Screening and identification of potential sex-associated sequences in Danio rerio.

Current knowledge on zebrafish (Danio rerio) suggests that sex determination has a polygenic genetic basis in this species, although environmental factors may also be involved. This study aimed to identify sex-associated genomic regions using two different marker systems: inter-simple sequence repeats (ISSRs) and random-amplified polymorphic DNA (RAPDs). Two bulks were constructed: one with DNA from zebrafish females and the other from males; then, a total of 100 ISSR and 280 RAPD primers were tested. Three DNA fragments presenting sexual dimorphism (female-linked: OPA17436 and OPQ191027 ; male-linked: OPQ19951 ) were determined from sequential analysis of the bulks followed by assessment in individuals. These fragments were cloned and convert into the following sequenced characterized amplified regions (SCAR): DrSM_F1, DrSM_F2, and DrSM_M, which share identities with sequences located in chromosomes 2, 3, and 11 (Zv9), respectively. Using these potential markers in zebrafish samples it was possible to correctly identify 80% of the males (DrSM_M) and 100% of the females (DrSM_F1 + DrSM_F2) in the analyzed population.

Molecular characterization of the citrate transporter gene TaMATE1 and expression analysis of upstream genes involved in organic acid transport under Al stress in bread wheat (Triticum aestivum).

In bread wheat, besides malate, the importance of citrate efflux for Al tolerance has also been reported. For better understanding the Al tolerance mechanism in bread wheat, here, we performed both a molecular characterization of the citrate transporter gene TaMATE1 and an investigation on the upstream variations in citrate and malate transporter genes. TaMATE1 belong to multidrug transporter protein family, which are located on the long arm of homoeologous group 4 chromosomes (TaMATE1-4A, TaMATE1-4B TaMATE1-4D). TaMATE1 homoeologues transcript expression study exhibited the preponderance of homoeologue TaMATE1-4B followed by TaMATE1-4D whereas homoeologue TaMATE1-4A seemed to be silenced. TaMATE1, particularly homoeologue TaMATE1-4B and TaALMT1 transcripts were much more expressed in the root apices than in shoots of Al tolerant genotype Barbela 7/72/92 under both control and Al stress conditions. In addition, in both tissues of Barbela 7/72/92, higher basal levels of these gene transcripts were observed than in Anahuac (Al sensitive). Noticeably, the presence of a transposon in the upstream of TaMATE1-4B in Barbela 7/72/92 seems to be responsible for its higher transcript expression where it may confer citrate efflux. Thus, promoter variations (transposon in TaMATE1-4B upstream and type VI promoter in TaALMT1) associated with higher basal transcript expression of TaMATE1-4B and TaALMT1 clearly show how different mechanisms for Al tolerance operate simultaneously in a single genotype. In conclusion, our results demonstrate that Barbela 7/72/92 has favorable alleles for these organic acids transporter genes which could be utilized through genomic assisted selection to develop improved cultivars for acidic soils.

Molecular characterization of TaSTOP1 homoeologues and their response to aluminium and proton (H(+)) toxicity in bread wheat (Triticum aestivum L.).

BACKGROUND: Aluminium (Al) toxicity is considered to be one of the major constraints affecting crop productivity on acid soils. Being a trait governed by multiple genes, the identification and characterization of novel transcription factors (TFs) regulating the expression of entire response networks is a very promising approach. Therefore, the aim of the present study was to clone, localize, and characterize the TaSTOP1 gene, which belongs to the zinc finger family (Cys2His2 type) transcription factor, at molecular level in bread wheat. RESULTS: TaSTOP1 loci were cloned and localized on the long arm of homoeologous group 3 chromosomes [3AL (TaSTOP1-A), 3BL (TaSTOP1-B) and 3DL (TaSTOP1-D)] in bread wheat. TaSTOP1 showed four potential zinc finger domains and the homoeologue TaSTOP1-A exhibited transactivation activity in yeast. Expression profiling of TaSTOP1 transcripts identified the predominance of homoeologue TaSTOP1-A followed by TaSTOP1-D over TaSTOP1-B in root and only predominance of TaSTOP1-A in shoot tissues of two diverse bread wheat genotypes. Al and proton (H(+)) stress appeared to slightly modulate the transcript of TaSTOP1 homoeologues expression in both genotypes of bread wheat. CONCLUSIONS: Physical localization of TaSTOP1 results indicated the presence of a single copy of TaSTOP1 on homoeologous group 3 chromosomes in bread wheat. The three homoeologues of TaSTOP1 have similar genomic structures, but showed biased transcript expression and different response to Al and proton (H(+)) toxicity. These results indicate that TaSTOP1 homoeologues may differentially contribute under Al or proton (H(+)) toxicity in bread wheat. Moreover, it seems that TaSTOP1-A transactivation potential is constitutive and may not depend on the presence/absence of Al at least in yeast. Finally, the localization of TaSTOP1 on long arm of homoeologous group 3 chromosomes and the previously reported major loci associated with Al resistance at chromosome 3BL, through QTL and genome wide association mapping studies suggests that TaSTOP1 could be a potential candidate gene for genomic assisted breeding for Al tolerance in bread wheat.

