Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L) / Conteúdo de heterocromatina revela polimorfismos importantes para o melhoramento genético do alho (Allium sativum L)

Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n= 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the "Branco Mineiro Piauí" accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Allium sativum L. é uma erva da família Alliaceae com sabor e aroma específicos e propriedades medicinais e nutracêuticas amplamente comercializada em diversos países. O Brasil é um dos maiores importadores de alho do mundo, apesar da sua produção ser restrita e limitada ao consumo interno. Assim, explorar a diversidade genética do alho comercial conservado em bancos de germoplasma é essencial para fornecer informações genéticas adicionais acerca dessa cultura economicamente importante. Uma ferramenta adequada para esse fim é a caracterização citogenética desses acessos. Este estudo teve como objetivo caracterizar a diversidade citogenética entre sete acessos de alho de um Banco de Germoplasma no Brasil. Os cariótipos foram obtidos por coloração convencional e com os fluorocromos de cromomicina A3 (CMA) e 4,6-diamidino-2-fenilindol (DAPI). Todos os acessos analisados apresentaram número cromossômico 2n = 16, fórmula cariotípica 6M + 2SM, cariótipos simétricos, núcleos reticulados em intérfase e cromossomos com condensação uniforme da cromatina da prófase para a metáfase. A coloração com fluorocromos mostrou diferenças na quantidade e distribuição de heterocromatina ao longo dos cromossomos e entre os acessos estudados. Com base no padrão de distribuição desses pequenos polimorfismos, foi possível separar os sete acessos em três grupos. Também foi possível diferenciar individualmente alguns dos acessos. Um dos resultados obtidos mostrou distensão heteromórfica da região organizadora nucleolar observada nos pares dos cromossomos 6 ou 7 com características peculiares. Foi sugerido, por exemplo, que o bloco heteromórfico de heterocromatina (CMA +++ / DAPI-) no cromossomo 6 do acesso “Branco Mineiro Piauí” pode ser usado como um marcador para identificar esse genótipo ou pode estar associado a algum caráter de interesse econômico.

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L.)

Abstract Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the Branco Mineiro Piauí accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Resumo Allium sativum L. é uma erva da família Alliaceae com sabor e aroma específicos e propriedades medicinais e nutracêuticas amplamente comercializada em diversos países. O Brasil é um dos maiores importadores de alho do mundo, apesar da sua produção ser restrita e limitada ao consumo interno. Assim, explorar a diversidade genética do alho comercial conservado em bancos de germoplasma é essencial para fornecer informações genéticas adicionais acerca dessa cultura economicamente importante. Uma ferramenta adequada para esse fim é a caracterização citogenética desses acessos. Este estudo teve como objetivo caracterizar a diversidade citogenética entre sete acessos de alho de um Banco de Germoplasma no Brasil. Os cariótipos foram obtidos por coloração convencional e com os fluorocromos de cromomicina A3 (CMA) e 4,6-diamidino-2-fenilindol (DAPI). Todos os acessos analisados apresentaram número cromossômico 2n = 16, fórmula cariotípica 6M + 2SM, cariótipos simétricos, núcleos reticulados em intérfase e cromossomos com condensação uniforme da cromatina da prófase para a metáfase. A coloração com fluorocromos mostrou diferenças na quantidade e distribuição de heterocromatina ao longo dos cromossomos e entre os acessos estudados. Com base no padrão de distribuição desses pequenos polimorfismos, foi possível separar os sete acessos em três grupos. Também foi possível diferenciar individualmente alguns dos acessos. Um dos resultados obtidos mostrou distensão heteromórfica da região organizadora nucleolar observada nos pares dos cromossomos 6 ou 7 com características peculiares. Foi sugerido, por exemplo, que o bloco heteromórfico de heterocromatina (CMA +++ / DAPI-) no cromossomo 6 do acesso Branco Mineiro Piauí pode ser usado como um marcador para identificar esse genótipo ou pode estar associado a algum caráter de interesse econômico.

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L. ) / Conteúdo de heterocromatina revela polimorfismos importantes para o melhoramento genético do alho (Allium sativum L. )

