Functional fusion expression of sunflower multicystatin in E. coli and its comparison with a single domain cystatin.

Identification of the molecular structure and novel biophysiological functions of plant cystatins or phytocystatins is of great interest in the field of molecular biology. The important requirements for these are the efficient production, purification and correctly folded forms of these proteins. We report here the cloning, easy expression and characterization of a sunflower multicystatin (SMC) as a functional fusion protein in E. coli. For the first time, the amplified cystatin coding region was expressed as a part of maltose-binding fusion protein using pMALc2X over-expression vector in TB1 strain of E. coli without affecting the recombinant bacterial growth. In comparison to the previously prepared recombinant SMC (rSMC), a high amount (~44 mg/L of bacterial cell culture) of purified fused SMC (fSMC) was obtained using single-step purification method. fSMC strongly inhibited papain activity in vitro as compared to Celosia single-domain cystatin. Purified fSMC may be used for basic biochemical, pharmacological or clinical studies without the cleavage of its fusion parts.

Detection of single copy sequences using BAC-FISH and C-PRINS techniques in sunflower chromosomes

Bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and cycling-primed in situ labeling (C-PRINS) techniques were evaluated for integration of physical and genetic maps of sunflower (Helianthus annuus L.). Single-site SSR markers were selected from three linkage groups of a high-density sunflower genetic map. This selection was based on previously identified QTL associated to S. sclerotiorum. These markers were used to select BACs contaning single copy sequences for BAC-FISH aplication. Blocking of highly dispersed repetitive sunflower sequences reduced unspecific hybridization, and allowed the detection of specific signals for BACs containing SSR markers HA4222 and HA2600, anchored to LG 16 and LG 10, respectively. Single-site FISH signal detection was optimized by adjusting the relative quantity and quality of unlabelled repetitive sequences present in the blocking DNA. The SSR marker ORS1247 anchored to the LG 17 was detected by C-PRINS, which yielded fluorescence signals that were specific and intense. This progress in localizing single-copy sequences using BAC-FISH and indirect C-PRINS strategies in sunflower will facilitate the integration of genetic and physical maps, allowing the identification of chromosomes containing key genes and/or QTL associated to agronomic important traits in sunflower.

Genetic mapping of EST-SSR, SSR and InDel to improve saturation of genomic regions in a previously developed sunflower map

In order to saturate a sunflower genetic map and facilitate marker-assisted selection (MAS) breeding for stress response, it is necessary to enhance map saturation with molecular markers localized in linkage groups associated to genomic regions involved in these traits. This work describes the identification and characterization of 1,134 simple sequence repeat (SSR) containing expressed sequence tags (EST) from unigenes available databases. Twelve of these functional markers as well as 41 public SSR markers were successfully localized in linkage groups, thus contributing to the saturation of specific regions on a reference genetic-linkage-map derived from recombinant inbred lines (RIL) mapping population from the cross between PAC2 x RHA266 lines. The enriched map includes 547 markers (231 SSR, 9 EST-SSR, 3 insertions/deletions (InDel) and 304 amplified fragment length polymorphisms (AFLP) distributed in 17 linkage groups (LG), spanning genetic size to 1,942.3 cM and improving its mean density to 3.6 cM per locus. As consequence, no gaps longer than 13.2 cM remain uncovered throughout the entire map, which increases the feasibility of detecting genes or traits of agronomic importance in sunflower.