Repression of Mitochondrial Citrate Synthase Genes by Aluminum Stress in Roots of Secale cereale and Brachypodium distachyon.

Aluminum (Al) toxicity in acid soils influences plant development and yield. Almost 50% of arable land is acidic. Plants have evolved a variety of tolerance mechanisms for Al. In response to the presence of Al, various species exudate citrate from their roots. Rye (Secale cereale L.) secretes both citrate and malate, making it one of the most Al-tolerant cereal crops. However, no research has been done on the role of the mitochondrial citrate synthase (mCS) gene in Al-induced stress in the rye. We have isolated an mCS gene, encoding a mitochondrial CS isozyme, in two S. cereale cultivars (Al-tolerant cv. Ailés and Al-sensitive inbred rye line Riodeva; ScCS4 gene) and in two Brachypodium distachyon lines (Al-tolerant ABR8 line and Al-sensitive ABR1 line; BdCS4 gene). Both mCS4 genes have 19 exons and 18 introns. The ScCS4 gene was located on the 6RL rye chromosome arm. Phylogenetic studies using cDNA and protein sequences have shown that the ScCS4 gene and their ScCS protein are orthologous to mCS genes and CS proteins of different Poaceae plants. Expression studies of the ScCS4 and BdSC4 genes show that the amount of their corresponding mRNAs in the roots is higher than that in the leaves and that the amounts of mRNAs in plants treated and not treated with Al were higher in the Al-tolerant lines than that in the Al-sensitive lines of both species. In addition, the levels of ScCS4 and BdCS4 mRNAs were reduced in response to Al (repressive behavior) in the roots of the tolerant and sensitive lines of S. cereale and B. distachyon.

Genetic variation in natural populations of Stipa tenacissima from Algeria.

Intermicrosatellite PCR [inter-simple sequence repeat (ISSR)-PCR] markers and cytogenetics criteria were used to assess the level of genetic diversity and genetic structure in 17 populations of Stipa tenacissima (Gramineae) from Algeria. All populations sampled in the steppe area were diploids (2n = 2x = 24), and those sampled in the dry area were hexaploids (2n = 6x = 72). The dendrogram based on ISSR-PCR showed homogeneity within populations and large variability among populations. All individuals of the same population were gathered and formed groups clearly separated in all populations. These groups were separated into two clusters related to biotope, one from the steppe area and the other from the dry area. AMOVA indicated low genetic diversity among populations (30% of variation) and high within populations (70%). This variation pattern would constitute an adaptive strategy to grow in various ecological conditions.

Sulfate nutrition improves short-term Al3+-stress tolerance in roots of Lolium perenne L.

