José Amauri Buso

Development of microsatellite markers from an enriched genomic library for genetic analysis of melon (Cucumis melo L.)

BackgroundDespite the great advances in genomic technology observed in several crop species, the availability of molecular tools such as microsatellite markers has been limited in melon (Cucumis melo L.) and cucurbit species. The development of microsatellite markers will have a major impact on genetic analysis and breeding of melon, especially on the generation of marker saturated genetic maps and implementation of marker assisted breeding programs. Genomic microsatellite enriched libraries can be an efficient alternative for marker development in such species.ResultsSeven hundred clones containing microsatellite sequences from a Tsp-AG/TC microsatellite enriched library were identified and one-hundred and forty-four primer pairs designed and synthesized. When 67 microsatellite markers were tested on a panel of melon and other cucurbit accessions, 65 revealed DNA polymorphisms among the melon accessions. For some cucurbit species, such as Cucumis sativus, up to 50% of the melon microsatellite markers could be readily used for DNA polymophism assessment, representing a significant reduction of marker development costs. A random sample of 25 microsatellite markers was extracted from the new microsatellite marker set and characterized on 40 accessions of melon, generating an allelic frequency database for the species. The average expected heterozygosity was 0.52, varying from 0.45 to 0.70, indicating that a small set of selected markers should be sufficient to solve questions regarding genotype identity and variety protection. Genetic distances based on microsatellite polymorphism were congruent with data obtained from RAPD marker analysis. Mapping analysis was initiated with 55 newly developed markers and most primers showed segregation according to Mendelian expectations. Linkage analysis detected linkage between 56% of the markers, distributed in nine linkage groups.ConclusionsGenomic library microsatellite enrichment is an efficient procedure for marker development in melon. One-hundred and forty-four new markers were developed from Tsp-AG/TC genomic library. This is the first reported attempt of successfully using enriched library for microsatellite marker development in the species. A sample of the microsatellite markers tested proved efficient for genetic analysis of melon, including genetic distance estimates and identity tests. Linkage analysis indicated that the markers developed are dispersed throughout the genome and should be very useful for genetic analysis of melon.

Garlic viral complex: identification of Potyviruses and Carlavirus in Central Brazil

Garlic viruses often occur in complex infections in nature. In this study, a garlic virus complex, collected in fields in Brazil, was purified. RT-PCR was performed using specific primers designed from the consensus regions of the coat protein genes of Onion yellow dwarf virus, a garlic strain (OYDV-G) and Leek yellow stripe virus (LYSV). cDNA of Garlic common latent virus (GCLV) was synthesized using oligo-dT and random primers. By these procedures individual garlic virus genomes were isolated and sequenced. The nucleotide sequence analysis associated with serological data reveals the presence of two Potyvirus OYDV-G and LYSV, and GCLV, a Carlavirus, simultaneously infecting garlic plants. Deduced amino acid sequences of the Brazilian isolates were compared with related viruses reported in different geographical regions of the world. The analysis showed closed relations considering the Brazilian isolates of OYDV-G and GCLV, and large divergence considering LYSV isolate. The detection of these virus species was confirmed by specific reactions observed when coat protein genes of the Brazilian isolates were used as probes in dot-blot and Southern blot hybridization assays. In field natural viral re-infection of virusfree garlic was evaluated.

Detection of three Allexivirus species infecting garlic in Brazil

Garlic viruses often occur in mixed infections under field conditions. In this study, garlic samples collected in three geographical areas of Brazil were tested by Dot-ELISA for the detection of allexiviruses using monoclonal specific antibodies to detect Garlic virus A (GarV-A), Garlic virus B (GarV-B), Garlic virus C (GarV-C) and a polyclonal antiserum able to detect the three virus species mentioned plus Garlic virus D (GarV-D). The detected viruses were biologically isolated by successive passages through Chenopodium quinoa. Reverse Transcriptase Polimerase Chain Reaction (RT-PCR) was performed using primers designed from specific regions of the coat protein genes of Japanese allexiviruses available in the Genetic Bank of National Center of Biotechnology Information (NCBI). By these procedures, individual garlic virus genomes were isolated and sequenced. The nucleotide and amino acid sequence analysis and the one with serological data revealed the presence of three distinct allexiviruses GarV-C, GarV-D and a recently described allexivirus, named Garlic mite-borne filamentous virus (GarMbFV), in Brazil.

