Om Parkash

Christophe Billette

POSITIONING OF INTRONS IN DIFFERENT LACCASE GENES, A RELEVANT TOOL FOR SOLVING PHYLOGENETIC POSITION AMBIGUITY OF VOLVARIELLA VOLVACEA LACCASE GENES

Keywords: Volvariella volvacea, Phylogenetic, Laccase, Introns, Heterozygotic

Volvariella volvacea (paddy straw mushroom) is a high temperature-loving mushroom with the shortest cropping cycle in the basidiomycete family Pluteaceae. This mushroom is cultivated extensively in many South-East Asian and African countries by employing both the outdoor and indoor cultivation techniques. The objective of this study is to analyze intron positions in different laccase genes of this mushroom. Primer pairs for laccase 1, 2, 3, 4, 5 and 6 were designed by using the mRNA sequences of these genes available in NCBI nucleotide database and the Primer 3 free software. These primers were used to amplify different laccase genes in one heterozygotic strain, Vv-01 and one putative homozygotic strain, BBSR-003 at an annealing temperature range of 53 to 58ºC to obtain amplicons of requisite sizes. PCR products were then sequenced and annotated using the BioEdit and Artemis softwares. Out of 6 laccase genes, partial sequences of laccase 1, 2, 3 and 6 in the heterozygotic strain Vv-01 and partial sequence of laccase 3 in homozygotic strain BBSR-003 were obtained. The lcc 3 sequences of two strains, Vv-01 and BBSR-003 have been submitted to NCBI GenBank with accession numbers HQ687205 and JF313903, respectively and they showed differences at 10 different base positions. In lcc 3, there was an intron 8 found earlier in laccase genes from Coprinopsis cinerea, but same has not been spliced in published mRNA sequence of V.volvacea. This intron has to be spliced in strain Vv-01 to give a functional protein and by virtue of which, the phylogenetic position of lcc3 in laccase dendrograms is probably wrong. In lcc 1, 16 different introns have been recorded and the amino acid sequence of Vv-01 strain differs by one amino acid less than the published sequence of lcc1. No evidence of heterozygocity has been recorded in this sequence. The lcc 2 sequence also revealed 16 different introns. Although only a small portion of sequence could be validated but evidence for heterozygocity was found in strain Vv-01. The small sequence of lcc 2 in strain BBSR-003 also differed at some bases from strain Vv-01. At the protein sequence level, 26 amino acids of the published mRNA seemed to be derived from the wrong reading frame. This could be due to errors in sequencing, or that the strain used had a pseudo gene or a very original allele for lcc 2. Based on these observations, the position of laccase 2 in the dendrogram is likely to be changed. The sequence of lcc 6 helped in identifying 5 introns. The positions of introns in different laccase genes (lcc 1, lcc 2 and lcc 3) of V. volvacea grouped them to sub-family A.

INTRODUCTION

Volvariella volvacea (Bull ex Fr.) Sing. belongs to the family Pluteaceae Kotl. & Pouz. of the Basidiomycetes [START_REF] Singer | The Agaricales in modern taxonomy[END_REF]. It is an important edible mushroom of the tropics and subtropics. Literature available on this mushroom has emphasized more on its cultivation technology than its morphology [START_REF] Chang | Volvariella volvacea and its life history[END_REF], cytology [START_REF] Li | Variation in the homothallic basidiomycetes Volvariella volvacea[END_REF], physiology [START_REF] Philippoussis | Bioconversion of agricultural lignocellulosic wastes through the cultivation of edible mushroom Agrocybe aegerita, Volvariella volvacea and Pleurotus spp[END_REF][START_REF] Akinyele | Effect of agro wastes, pH and temperature variation on the growth of Volvariella volvacea[END_REF] and molecular biology [START_REF] Buswell | Cultivation, biochemical, molecular biological and medicinal aspects of the culinary-medicinal straw mushroom Volvariella volvacea (Bull.:Fr.) Singer (Agaricomycetideae)[END_REF]. This mushroom can produce an array of extracellular hydrolytic and oxidases enzymes -e.g. endo-1,4-β-gucanase (EC 3.2.1.4), cellobiohydrolase (EC 3.2.1.91), β-glucosidase (EC 3.2.1.21) and laccase (EC 1.10.3.2) [START_REF] Buswell | Cultivation, biochemical, molecular biological and medicinal aspects of the culinary-medicinal straw mushroom Volvariella volvacea (Bull.:Fr.) Singer (Agaricomycetideae)[END_REF][START_REF] Cai | Cellulases and hemicellulases of Volvariella volvacea and the effect of Tween 80 on enzyme production[END_REF][START_REF] Cai | β-Glucosidase components of the cellulolytic system of the edible straw mushroom, Volvariella volvacea[END_REF][START_REF] Cai | Production and distribution of endoglucanase, cellobiohydrolase, and β-glucosidase componenets of the cellulolytic system of Volvariella volvacea, the edible straw mushroom[END_REF][START_REF] Thurston | The structure and function of fungal laccases[END_REF][START_REF] Ahlawat | Profile of the extra cellular lignocellulolytic enzymes activities as a tool to select the promising strains of Volvariella volvacea (Bull. ex Fr.) Sing[END_REF]. The copper binding regions and the N-terminal amino acid sequences have also been used to generate complete sequence of six laccases from this mushroom [START_REF] Chen | Biochemical and molecular characterization of laccase from edible straw mushroom, Volvariella volvacea[END_REF][START_REF] Chen | Molecular cloning of a new laccase from the edible straw mushroom Volvariella volvacea: Possible involvement in fruit body development[END_REF] and out of these, lac1 and lac4 have been suggested to play roles in morphogenesis in this mushroom [START_REF] Chen | Biochemical and molecular characterization of laccase from edible straw mushroom, Volvariella volvacea[END_REF][START_REF] Chen | Molecular cloning of a new laccase from the edible straw mushroom Volvariella volvacea: Possible involvement in fruit body development[END_REF]. The complete mRNA sequences of six laccases in this mushroom have also been obtained in the past, which are also available in NCBI database.