Avaliação pela técnica semiautomática de produção de gases das silagens de quatro genótipos de girassol (Helianthus annuus) (Rumbosol 91, Victoria 627, Victoria 807 e Mycogen 93338) / Evaluation of four sunflower (Helianthus annuus) genotypes (Rumbosol 91, Victoria 627, Victoria 807, and Mycogen 93338) silages by semi-automated in vitro gas production technique

Foram avaliadas as silagens de quatro genótipos de girassol (Helianthus annuus) (Rumbosol 91, Victoria 627, Victoria 807 e Mycogen 93338), pela técnica semiautomática de produção de gases. O delineamento experimental utilizado foi o de blocos ao acaso, em esquema de parcelas subdivididas, sendo as médias comparadas pelo teste SNK (p<0,05). O genótipo Rumbosol 91 apresentou a maior produção acumulativa de gases para o tempo de 96 horas de incubação com valor de 128,47mL/g de MS, e o menor valor foi observado para o genótipo Victoria 807 com 92,88. Não foram observadas diferenças entre os valores de degradabilidade da matéria seca (DMS) para as silagens avaliadas nos diferentes tempos. O potencial máximo de produção de gases variou de 91,67 para o genótipo Victoria 807 a 125,46mL/g de MS para a silagem do genótipo Rumbosol 91. O genótipo Rumbosol 91 apresentou a maior DMS para as taxas de passagem de 2 e 5 por cento com valores de 46,39 e 43,26 por cento. O maior valor para taxa de passagem (8 por cento) foi observado para o genótipo Victoria 627. As silagens dos genótipos Rumbosol 91 e Victoria 627 mostraram destacado potencial para produção de gases e taxa de produção de gases.

Four silages from distinct sunflower (Helianthus annuus) genotypes (Rumbosol 91, Victoria 627, 807 Victoria, and Mycogen 93338) were evaluated by semi-automated in vitro gas production technique in a completely random block design in a split plot scheme. The means were compared by the SNK test (P<0.05). Rumbosol 91 genotype showed the highest cumulative gas production after 96 hours of incubation, reaching 128.47mL/g of dry matter (DM), and the lowest value, 92.88mL/g, was observed for Victoria 807 genotype. No differences between silage dry matter digestibility (DMD) values at different times were observed (P>0.05). Maximum potential gas production ranged from 91.67 for Victoria 807 to 125.46mL/g of DM Rumbosol 91. Genotype Rumbosol 91 showed the highest DMD for fermentation rate of 2 (46.39 percent) and 5 percent (43.26 percent), and the highest values for fermentation rate of 8 percent was observed for Victoria 627 genotype. Silages from Rumbosol 91 and Victoria 627 genotypes showed outstanding potential of gases production and rate of gases production.

An optimized mini-preparation method toobtain high-quality genomic DNA from mature leaves of sunflower

In order to investigate the mutation characteristics and to further examine the genetic variation of mutant sunflower (Helianthus annuus) obtained in plants grown from seeds exposed to space conditions, only the mature tissues such as leaf and flower could be used for DNA extraction after mutation characteristics were confirmed. The rich contents of polysaccharides, tannins, secondary metabolites, and polyphenolics made it difficult to isolate high-quality DNA from mature leaves of sunflower according to previous reports. Based on the comparison of the differences in previously reported protocols, a modified method for the extraction of high-quality DNA was developed. Using this protocol, the DNA isolated from sunflower was high in quality and suitable for restriction digestion (EcoRI, HindII, BamHI), random amplified polymorphic DNA study and further molecular research. Therefore, the modified protocol was suitable for investigating the genetic variation of sunflower using mature leaf DNA.

Selection and estimation of the heritability of sunflower (Helianthus annuus) pollen collection behavior in Apis mellifera colonies

We selected honey bee colonies (Apis mellifera L.) with a high tendency to collect sunflower pollen and estimated the heritability of this trait. The percentage of sunflower pollen collected by 74 colonies was evaluated. Five colonies that collected the highest percentages of sunflower pollen were selected. Nineteen colonies headed by daughters of these selected queens were evaluated for this characteristic in comparison with 20 control (unselected) colonies. The variation for the proportion of sunflower pollen was greater among colonies of the control group than among these selected daughter colonies. The estimated heritability was 0.26 +/- 0.23, demonstrating that selection to increase sunflower pollen collection is feasible. Such selected colonies could be used to improve sunflower pollination in commercial fields.