Viral reinfection affecting bulb production in garlic after seven years of cultivation under field conditions

Six sequential field experiments were conducted from 1999 to 2002 to evaluate virus reinfection in garlic cv. Amarante and its effect on bulb production. The treatments in the year 1999 were: T1 – virus-free garlic-seed obtained by thermotherapy and meristem-tip culture and indexed for virus by immuno-sorbent electron microscopy (ISEM) under its first field cycle; T2, T3 and T4 – garlic-seed in the second, third and fourth field cycles, respectively; and C – standard garlic-seed from the grower (with no control of virus infection) as a control. In the years 2000 to 2002, a new plot of virus-free seed was added to the experiment and cloves from the previous treatments were again grown under field conditions. In the fourth year of experiments, the treatments comprised T1 (virus-free garlic seeds under the first field cycle) to T7 (garlic-seed in the sixth field cycle) and C. Two experiments were conducted in the years 1999 and 2000, at two locations and in the years 2001 and 2002 only one experiment per year at one location. The combined analysis of variance for all experiments indicated a significant difference between the treatments for plant height and yield. The bulbs were classified into commercial classes according the Brazilian regulation and the commercial classes 4–7 were 72 %, 60%, 59%, 53% and 35% of the total number of bulbs harvested, from T1 to T5, respectively. Virus reinfection at the end of the second field cycle, determined by serology using antisera against the most common viruses of garlic in Brazil reached 83%. Treatments T1–T7 yielded 118%, 79%, 57%, 51%, 39%, 33% and 31% higher than the control.

Seleção de clones de batata para microclimas de altitude no Planalto Central

A potato selection program was done in Anapolis, State of Goias, Brazil, beginning in 1986 with 15,000 genotypes, resulting from 200 crosses obtained by Embrapa Hortalicas in 1985 and 1986. In the first selection cycle, in 1986, 5,000 genotypes were selected, considering plant growth and development, disease incidence, tuber quality and yield potential. These criteria were also adopted in further generations, when 15 to 20% of the best genotypes were yearly selected. In 1990 52 of the selected genotypes were evaluated in comparison with the cultivars Achat and Bintje. The best 28 clones were then named BAT, cleaned by meristem tip culture and evaluated from 1995 to 1997 at Anapolis, Morrinhos, Pirenopolis, Urutai and Jaboticabal. All data were submitted to the analysis of variance. In addition, data from fourteen genotypes in seven environments were analyzed by the linear regression technique of Ebehart & Russell. Clones BAT 2, BAT 3, BAT 4, BAT 19, BAT 27 and BAT 28 ranked best among the highest yielding ones, in addition to good tuber quality. Genotypes had similar behavior regarding adaptability, responding in a proportional way to environment improvement. BAT 19 was the most stable clone.

Shoot tip culture and thermotherapy for recovering virus-free plants of garlic.

Garlic shoot tip culture associated with dry heat thermotherapy (cloves exposed to 37°C for 35 days) were essential for recovering virus free plants of the cv Amarante. In this condition 70% of the explants developed in vitro and produced plants. A total of 77% of those plants was virus free when indexed by ISEM, which resulted in a final index of 54% of virus free plants from treated cloves. The percentage of regeneration decreased to 20% as the temperature increased up to 40°C. However 90% of those plants were virus free, leading to a final index of 18% virus free plants out of treated cloves.

BRS Ana: cultivar de batata de duplo propósito

The genotype BRS Ana is a new potato cultivar adequate for French fries, with potential for processing into frozen French fries and flakes, released in 2007. It was developed by the Embrapa Potato Breeding Program (Embrapa Temperate Agriculture, Pelotas-RS; Embrapa Transference of Technology, Office of Canoinhas-SC; and Embrapa Vegetables, Brasilia-DF), based on tuber appearance and yield, specific gravity and French fries quality. Tubers are red-skinned, lightly rough, oval shaped with shallow eyes. The pulp is white. Cultivar BRS Ana has high yield potential. In the subtropical ecosystem, cultivar BRS Ana showed higher yield (31.2 t ha-1) than the most used cultivars in Brazil when grown in autumn, and did not differ from them in the spring. In the tropical ecosystem, under irrigation, BRS Ana did not differ from both control cultivars. It produced higher percentage of marketable tubers (55.6%) and average tuber weight (108.4 g) than the controls in the fall crop of subtropical ecosystem. In both ecosystems, cultivar BRS Ana presented high specific gravity (1.086) and dry matter content (19.7%). The sensorial analysis showed that cultivar BRS Ana is adequate for home made French fries as well as for industrial processing. It is moderately susceptible to late blight (Phytophthora infestans) and presents good resistance to early blight (Alternaria solani). The reaction to the tuber soft rot (Pectobacterium sp.) is similar to the most used cultivars. It has low seed degeneration conferred by moderate resistance to PVY and low incidence of PLRV. Susceptibility to tuber physiological disorders has not been observed. It seems that BRS Ana has lower fertilizer and water requirements than the most planted cultivars, meaning reduction of crop cost and risk. In the subtropical ecosystem, tuberization starts later in spring, therefore BRS Ana should be planted earlier in the season.