The genus Volvariella comprises about 50 species worldwide [START_REF] Kirk | Anisworth & Bisby's dictionary of fungi[END_REF], including the cultivated V. volvacea (paddy straw mushroom). Using the 'strips' recognized by Singer [START_REF] Singer | The Agaricales in modern taxonomy[END_REF], several morphological/ecological groups have been differentiated within this genus. Specifically, the V. volvacea-group has been identified with pileus >50 mm in diameter, with darkly colored and usually grey-brown basidiospores. Based upon the nLSU data, Moncalvo et al. [START_REF] Moncalvo | One hundred and seventeen clades of euagarics[END_REF] placed Volvariella in a very distinct position to Pluteus, clustering with Fistulina Bull. and Schizophyllum Fr. Similarly in a later study [START_REF] Matheny | Major clades of Agaricales: a multigenetic overview[END_REF] using six genes, Volvariella (V. gloiocephala) was placed as the sister group of Pluteus along with Melanoleuca, traditionally classified in the Tricholomataceae R. Heim ex Pouzar [START_REF] Singer | The Agaricales in modern taxonomy[END_REF], and were together placed with members of Amanitaceae, Pleurotaceae Kuhner, the aquatic basidiomycete Limnoperdon G.A. Escobar and some other 'orphan' agaric genera (Tricholomopsis Singer, Cantharocybe H.E. Bigelow & A.H. Sm., Macrocystidia Joss) in one major group named the 'Pluteoid' clade. Likewise Binder et al., [START_REF] Binder | Amylocorticiales ord. Nov. and Jaapiales or. Nov.: early-diverging clades of Agaricomycetidae were dominated by corticioid forms[END_REF] got a similar topology for Pluteus, V. gloiocephala and Melanoleuca with Amanita Pers. as members of the core Pluteoid clade with Tricholomopsis and Cantharocybe outside of the clade. In a recent study based upon molecular data from nuclear ribosomal genes (nSSU, ITS, nLSU), Volvariella has been described as polyphyletic, and Volvariella sensu stricto has been placed with some hygrophoroid genera (Camarophyllus, Cantharocybe), representing as a sister group of the Pluteoid clade and a new generic name, Volvopluteus has been proposed with species as earlei and gloiocephalus, keeping these last ones in Pluteoid clade [START_REF] Justo | Phylogeny of the Pluteaceae (Agaricales, Basidiomycota): taxonomy and character evolution[END_REF]. Compared to the Agaricoid clade, the respective positions of Pleurotus ostreatus and V.volvacea are still unclear.

