing the expected range of genetic variation under different degrees of kinship (Congiu et al., 2011). In fact, knowing the inheritance model at microsatellite loci, al-lows simulating full or half sibs genotypes that can be used for estimat-ing expected distribution of pairwise relatedness, under different degrees of kinship. This might be especially useful when comparing in-dividuals whose parents are not sampled or no longer exist (Rodzen et al., 2004). The modality of inheritance has never been investigated be-fore, in either the Adriatic sturgeon or in other Palearctic polyploid stur-geons. The only available studies were conducted on two North American tetraploid species: the white sturgeon (Acipenser transmonta-nus) (Rodzen and May, 2002) and the lake sturgeon (Acipenser fulves-cens) (McQuown et al., 2002; Pyatskowit et al., 2001) by comparing microsatellite genotypes of parents and progeny. These authors inferred allele dosage from band intensity and compared the frequencies of ob-served genotypes with the ones expected under the pure tetrasomic or disomic inheritance patterns. However, as better developed in the Dis-cussionsection, this approach may present some ambiguities due to in-ference of allele dosage, presence of null alleles and interin-ference between parental genotypes due to allele sharing. Furthermore, since
In the present study, we analyzed microsatellite segregation pat-terns in complete families; however, in order to avoid the possible ambiguities cited above, we performed a preliminary selection of the loci to be analyzed. Only loci for which each single allele could be identified and followed in the progeny were used. For this purpose, all parents of the available families were genotyped in advance at 24 loci, and for each family, only the loci that satisfied the conditions explained in the following section were further analyzed in the prog-eny. This permitted obtaining, for the first time, unambiguous results about the inheritance pattern in a sturgeon species.
2. Materials and methods
2.1. Preliminary selection of families and loci
All samples analyzed in the present study were obtained from the aquaculture plant Azienda Agricola VIP located in Orzinuovi (Brescia, Italy), where all the breeders of wild origin are reared and where some of them are reproduced every year. We focused on the mating pairs used for reproduction in 2007 and 2010, for which progenies had already been collected or were already available, respectively.
All the breeders had been genotyped at 24 loci (Congiu et al., 2011) and the genotypes screened for the selection of loci to be analyzed.
A locus was considered to be informative when the following condi-tions were satisfied: (a) at least one of the parents (the informative one) had a complete heterozygote genotype (four different alleles);
(b) no more than one allele was shared by the two parents. In this way (point a), the four alleles of the informative parent are known to be present in a single copy, thus avoiding ambiguities due to allele dosage. Moreover, two of the four alleles are always expected to seg-regate in every F1animal, thus excluding the presence of null alleles.
Accordingly, the transmission of each allele could be unambiguously followed in the progeny. With regard to point b, alleles transmitted by the informative parents can also be identified in the progeny when one allele is shared with the mating partner. An example is shown inFig. 1in which allele 1 is shared by the two parents. After preliminary selection, 7 families and seven loci were selected for fur-ther analyses. Since some families were informative at more than one locus, a total of 12 family-locus combinations were analyzed. In one family group (NaccS17♂× NaccS19♀) we also had the opportunity to follow the genetic contribution of both parents, both complete het-erozygotes without shared alleles.
2.2. Sample collection and laboratory procedures
Genomic DNA was extracted from breeders' fin clips (10–100 mg) and from F1larvae, using the DNA Easy Tissue Extraction Mini kit (Euroclone) and stored at−4 °C. A total of 174 F1animals were ana-lyzed (Table 1). Loci anaana-lyzed are listed inTable 1, together with the optimized annealing temperatures, allelic size range and the number of fingerlings genotyped for each family.
Microsatellite loci were amplified from genomic DNA in 25 μl reac-tions containing: Taq buffer 1X (GE Healthcare), 1.5 mM MgCl2, 0.4 μM of each primer, 200 μM dNTPs, 0.5 units of Taq (GE Healthcare), and about 50 ng of genomic DNA. All amplifications were performed on GeneAmp PCR System 9700 thermal cyclers (Applied Biosystems).
After checking amplifications on 1.8% agarose gel, genotyping was performed on ABI PRISM 3730XL or ABI 3100 automatic sequencers (external service, BMR Genomics). Scoring was conducted using the software Genotyper version 3.7 (Applied Biosystems).
2.3. Statistical analyses
224 E. Boscari et al. / Aquaculture 321 (2011) 223–229
Stift et al. (2008). For each cross-locus combination, we obtained log-likelihood estimates from constrained nonlinear regression models of the alternative inheritance models, using SPSS syntax provided by Stift et al. (2008). Significance levels for multiple comparisons across loci and families were adjusted using the sequential Bonferroni tech-nique (Rice, 1989).
3. Results
At 23 of 24 loci previously analyzed in the F0animals, no individ-uals showed more than four alleles (data not shown). The only excep-tion was represented by locus D3 in which 23 of 42 F0 animals showed 5, 6 or 7 alleles. Locus D3 was already known to be duplicated (Forlani et al., 2008) and was accordingly discarded from this study.
Of the remaining 23 loci, 20 were potentially useful in investigat-ing allele transmission patterns, havinvestigat-ing four or more alleles. The max-imum number of alleles per locus observed was 16 with an average of 7.96 (SD=3.78) alleles per locus.