Trivalent aluminum ions (Al3+) in acidic soils are a major constraint for crop productivity inhibiting root elongation and promoting cell death. Al3+-toxicity has adverse biochemical and physiological effects on plant root growth. Sulfur is an essential macronutrient assimilated from the soil in the form of sulfate. However, the implication of sulfate nutritional status in the modulation of short-term Al3+-tolerance mechanisms in plant roots has not been previously reported. Here, we evaluated the effects of increased sulfate supply on short-term Al3+-toxicity in roots of Lolium perenne, measuring Al, Ca, Mg and S uptake, lipid peroxidation, total SOD activity, and transcriptional levels of Cu/Zn and Fe-SOD genes. First, the nitrogen sulfur ratio (N/S) in the TF nutrient solutions used in this study were computed to confirm that L. perenne plants were grown in sulfate deficiency (120 µM), optimal supply (240 µM), or overdoses conditions (360 µM), without affecting dry root biomass. Sulfate supplementation (>240 µM, and N/S ratio < 16) played a significant protection to Al3+-stress that prevents morphological changes in root tips, inhibits lipid peroxidation and differentially up-regulates total SOD activity, due changes in SOD gene expression. The results support the importance of sulfate nutritional status, on plant tissue homeostasis, enhancing the physiological tolerance mechanisms modulating lipid peroxidation damage induced by short-term Al3+-toxicity.

The molecular diversity of different isolates of Beauveria bassiana (Bals.) Vuill. as assessed using intermicrosatellites (ISSRs).

Inter-microsatellite PCR (ISSR-PCR) markers were used to identify and to examine the genetic diversity of eleven Beauveria bassiana isolates with different geographic origins. The variability and the phylogenetic relationships between the eleven strains were analyzed using 172 ISSR-PCR markers. A high level of polymorphism (near 80%) was found using these molecular markers. Seven different isolates showed exclusive bands, and ISSR primer 873 was able to distinguish between all the strains. The dendrogram obtained with these markers is robust and in agreement with the geographical origins of the strains. All the isolates from the Caribbean region were grouped together in a cluster, while the other isolates grouped in the other cluster. The similarity exhibited between the two clusters was less than 50%. This value of homology shows the high genetic variability detected between the isolates from the Caribbean region and the other isolates. ISSR-PCR markers provide a quick, reliable and highly informative system for DNA fingerprinting, and allowed the identification of the different B. bassiana isolates studied.

Neutral molecular markers support common origin of aluminium tolerance in three congeneric grass species growing in acidic soils.

Aluminium (Al) toxicity is the main abiotic stress limiting plant productivity in acidic soils that are widely distributed among arable lands. Plant species differ in the level of Al resistance showing intraspecific and interspecific variation in many crop species. However, the origin of Al-tolerance is not well known. Three annual species, difficult to distinguish phenotypically and that were until recently misinterpreted as a single complex species under Brachypodium distachyon, have been recently separated into three distinct species: the diploids B. distachyon (2n = 10) and B. stacei (2n = 20), and B. hybridum (2n = 30), the allotetraploid derived from the two diploid species. The aims of this work were to know the origin of Al-tolerance in acidic soil conditions within these three Brachypodium species and to develop new DNA markers for species discrimination. Two multiplex SSR-PCRs allowed to genotype a group of 94 accessions for 17 pentanucleotide microsatellite (SSRs) loci. The variability for 139 inter-microsatellite (ISSRs) markers was also examined. The genetic relationships obtained using those neutral molecular markers (SSRs and ISSRs) support that all Al-tolerant allotetraploid accessions of B. hybridum have a common origin that is related with both geographic location and acidic soils. The possibility that the adaptation to acidic soils caused the isolation of the tolerant B. hybridum populations from the others is discussed. We finally describe a new, easy, DNA barcoding method based in the upstream-intron 1 region of the ALMT1 gene, a tool that is 100 % effective to distinguish among these three Brachypodium species.

Leishmaniasis in the major endemic region of Plurinational State of Bolivia: Species identification, phylogeography and drug susceptibility implications.