Recently, the laccase genes have been used for species identification and establishment of evolutionary relationships, where other molecular markers have failed [START_REF] Hirschhauser | Multiplex PCR for species discrimination of Sclerotiniaceae by novel laccase introns[END_REF]. This group of enzymes (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.) play important role in catalyzing the single electron transfer from various organic compounds and concomitant four-electron reduction of oxygen to water, and they are found widely spread in plants, bacteria, insects and fungi. Laccase gene has also been used for studying presence of basidiomycetes in a forest soil and saprotrophic fungi have been reported to be less spread through the soil horizons than the mycorrhizal ones. [START_REF] Luis | Diversity of laccase genes from basidiomycetes in a forest soil[END_REF]. The diversity of the laccase genes have again been used in phylogenetic perspectives of Botryosphaeria rhodina (Ascomycota: fungi) and some related taxa [START_REF] Castilho | On the diversity of laccase gene: A phylogenetic perspective from Botryosphaeria rhodina (Ascomycota: Fungi) and other related taxa[END_REF], and likewise the multi-gene family of Coprinopsis cinerea has been studied, which has been divided into two sub-families A and B [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide in to two distinct sub-families[END_REF]. In the laccase phylogeny, three principal clades, comprised Section: Diversity and Taxonomy of sub-family A and B from C. cinerea, and sub-family C from Pleurotus ostreatus PoxA3 and A. bisporus lcc1 and lcc2 laccases have been proposed [START_REF] Hoegger | Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences[END_REF][START_REF] Courty | Phylogenetic analysis, genomic organization, and expression analysis of multicopper oxidases in the ectomycorrhizal basidiomycete Laccaria bicolor[END_REF][START_REF] Lettera | Identification of a new member of Pleurotus ostreatus laccase family from mature fruiting body[END_REF]. In clade A, the position of V. volvacea is very peculiar, which suggests that these laccases can belong to a new clade, distinct from clade A and supports the idea of an early divergence of these genes from the other laccase genes of clade A. The present study was conducted to determine the variations in the positions of introns in laccase genes of V. volvacea, and identify their relationship with the positions of introns in laccase genes of other related species, and to draw the evolutionary relationships between the laccase genes of related species.

MATERIALS AND METHODS

V. volvacea strains and their DNA extraction. The strains Vv-01 and BBSR-003, originally collected from Coimbatore (Tamil Nadu) and Bhubaneswar (Orissa), the Southern costal region of India, were used in the study. The strains were grown separately at 32 ± 2ºC for 5 days on Malt Extract Agar medium petridishes and the well grown mycelia was scarped and used for genomic DNA extraction. DNA was extracted by using Illustra DNA extraction Kit from GE Healthcare, UK by following the protocol provided by the manufacturer.

Primers for laccase gene amplification:

The primer pairs for six laccase genes (lcc1, lcc2, lcc3, lcc4, lcc5 and lcc6) of V. volvacea were designed based on the mRNA sequences of these genes (Accession Nos. lac1-AY249052.1, lac2-AY338483.1, lac3-AY338484.1, lac4-AY338486.1, lac5-AY338485.1 and lac6-AY338487.1) available in NCBI nucleotide database using Primer 3.0 (version 0.4.0) free software available on internet. These primers were synthesized from Sigma Life Science, France (Table 1).

PCR amplification of laccase genes: PCR amplification of different genes was performed in reaction mixture of 25 µl; comprising 5X PCR buffer 5 µl (Promega, Medison, WI), dNTP 3.5 µl (1.2 mM each), bovine serum albumin 0.5 µl (10 mg/ml), Taq DNA polymerase 0.2 µl (5 U/µl, Promega M830B), primer pairs @ 2 µl each (10 µM) and DNase/RNase-free water 10.8 µl and template DNA 1 µl (50 ng/µl). The PCR reaction was performed in Master Cycler Gradient (Eppendorf) with initial denaturation at 95ºC for 5 min, followed by 35 cycles of 95ºC for 1 min, 55ºC for 5 min and 72ºC for 2 min 30 sec, and by final extension at 72ºC for 4 min, with a lid heating option at 110ºC. The PCR amplified products (amplicons) were run on 1.2% agarose gel prepared in 1.0% TBE buffer and the gel was run at 70 V for 1 hour 30 min in 0.5% TBE buffer. The gel was visualized under gel doc system from BioRad.

PCR amplification of different genes was optimized by using the annealing temperature gradients of 2 to 6ºC depending on the performance of the initial PCR amplification of the different laccase genes. The annealing temperature giving the highest product intensity at appropriate position (product size) under UV light was selected as the annealing temperature for further PCR reactions.

Sequencing of PCR amplicons:

The amplicons along with the corresponding primer pairs (forward and reverse) were sent to BECKMAN COULTER GENOMICS, UK for sequencing and the received sequences were further processed.