Abstract Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the "Branco Mineiro Piauí" accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Resumo Allium sativum L. é uma erva da família Alliaceae com sabor e aroma específicos e propriedades medicinais e nutracêuticas amplamente comercializada em diversos países. O Brasil é um dos maiores importadores de alho do mundo, apesar da sua produção ser restrita e limitada ao consumo interno. Assim, explorar a diversidade genética do alho comercial conservado em bancos de germoplasma é essencial para fornecer informações genéticas adicionais acerca dessa cultura economicamente importante. Uma ferramenta adequada para esse fim é a caracterização citogenética desses acessos. Este estudo teve como objetivo caracterizar a diversidade citogenética entre sete acessos de alho de um Banco de Germoplasma no Brasil. Os cariótipos foram obtidos por coloração convencional e com os fluorocromos de cromomicina A3 (CMA) e 4,6-diamidino-2-fenilindol (DAPI). Todos os acessos analisados ​​apresentaram número cromossômico 2n = 16, fórmula cariotípica 6M + 2SM, cariótipos simétricos, núcleos reticulados em intérfase e cromossomos com condensação uniforme da cromatina da prófase para a metáfase. A coloração com fluorocromos mostrou diferenças na quantidade e distribuição de heterocromatina ao longo dos cromossomos e entre os acessos estudados. Com base no padrão de distribuição desses pequenos polimorfismos, foi possível separar os sete acessos em três grupos. Também foi possível diferenciar individualmente alguns dos acessos. Um dos resultados obtidos mostrou distensão heteromórfica da região organizadora nucleolar observada nos pares dos cromossomos 6 ou 7 com características peculiares. Foi sugerido, por exemplo, que o bloco heteromórfico de heterocromatina (CMA +++ / DAPI-) no cromossomo 6 do acesso "Branco Mineiro Piauí" pode ser usado como um marcador para identificar esse genótipo ou pode estar associado a algum caráter de interesse econômico.

Variations in heterochromatin content reveal important polymorphisms for studies of genetic improvement in garlic (Allium sativum L.).

Allium sativum L. is an herb of the Alliaceae family with a specific taste and aroma and medicinal and nutraceutical properties that are widely marketed in several countries. Brazil is one of the largest importers of garlic in the world, despite of its production is restricted and limited to internal consumption. Thus, explore the genetic diversity of commercial garlic conserved at germplasm banks is essential to generate additional genetic information about its economically important crop. A suitable tool for this purpose is the cytogenetic characterisation of these accessions. This study aimed to characterise the cytogenetic diversity among seven accessions of garlic from a Germplasm Bank in Brazil. The karyotypes were obtained by conventional staining and with chromomycin A3 (CMA) and 4,6-diamidino-2-phenylindole (DAPI) fluorochromes. All accessions analysed showed chromosome number 2n = 16, karyotype formula 6M+2SM, symmetrical karyotypes, reticulate interphase nuclei, and chromosomes with uniform chromatin condensation from prophase to metaphase. The fluorochromes staining showed differences in the amount and distribution of heterochromatin along the chromosomes and between accessions studied. Based on the distribution pattern of these small polymorphisms, it was possible to separate the seven accessions into three groups. It was also possible to differentiate some of the accessions individually. One of the results obtained showed a heteromorphic distension of the nucleolar organiser region observed on the chromosome pairs 6 or 7 with peculiar characteristics. It was suggested for example, that the heteromorphic block of heterochromatin (CMA+++/DAPI-) on chromosome 6 of the "Branco Mineiro Piauí" accession can be used as a marker to identify this genotype or may be associated with some character of economic interest.

Chloroplast diversity of Casearia grandiflora in the Cerrado of Piauí State.

Casearia grandiflora (Salicaceae) is a typical Cerrado species adapted to disturbed environments, making it useful for restoration projects. Knowledge of genetic diversity is important for establishing conservation strategies for this species. This study aimed to compare chloroplast haplotype diversity and structure of C. grandiflora, under the assumption that protected areas hold greater genetic diversity than disturbed areas. The populations studied are from Parque Nacional de Sete Cidades Conservation Unit and from the surroundings of the city of Cocal de Telha, both located in the State of Piauí. Molecular analyses were performed with seven chloroplast microsatellite loci. The number of private haplotypes and haplotype diversity were higher in the conservation unit, which reinforces the importance of these areas in maintaining biodiversity. Analysis of molecular variance showed that most of the genetic variation is found within populations, with a moderate divergence between them (FST = 0.14). The Bayesian analysis and discriminant analysis of principal components suggested that populations are not structured, revealing that a set of individuals from Parque Nacional de Sete Cidades were more divergent within populations than between them. Since literature has little information on C. grandiflora, the results of this study provide important contribution to a better understanding of the specie's genetic diversity.

DNase concentration assay to obtain DNA-free RNA from sugarcane leaves.