The Plurinational State of Bolivia is one of the Latin American countries with the highest prevalence of leishmaniasis, highlighting the lowlands of the Department of La Paz where about 50% of the total cases were reported. The control of the disease can be seriously compromised by the intrinsic variability of the circulating species that may limit the efficacy of treatment while favoring the emergence of resistance. Fifty-five isolates of Leishmania from cutaneous and mucocutaneous lesions from patients living in different provinces of the Department of La Paz were tested. Molecular characterization of isolates was carried out by 3 classical markers: the rRNA internal transcribed spacer 1 (ITS-1), the heat shock protein 70 (HSP70) and the mitochondrial cytochrome b (Cyt-b). These markers were amplified by PCR and their products digested by the restriction endonuclease enzymes AseI and HaeIII followed by subsequent sequencing of Cyt-b gene and ITS-1 region for subsequent phylogenetic analysis. The combined use of these 3 markers allowed us to assign 36 isolates (65.5%) to the complex Leishmania (Viannia) braziliensis, 4 isolates (7, 27%) to L. (Viannia) lainsoni. and the remaining 15 isolates (23.7%) to a local variant of L. (Leishmania) mexicana. Concerning in vitro drug susceptibility the amastigotes from all isolates where highly sensitive to Fungizone® (mean IC50 between 0.23 and 0.5µg/mL) whereas against Glucantime® the sensitivity was moderate (mean IC50 ranging from 50.84µg/mL for L. (V.) braziliensis to 18.23µg/mL for L. (L.) mexicana. L. (V.) lainsoni was not sensitive to Glucantime®. The susceptibility to miltefosine was highly variable among species isolates, being L. (L.) mexicana the most sensitive, followed by L. (V.) braziliensis and L. (V.) lainsoni (mean IC50 of 8.24µg/mL, 17.85µg/mL and 23.28µg/mL, respectively).

Reduced wound infection in colorectal resection by using a wound auto-retractor.

Surgical site infection (SSI) remains an important problem in colorectal surgery. The aim of this study is to determine whether the use of a wound protection system can be effective in reducing the incidence of wound infection after colorectal resection. Ninety-five consecutive patients underwent colorectal resection, carried out by one single surgeon during a six-year period (2009-2015). A laparotomy auto-retractor was used in all cases (Alexis Wound Retractor; Applied Medical, Rancho Santa Margarita, CA). Forty-two resections (44%) were made by laparoscopy. Anastomoses for laparoscopic right colectomies, section of left colon and insertion of the anvil of CEEA for laparoscopic left colectomies were made extracorporeally. No colon cleansing was used in 67 patients (72%). The median age for those undergoing colectomy was 67 (range 41-90). The median Body Mass Index was 25.04 (range 18- 36.76). Three patients (3%) were operated on an emergency basis because of bowel obstruction or perforation. Fifty-three patients were classified ASA I-II (56%). There were six re-operations, for anastomotic dehiscence, peri-ostomal cellulitis and postoperative bleeding. The median postoperative stay was eight days (range 3-28). Only one patient (1%) developed wound infection. Due to the significantly reduced incidence of postoperative wound infection, this study suggests that the Alexis retractor be considered for routine use.

Brachypodium distachyon: a model species for aluminium tolerance in Poaceae.

Aluminium (Al) toxicity is the main abiotic stress limiting plant productivity in acidic soils. Studies on Al tolerance have been conducted in Poaceae but their genomes are very complex. Fifty-nine diploid lines (2n=10) of Brachypodium distachyon (L.) P. Beauv. and 37 allotetraploid samples (2n=30) of Brachypodium hybridum Catalán, Joch. Müll., Hasterok & Jenkins sp. nov. were used to evaluate their tolerance to different Al concentrations. B. distachyon is Al-sensitive compared with oat, rice and rye. The diploid lines (except ABR8) were sensitive like barley and Arabidopsis; however, 10 allotetraploid samples were Al-tolerant. Four different root-staining methods were used to detect Al accumulation, cell death, lipid peroxidation and H2O2 production in diploid and allotetraploid plants. The roots treated with Al showed more intense staining in sensitive than tolerant lines. Also, without any staining, the Al treated roots of sensitive plants appear darker than roots from tolerant ones. The study concerning to the organic acids exudation shows that the exudation of citrate and malate was induced only in the roots from tolerant diploid line (ABR8) and tolerant allotetraploid samples. In contrast, the mRNA expression changes of several candidate genes for Al-activated transporters belonging to the ALMT and MATE families were analysed by quantitative PCR (qRT-PCR). The data obtained indicate that the transcripts from BdALMT1, BdMATE1 and BdMATE2 were present mainly in roots and, moreover, that the BdALMT1 transcript is present in higher amounts in the tolerant ABR8 than in the sensitive ABR1 plants indicating that this gene may be involved in Al tolerance. Finally, an insertion was detected in the promoter region of the BdALMT1 of tolerant diploid and allotetraploid plants.