Section: Diversity and Taxonomy

Analysis of sequences and annotation for intron positioning:

The received sequences were edited using the BioEdit free software. This software facilitates visualization of the nucleotide sequences along with their peaks on chromatogram. The poor quality sequences were first deleted, followed by blast searches to verify the genes. The confirmed cleaned sequences were saved.and compared with the available mRNA sequence using CAP contig. The comparison involved opening of the cleaned sequences from both forward and reverse primers of respective laccase genes along with mRNA sequence together inside the same window. The sequences obtained from both forward and reverse primer were matched with the sequence of mRNA to identify the location of different introns in the laccase genome. Further annotations for finding amino acid sequences and the intron positions at the amino acid level were performed using the Artemis free Software. The annotated nucleotide and amino acid sequence obtained from different laccase genes were prepared in Sequin for their submission to NCBI data base as the new sequences.

Statistical Analysis:

The intron profile of each laccase gene was scored as '1' for presence and '0' for absence of intron. A putative ancestor laccase gene with no intron was added. A Section: Diversity and Taxonomy combined binary data matrix for all the laccase genes was constructed. The binary data matrix was entered in the Paup package version 4.0 beta [START_REF] Swofford | PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods)[END_REF] and data was analysed using DOLLO parsimony [START_REF] Young | Are the Angiosperms Primitively Vesselless[END_REF]. Bootstrapping was carried out with 1000 replications.

RESULTS AND DISCUSSION

Amplification of laccase genes: Out of the six pairs of primers corresponding to the six different laccase genes of strain Vv-1, unique single bands of desired sizes were obtained only for laccase genes 1 and 3. For laccase 2, only low molecular weight fragments (probably primers dimers) were obtained, while in the case of laccase 4, multiple amplicons were obtained, which indicates non-specificity of the primers. For laccase 5 and 6 genes, the amplicons of desirable size were obtained but were in very low concentration. In order to improve the amplification efficiency, the PCR was performed at an annealing temperature of 57 ± 2ºC and amplicon's band intensities increased with the increase in the annealing temperature (Fig 1). Similarly, attempts were made for laccase 4 and 5 genes at annealing temperatures of 57 ± 2ºC and 54 ± 1ºC, respectively but no further improvement in amplicon quality (intensity /purity) was recorded, hence the primer pairs were rejected due to low specificity. The redesigned primers of laccase 2 and 6 genes were tried at annealing temperatures of 58 ± 2ºC and laccase 2 exhibited fairly good amplification at almost all temperatures (56-60ºC), while laccase 6 gene exhibited amplification at only 59 and 60ºC annealing temperatures. In case of homozygotic strain BBSR-003, out of 4 pair of primers (laccase1, 2, 3 & 6) attempted at 57 ± 1ºC annealing temperatures, appropriate amplification were obtained only for laccase 3 gene.

Intron positions in different laccase genes:

The CAP contig of laccase 1 gene revealed a total of sixteen introns in addition to the possibility of additional introns at the beginning of the gene. The number of introns was also sixteen in laccase 2 gene. However, in same stretch of gene length, it had intron 12, which was absent in lcc 1. The three introns (70, 72 and 75), which existed in lcc1 were missing in this gene. Laccase 2 exhibited two introns (3 and 4) in the genome region, where sequence is missing in lcc1 and lcc3 (Fig. 2). A total of eleveen introns could be documented in the same gene length of laccase 3 from strain Vv-1, out of which only six matched with the positions of introns in lcc1 and seven in lcc2 (Fig. 3). The small partial sequence of lcc6 we obtained exhibited only five introns (position 72, 39, 73, 74 and 77, a new position between 62 and 75) and their location was not considered in laccase gene classification nor in the phylogenetic analyses between laccases of different mushroom species. In a similar study carried out on Coprinopsis cinerea, the number of introns in different laccase genes varied from what we have recorded in the present study [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide in to two distinct sub-families[END_REF]. It was 7 in lcc1, while 19 in lcc17 [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide in to two distinct sub-families[END_REF][START_REF] Hoegger | The laccase gene family in Coprinopsis cinerea (Coprinus cinereus)[END_REF]. The position of introns in different laccase genes of different mushrooms was compared and the laccases belonging to sub-family A (Lcc3 and Lcc9) from Coprinopsis cinerea exhibited a closer relationship with respect to positions of introns in their genes than laccase genes belonging to sub-family B or C (Fig. 2). Fig. 3 presents the comparative sequences of laccase 2 gene of strain Vv-01 of V. volvacea with positions of 16 introns to the published mRNA sequence from some other strain. In this figure the gray boxes indicate bases that still need to be confirmed. Some of the ambiguous results were due to the heterozygocity of this gene (two sequences of gene) in the heterokaryotic strain Vv-01.