Table 2reports the different allele combinations (with the corre-sponding frequencies) observed in the progenies of all families at analyzed loci. For each family, the allelic profiles of both parents are reported along with six F1profiles, representative of the different allele combinations observed. Alleles inherited from the informative hetero-zygote parent are marked in bold. The alleles inherited from the
other parent were not informative, except for the case of family NaccS17 × NaccS19 at locus AoxD234, in which both parents were completely heterozygote.
At all loci analyzed, the six allele combinations expected in the prog-eny under tetrasomic inheritance were observed. The only exception is represented by locus AoxD241, which showed only 5 allele combina-tions in the progeny of the family NaccS29×NaccS26, probably due to the limited number of F1individuals available. However, the presence of five combinations is sufficient to exclude a disomic inheritance pat-tern. Moreover, at the same locus, all six allele combinations were ob-served in the progeny of NaccS30×NaccS16, confirming tetrasomy.
Results of Likelihood ratio tests are reported inTable 3in which the best fitting model among the ones tested is compared with the null hy-pothesis of tetraploidy. Strict disomic inheritance was rejected at all loci and no intermediate pattern fitted the observed allele combination fre-quencies significantly better than the null model. The number of indi-viduals analyzed did not allow us to significantly reject intermediate inheritance models, however, also in the case of a certain degree of pref-erential chromosome pairing, the rejection of the strict disomic model indicates that all possible pair combinations of parental chromosomes can be expected in the gametes.
4. Discussion
4.1. Inheritance pattern
Preliminary genotyping performed on F0animals at 24 loci provid-ed information useful for inferring the ploidy level of the Adriatic sturgeon. As mentioned above, the level of ploidy to be ascribed to sturgeon species with about 120 or 240 chromosomes has been de-bated. The observed presence of no more than four alleles per individ-ual at 19 loci (out of the 20 loci with more than four alleles in the population) suggests that the Adriatic sturgeon should be considered functionally a tetraploid. Only one locus (D3) previously reported to be duplicated (Forlani et al., 2008), presented up to seven alleles per individual (data not shown). Locus D3 is not the one with the highest number of alleles among the ones analyzed in this study, however, more than half the individuals showed five, six or seven al-Table 1
Technical details of microsatellite screenings of Acipenser naccarii families.
Parental pairs
NaccS30 ♂×NaccS16 ♀ AnacE4 57 °C 326–354 28
AoxD241 57 °C 156–198 25
NaccS31 ♂×NaccS19 ♀ AoxD234 52 °C 215–275 29
AnacB10 62 °C 212–258 30
NaccS9 ♂×NaccS33 ♀ AoxD234 52 °C 215–275 31
NaccS29 ♂×NaccS26 ♀ AoxD241 57 °C 156–198 20
NaccS31 ♂×NaccS8 ♀ AoxD234 52 °C 215–275 28
NaccS17 ♂×NaccS19 ♀ AoxD234 52 °C 215–275 14
NaccS23 ♂×NaccS28 ♀ AoxD64 60 °C 216–252 23
AnacE4 57 °C 326–354 22
AnacA6 62 °C 289–313 23
Fig. 1. Expected allele segregation under disomic and tetrasomic inheritance. The figure represents the possible expected phenotypes if one parent presents a completely hetero-zygote genotype and shares one single allele with the mating partner. Under the hypothesis of disomy, we have supposed pairs 1,2 and 3,4 to be homolog. Consequently allele com-binations 12 and 34 are not expected under disomic inheritance while under tetrasomic inheritance all pairwise comcom-binations of alleles are possible. Underlined numerals indicate biparental inheritance of allele 1.
225 E. Boscari et al. / Aquaculture 321 (2011) 223–229
Table 2
Single-locus microsatellite inheritance for each sturgeon family. A total of 12 family/locus combinations were studied. Phenotypic profiles of parents and six representative F1individuals are reported for each cross. Alleles are reported as allele sizes in bp. The 4 alleles of the complete heterozygote parents are highlighted in bold and marked with capital letters A, B, C or D, also used to label allele combinations observed in the progeny. The missing combination for the family NaccS29 × NeccS26 at locus AoxD241 is marked in gray. Since both NaccS17 and NaccS19 parents were informative at locus AoxD234, alleles observed in NaccS17 are underlined and labeled with lowercase letters. The umber of individuals observed for each allele combination is reported in brackets.
Locus AoxD234 Phenotypes Locus AoxD241 Phenotypes
A
Observed combinationsObserved combinationsObserved combinations Observed combinationsObserved combinations
Observed combinations
CD (3) 223 227 247 275 CD (4) 172 176 180 188
Locus AoxD234 Phenotypes Locus AnacB10 Phenotypes
215 219 215 249 252
Locus AoxD234 Phenotypes Locus AoxD234 Phenotypes
215 219 a
Locus AnacE4 Phenotypes Locus AoxD241 Phenotypes
332 344 A
226 E. Boscari et al. / Aquaculture 321 (2011) 223–229
with a comparable or higher level of polymorphism. The absence of individuals with more than four alleles at all remnant loci suggests that locus D3 maps to a duplicated region in the genome, as already reported byForlani et al. (2008).
with a comparable or higher level of polymorphism. The absence of individuals with more than four alleles at all remnant loci suggests that locus D3 maps to a duplicated region in the genome, as already reported byForlani et al. (2008).