Allelic frequencies of the 15 STR loci included in the AmpFlSTR Identifiler PCR Amplification Kit in an autochthonous sample from Spain.

The aim of this study was to estimate the allelic frequencies of the 15 STR loci included in the AmpFlSTR Identifiler PCR Amplification Kit in a sample of 342 unrelated Caucasian individuals autochthonous from Spain to be used for forensic purposes and population studies. The combined power of discrimination and the combined power of exclusion for all of the 15 loci were 5.68x10(-18) and 0.9999964, respectively. According to the obtained data, the D18S51 locus may be considered the most informative among the tested loci.

Genic heterozygosity, chromosomal interchanges and fitness in rye: any relationship?

Relationship between heterozygosity at allozyme loci, chromosomal interchanges and fitness was analyzed in a rye cultivar showing a polymorphism for such rearrangements. Nine allozyme systems (ACO, ACPH, GOT, GPI, LAP, MDH, PER, PGD and PGM) and five components of fitness (number of fertile tillers, total offspring, egg cell fertility, flowers/ear and seeds/ear) were studied. The estimated selection coefficients against interchange heterozygotes ranged from s = 0.12 to s = 0.34. A significant effect of the genic heterozygosity on some fitness components was observed in interchange heterozygotes (tillering and total offspring), in their standard homozygous sibs (flowers/ear and seeds/ear) and in the descendants of the crosses between standard karyotypes (flowers/ear, seeds/ear and egg cell fertility). However, the main effect was linked to genetic background associated to different crosses. Significant differences for Acph-1, Gpi-1, Lap-1, Mdh-1, Mdh-4, Pgd-2 and Pgm-1 loci were also found in some of these crosses although these differences were inconsistent. This suggests that probably the allozyme loci analyzed were not directly contributing to the fitness and that they are linked, in some cases, to different deleterious alleles depending on both cross and locus. This fact could support the local effect hypothesis as explanation although we do not discard the existence of some inbreeding level (general effect hypothesis) since all crosses and loci studied show a overall consistent trend of increased fitness with increased heterozygosity.

A simple method for the estimation of recombination frequencies and genetic distances.

We have developed an alternative approach to the maximum likelihood method for calculating recombination values and linkage intensities. This new method is simpler than those in current use in that it obviates the need for formulae and tables. Maximum likelihood methods imply the use of iterative procedures over highly complicated estimation equations (at least second degree polynomials), whereas simplified methods use single-step procedures involving simple linear equations. The proposed method uses the frequencies of the distinguishable phenotypes that came from the union of at least one recombinant gamete with another gamete. We performed Monte-Carlo simulations for various combinations of genetic distance and offspring size. The recombination values obtained using the new method were compared with those derived by the maximum likelihood method on both simulated and experimental data. They were found to be nearly identical. The observed distribution of the recombination frequency values does not differ significantly from the Normal distribution, except for extreme values of the mean, as the Skewedness and kurtosis coefficients do not differ from zero. Our method has a similar accuracy to the maximum likelihood methods for recombination frequencies smaller than 25 cM and the difference does not increase greatly for greater frequencies.

Chromosomal location of 46 new RAPD markers in rye (Secale cereale L.).

The polymerase chain reaction (PCR) was used to locate RAPD markers using disomic wheat-rye addition lines in order to develop a set of molecular markers distributed on the seven rye chromosomes. We carried out RAPD amplifications on genomic DNA of wheat 'Chinese Spring' (CS), rye 'Imperial' (I), the amphiploid wheat-rye and the seven disomic wheat-rye addition lines (1R-7R) using 140 different 10-mer oligonucleotides. Forty six new RAPD markers were located on the seven rye chromosomes and all the disomic wheat-rye addition lines were identified on the basis of their amplification patterns. The number of RAPD bands located on 1R, 2R, 3R, 4R, 5R, 6R and 7R chromosomes were 5, 8, 11, 8, 8, 10 and 6, respectively. The seven wheat-rye addition lines can be distinguished using only the following three 10-mer oligonucleotides: OPA16, OPF19 and GEN3-605, the other RAPD primers being useful for this purpose. The use of these RAPDs as a source of molecular markers that could be linked to interesting genes or other important agronomic traits is discussed.