VV1_lcc2

~~~~~~~~~~ Eight most parsimonious cladograms deduced from the positions of introns in twelve laccase genes belonging to three sub-families from four different basidiomycetes, revealed formation of eleven different clades (Fig. 4). In this dendrogram, Lcc3 from V. volvacea occupied the same clade as Lcc3 from C. cinerea in 97% of the bootstraps. Lcc1 and Lcc2 from V. volvacea also formed one clade with more than 98% of the bootstraps. Lcc9 from C. cinerea belonging to sub-family A formed a separate clade from Lcc3 from same mushroom [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide in to two distinct sub-families[END_REF] similarities between laccases of one sub-family belonging to a particular species and their distinctness from laccases of other sub-families either from the same or other related species. Laccases from V. volvacea share into two groups as already shown with protein sequences. In neighbour joining tree of the deduced amino acid sequences of C. cinerea, lcc2, lcc3, lcc12, lcc13 and lcc14 were placed in one clade [START_REF] Kilaru | The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide in to two distinct sub-families[END_REF]. In Hoegger's study [START_REF] Hoegger | Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences[END_REF], the neighbour joining tree of basidiomycete laccases based on realigned sequences has also clustered Vvo lac3 with Cci Lac3, as in present study. In Fig. 4, VvLcc2 and VvLcc1 clustered outside of the clade along with CcLcc9, VvLcc3 and CcLcc3, but another of the eight most parsimonious cladograms obtained, have placed these two genes as the sister group of the remainder of the sub-family A laccases.

Sequence variations in lcc3 of two strains:

The nucleotide sequence obtained for laccase 3 genes of two strains (Vv-01 and BBSR-003) varied at ten different nucleotide positions. Strain Vv-01 exhibited heterozygocity at 10 different places, which are presented as R, Y, R, Y, R, Y, R, Y, R and Y, which mean A or G for R and C or T for Y. However, at these locations, the nucleotides in strain BBSR- 003 were A, T, G, T, G, C, A, C, G andC respectively. This proves heterozygocity of this mushroom at the nucleotide level of this laccase gene (Fig 5). However, at the amino acid sequence level, there was only one difference: strain Vv-01 had D or N (i.e. Aspartic acid or Asparagine) noted X (i.e. any amino acid) in Fig. 6, while strain BBSR-003 had N means Asparagine.

The present study has mainly analyzed the variations in number and position of introns in different laccases of V. volvacea and compared that variation to other known laccases of A. bisporus, P. ostreatus and C. cinerea. The study has generated some valuable information like the grouping of V. volvacea laccases (lcc1, lcc2 and lcc3) in sub-family A and confirmation of clustering VvLcc3 with CcLcc3. In the present study, there was an intron 8, found earlier in lcc3 from C. cinerea but has not been spliced in the published mRNA sequence. This intron has to be spliced in strain Vv-01 to give a functional protein and by virtue of which, the published phylogenetic position of lcc3 in the laccase dendrograms is probably wrong. In lcc1, 16 different introns have been recorded and the sequence of Vv-01 strain differs by one amino acid from the published sequence of lcc1. No evidence of heterozygocity has been recorded in this sequence. The lcc2 gene also revealed 16 different introns. Although only a small portion of sequence could be validated but that also proved some heterozygocity in strain Vv-01. The small sequence of lcc 2 in strain BBSR-003 also revealed differences at some bases from strain Vv-01. At the protein sequence level, there were clearly 26 amino acids of the published mRNA, which were on the wrong reading frame. Maybe it is due to errors in sequencing, or the strain used had a pseudo gene or a very original allele for lcc 2. Due to these factors, the position of laccase 2 in the dendrogram will be changed. The sequence of lcc6 has helped in identification of 5 introns. The positions of introns in different laccase genes (lcc1, lcc2 and lcc3) of V. volvacea showed that they belong to sub-family A. Although not the main objective, the present study has proved heterozygocity in strain Vv-01 of V.volvacea, which has not been shown in earlier studies carried out on this species. . We compared intron positions present in sub-family A laccase genes in A. bisporus and C. cinerea from Agicoid clade (VI) and P. ostreatus and V. volvacea from Pluteoid clade (III) [START_REF] Matheny | Major clades of Agaricales: a multigenetic overview[END_REF]. From this comparison, it is clear that only intron position 2 was shared exclusively by P. ostreatus and the Agaricoid clade laccases. However, data in the present study is insufficient to indicate whether this intron position is present in V. volvacea. In contrast, intron positions 5, 6 and 10 are shared exclusively by V. volvacea and Agaricoid clade laccases. VvLcc3 clustered with Agaricoid clade VI sub-family A genes while P. ostreatus laccase genes formed a sister clade. This could indicate that ancestor genes from sub-family A laccases from V. volvacea and Agaricoid species could have diverged more recently than from ancestor genes of P. ostreatus sub-family A laccases. However VvLcc1 and VvLcc2 share intron position 37 and 39, which are present in laccase sub-family C and in Polyporus sub-family A laccases (Billette C. unpublished result). Then they could belong to the clade containing Polyporus laccase in sub-family A. Moreover, position of these two genes vary in the different cladograms we have obtained. Present finding is not sufficient to elucidate the respective phylogenetic positions of V. volvacea, P. ostreatus and Agaricoid clade species. To build further species phylogenetic trees, it is important to use sequences from V. volvacea and from Volvopluteus gloiocephalus, since they don't cluster together as demonstrated by Justo et al. [START_REF] Justo | Phylogeny of the Pluteaceae (Agaricales, Basidiomycota): taxonomy and character evolution[END_REF].

The present findings have implications on other issues such as the role of intron positions in phylogenetic studies and the use of nucleotides sequence variability in exons flanking the specific introns for species identification, as it was done in species discrimination in Sclerotiniaceae [START_REF] Hirschhauser | Multiplex PCR for species discrimination of Sclerotiniaceae by novel laccase introns[END_REF]. The sequence of conserved copper binding amino acids of novel laccase genes also has the potential to help in resolving speciation conflict in several closely related species, where other molecular techniques have yielded confusing inferences, like in the case of Sclerotiniaceae [START_REF] Hirschhauser | Multiplex PCR for species discrimination of Sclerotiniaceae by novel laccase introns[END_REF]. The concept suggests redefining different genera by identifying these genes and concomitantly supports reclassification of misleading fungal species [START_REF] Hawksworth | Fungal diversity and its implications for genetic resource collections[END_REF]. The study carried out by Castilho et al. [START_REF] Castilho | On the diversity of laccase gene: A phylogenetic perspective from Botryosphaeria rhodina (Ascomycota: Fungi) and other related taxa[END_REF] has reinforces the laccase diversity and interspecific variation in this genetic pool. As one of the most important criteria in phylogenetic systematics is the issue of homology [START_REF] Henning | Phylogenetic systematic[END_REF], and out of different types of homologies, laccases fall in the category of paralogy, which means that they originate from gene duplication events. For example, the laccase amino acid sequences have been used for phylogenetic study and the molecular evolution studies can still further help in finding the laccases of industrial importance even in still unknown species.

Figure 1 :

 1 Figure 1: PCR amplification of different laccase genes of V.volvacea. a) From left to right, surrounded by 1 Kb ladder (Life Technologies), Laccase 1 to 6. b) DNA ladder, Laccase 1 annealing temperature from 55 to 59ºC, Laccase 3 annealing temperature from 55 to 59ºC

Figure 2 .

 2 Figure 2. Relative positions of introns in different laccase genes of different sub-families. Nomenclature of introns 2 to 35 [23], 36 to 44 (A.bisporus lcc1 and lcc2), 45 to 68 (P. ostreatus Poxa3, Pox1-2-4, Pox3, Pox5), 69 to 76 (V.volvacea lcc1 and lcc3). P. ostreatus gene nomenclature corresponds to: Pox1(LACC9), Pox2 (LACC10), Pox3 (LACC4), Pox4 (LACC1), Pox5 (LACC11), PoxA1b (LACC6) and Poxa3 (LACC2)

Figure 3 :

 3 Figure 3: Sequence of laccase 2 gene of strain Vv-01 of V. volvacea with positions of 16 introns, compared to published mRNA sequence. a) Letters in bold indicate the position of forward and reverse primers (Vv1FL2 and Vv42RL2). b) Grey boxes indicate bases that must be confirmed

Figure 4 :

 4 Figure 4: One of the eight most parsimonious cladograms obtained by Dollo parsimony deduced out of 75 introns positions in laccases genes of three subfamilies of four basidiomycetes. Thirty intron positions were parsimony-informative characters. Bootstrap values are from 50 % majority-rule consensus tree. Sub-family B -CcLcc17, sub-family C-Poxa3 and AbLcc1-2, sub-family A -all other genes, ANCEST -hypothetical ancestor gene with no intron.

  . The four laccases (Pox1-2-4, Pox5, PoxA1b and Pox3) of P. ostreatus belonging to sub-family A formed a clade in 97% of the bootstraps. All sub-family A laccase genes formed a clade supported by 92 bootstraps and sub-family A and C laccases formed clade supported by 94 bootstrap value. The only laccase (Lcc17) from C. cinerea belonging to sub-family B shared only 3 intron positions with genes of sub-family A and C (Fig. 2). The results indicate high Section: Diversity and Taxonomy

Figure 5 :Figure 6 :

 56 Figure 5: Variations in partial nucleotide sequences of laccase 3 from strains Vv-01 and BBSR-003 of V. volvacea

Table 1 :

 1 Source and sequences of primers designed for different laccase genes

Laccase gene mRNA Accession No. in NCBI GenBank mRNA GI No. in NCBI GenBank Primers Sequence (5´ to 3´)

	Laccase 1	AY249052.1		37732219	Vv1FL1	CCGATGAAGTTGGGACATTC
					Vv4RL1	TCGCAATCACAATCACAGTTC
	Laccase 2	AY338483.1		37791150	Vv1FL2	CCTTGCTCAACACCCTTACC
					Vv4RL2	GCCAGAGACTGGGTTAGACG
					Vv12FL2	CTGCTGCACAACAGAGCTTC
					Vv42RL2	AATACCAGAGGACGCGAGAC
	Laccase 3	AY338484.1		37791152	Vv1FL3	TGCGAGCGTAACTCAATGTC
					Vv4RL3	GCAAAGCTCATCCCAAGAAG
	Laccase 4	AY338486.1		37791156	Vv1FL4	ATATCTGCTCGGCCATCTTG
					Vv4RL4	GACAGAGCTGCTTCCATTCC
					Vv15FL4	TCATTCTGGGGGTTATCCTG
					Vv45RL4	TAGAAGCGGGTTTCTCCTTG
					Vv05FL4	CTCGTCGCGGGTAAAATATC
	Laccase 5	AY338485.1		37791154	Vv1FL5	CAGTGCAATTTTGGTCAACG
					Vv4RL5	AAGTGTCCGATCCACTGTCC
	Laccase 6	AY338487.1		37791158	Vv1FL6	TGGAACAGCCTCTCACACAC
					Vv4RL6	ATTCGACACCAGGTCTGAGG
					Vv01FL6	CAACACCCTCACTGTCGTTC
					Vv35RL6	CACAAAGGTCATCCCACTCC
	Laccase 3	Received	genome	NA	Vv2FL3	AGCCCGTTGACAACTACTGG
	(for	sequence of lcc3		Vv3RL3	GGGACGGCATTTTCTATGAG
	remaining				Vv2RL3	AGTTGTCAACGGGCTGATTG
	portion)				Vv3FL3	TTGATTGGGATATGCTGCAC
	Laccase 1(for	Received	genome	NA	Vv18FL1	GGCTGATTTCGATCTCTTGC
	remaining	sequence of lcc1		Vv2FL1	CCGTTATCAATAGGCCAACG
	portion)				Vv18RL1	AATCAGCCTCATGCGATACC
					Vv2RL1	TTGATAACGGAATGCGTCAG
					Vv3RL1	CCTAGGGTCTTGCTCGAGTTC
					Vv08FL1	TTGAGCTCGCATACGTTGAC
					Vv45RL1	TCTGAAGCGGGGATAGAAAA

Proceedings of the 7 th International Conference on Mushroom Biology and Mushroom Products (ICMBMP7) 2011

Section: Diversity and Taxonomy


est destinée au dépôt

Trade-off between reproductive success and physiological status

Zoé Gauthey, Marine Freychet, Cédric Tentelier, Jacques Labonne, A. Elosegi

To cite this version:

Zoé Gauthey, Marine Freychet, Cédric Tentelier, Jacques Labonne, A. Elosegi. Trade-off between reproductive success and physiological status. Nowpas 2013, Apr 2013, Machynlleth, United Kingdom. 45 p. hal-02745466

Trade-off between reproductive success and physiological status Gauthey Z. (1) , Freychet M. (3) , Tentelier C. (2) , Labonne J. (3) , Elosegi A. (4)

(1) INRA, Quartier Ibarron, 64310 Saint-Pée sur Nivelle, France, zgauthey@st-pee.inra.fr

(2) INRA, Quartier Ibarron, 64310 Saint-Pée sur Nivelle, France, cedric.tentelier@st-pee.inra.fr

(3) INRA, Quartier Ibarron, 64310 Saint-Pée sur Nivelle, France, labonne@st-pee.inra.fr

(4) University of the Basque country, 48080 Bilbao (Spain), arturo.elosegi@ehu.es Key words: reproductive success, sexual selection, trade-off, metabolic status

Abstract:

Reproductive success is a central concept in evolutionary biology. It measures the number of offspring produced by an individual to the next generation and indirectly helps to assess adaptive value of individual choices and strategies in the face of natural and sexual selections. Yet reproductive strategies are time-consuming and energetically costly: both parents invest in gametes production, intrasexual competition and parental care. Considering that energetic stores are limited, we can wonder about how individuals can balance out in the tradeoff between investment in the reproduction and their energetic status. In Salmo trutta, intrasexual competition and parental care are intense; hence, the energetic status should be determinant for the access to mating and gaining reproductive success.

To answer these questions, we designed an experiment where fifty fish were placed in an experimental channel during the reproductive season and their reproductive behaviour was monitored over this time.

By estimating offspring survival and by using parentage analysis, I assessed the reproductive success of each individual. To test the influence of energy expenditure on reproductive success, body condition and metabolic status inferred from plasma samples (fatty acid, triglycerides, glucose, amino acids) were measured before and after the reproduction. My first results showed that individuals that lost the largest proportion of plasmatic triglycerides and fatty acids are those that presented the higher reproductive success. Reproductive success was also influenced by body size. These results confirm the existence of a strong trade-off between energy expenditure and reproductive success, and open a way to assess both efficiency and cost of various behavioural tactics through statistical modelling.


New methodology to measure continuously the δ13C of soil CO2 efflux and concentration at different depths by laser spectrometry Florian Parent (1,2),Caroline Plain (1,2),Daniel Epron (1,2),Bernard Longdoz (1,2) (1) INRA, UMR 1137, Ecologie et Ecophysiologie Forestières, Centre de Nancy, F-54280 Champenoux, France, (2) Nancy Université, Université Henri Poincaré, UMR 1137, Ecologie et Ecophysiologie Forestières, Faculté des Sciences, F-54500 Vandoeuvre les Nancy, France

The use of the carbon isotopic composition of CO2 (δ13C-CO2) is a promising tool to improve our knowledge about the mechanisms of CO2 production within soil. Temporal fluctuations of δ13C-CO2 soil efflux reflect the influence of climatic and edaphic conditions on (i) partitioning between the respiration sources, (ii) CO2 production intensity and (iii) CO2 transport from soil layers to surface. A better understanding of fractionation processes affecting CO2 during this transport is required to obtain quantitative information about the CO2 production.

The differences in transport (diffusion, advection) properties between 13CO2 and 12CO2 cause a fractionation during CO2 transfer from sources to the surface, creating a decrease of δ13C-CO2 at surface compared to air contained in soil pores. Short-term fluctuations of this apparent fractionation is expected in case of (i) changes in diffusive properties of the soil (soil water content, temperature) and/or (ii) air pressure variations created by turbulence. As a consequence, at this time scale, the difference between δ13C-CO2 of soil atmosphere and δ13C-CO2 of soil CO2 efflux is very variable and difficult to interpret. With tunable diode laser spectrometer (TDLS) it is now possible to measure δ13C-CO2 with high frequency. TDLS offers then the possibility to quantify δ13C-CO2 of soil efflux (by coupling with soil chamber, Marron et al. 2009) and soil atmosphere if (i) soil air sampling doesn't disturb the natural environment and (ii) soil air collected is diluted to reduce the high soil CO2 concentrations (several thousand ppmv) to values measurable by the TDLS (inferior to 1000 ppmv). To solve these limitations, we have adapted a method (Gut et al. 1998) based on the diffusion of soil pore air within a tube through a membrane having a high permeability to gases and being hydrophobic. These tubes have been placed horizontally at different depths in the soil. An air flowing in tubes allows CO2 circulation up to a diluting system composed by mass flow controllers and free CO2 injection. The output of this diluting system is coupled to the TDLS and measurements permit then to establish a vertical profile of the isotopic signature of soil CO2 concentration at hourly scale.

Laboratory tests were performed to prove the absence of membrane influence on the δ13C-CO2. The membrane tube has been placed in a chamber where gases with different CO2 concentration and δ13C were introduced. Results didn't show any fractionation for 13C during diffusion through the membrane. The δ13C difference between gas inside and gas outside the tube was always inferior to the TDLS precision ( 0.2

). In addition, some tests in the field have proved (i) that soil CO2 concentrations and δ13C are not significantly affected by our system functioning (observation of natural evolution) and (ii) that the values obtained are coherents with results from IRMS (R 2 =0.98) and with efflux measurements. This new methodology seems to be a very effective technique for measuring the δ13C-CO2 vertical profile of soil atmosphere and to understand fractionation processes that occur during this transfer. Moreover, incorporation of data collected using our methodology in mechanistic models of CO2 transport processes will permit to assess the vertical profil of sources contributions to CO2 efflux and will provide news insights into soil carbon dynamics.