The success of gene expression studies, protein synthesis, and construction of cDNA libraries directly depends on the purity and integrity of the RNA used in these tests, as even minor amounts of contaminant DNA (<1%) can produce a false positive amplification signal in quantitative real-time PCR. For RNA contaminated with genomic DNA, an essential step in the studies on gene expression is the treatment of the RNA samples with DNase. This study was conducted to test three different concentrations of DNase I (0.02, 0.04, and 0.06 µL/​​ng of RNA), which were chosen based on the results of the RNA sample quantifications and as indicated by the manufacturer, to digest genomic DNA present in the RNA samples extracted from sugarcane leaves with the Concert™ Plant RNA Reagent. The results showed that all three concentrations of DNase significantly reduced DNA concentrations. However, RNA was also degraded on DNase I treatment. In addition, the amount of DNA present in the RNA samples after purification with DNase I was sufficient for its amplification in the tests conducted with conventional PCR. Furthermore, the condition of RNA samples obtained after the treatments allowed for real-time PCR. Therefore, we concluded that 0.02 µL DNase I was the ideal concentration for sugarcane RNA purification, as higher concentrations do not increase the efficiency of the genomic DNA digestion in RNA samples and only make the purification process more expensive. This study provides important information on the effect of high concentrations of DNase I and complements previous studies that have so far tested only the DNase concentration recommended by the manufacturer.

Investigating genetic diversity in sapucaia using inter simple sequence repeat markers.

Sapucaia is a tree species originating from the Brazilian Amazon and is widely distributed in Brazil, especially in the mid-north region (Piauí and Maranhão states). Its seeds are rich in calories and proteins, and possess great potential for commercialization. Little is known about the genetic variability in the germplasm of most Lecythis species. Here, 11 inter-simple sequence repeat primers were used to estimate the genetic variability among 17 accessions, and to determine the levels of genetic variation and the standards of population structure in sapucaia. The accessions were obtained from the active germplasm bank (AGB) of Embrapa Meio-Norte, Teresina, PI, Brazil, and corresponded to four occurrence areas. Ninety-six loci were analyzed among the studied individuals. High variation was found at the species level, where the percentage of polymorphic bands was 94.79%, Nei's genetic diversity (h) was 0.3110, and Shannon's index (I) was 0.4732. In the analyzed populations, the percentage polymorphism ranged from 20.83 to 94.79%, Nei's genetic diversity ranged from 0.0863 to 0.2969, and Shannon's index ranged from 0.1260 to 0.4457. Significant genetic differentiation was detected among the populations (ΦST = 10.66%); however, the greatest genetic differentiation was found within the populations (89.34%), between which there was an intermediate level of gene flow (Nm = 1.10). Accessions BGS 2 and BGS 4 were the most divergent, whereas accessions BGS 14 and BGS 15 were the most similar. Therefore, sapucaia analyzed from the AGB present an elevated level of genetic diversity and may have potential use in genetic breeding programs.

Comparison of eight methods of genomic DNA extraction from babassu.

Babassu (Orbignya phalerata Martius) is one of the most important palms in Brazil because of the largest morphological variation, wide geographic distribution, and high socio-economic importance. The diversity present in babassu germplasm should be protected against loss to ensure their use with high productivity. Study of the available variability in populations of babassu is necessary to develop conservation strategies. The study of genetic variability can be conducted using molecular markers and many of these studies require significant quantity of high-quality DNA. The present study aimed to effect comparison among eight DNA extraction methods in case of O. phalerata. The quality and concentration of nucleic acids were analyzed by spectrophotometry and integrity of DNA was ascertained by agarose gel electrophoresis. The spectrophotometry revealed that some methods resulted in high levels of concentration of nucleic acids, in which values of the ratio A260/280 and A260/230 were outside the range of purity. The agarose gel electrophoresis established the concentration and integrity of DNA. The methods of Murray and Thompson (1980) and Ferreira and Grattapaglia (1998) did not result in satisfactory quantities of DNA. Conversely, the method proposed by Khanuja et al. (1999) resulted in DNA of adequate quality and quantity that could be satisfactorily used for amplification reactions performed with two ISSR primers.

Comparison of methods to isolate DNA from Caesalpinia ferrea.

Molecular markers are important for characterizing the genetic diversity of plants and can provide the basis for strategies to protect and conserve endangered populations. However, numerous molecular techniques are used, requiring an evaluation of fast and efficient methods to extract DNA. Since molecular studies of Caesalpinia ferrea are rare, it is important to develop and/or adapt a DNA extraction protocol that produces quality DNA samples to enable the design of strategies for the conservation of this threatened species. This study aimed to compare five methods for DNA extraction and to determine the most efficient protocol for C. ferrea. Sufficient genomic DNA was obtained from the leaves of C. ferrea using all the tested protocols to perform techniques involving molecular markers. Two protocols based on the detergent cetyl trimethyl ammonium bromide, as well as a commercial kit, yielded high concentrations of pure DNA. However, when polymerase chain reaction amplifications were performed, DNA was only successfully amplified from extractions performed with the commercial kit, which produced sufficient genomic DNA of good quality from the leaves of C. ferrea to perform techniques involving molecular markers.

RAPD analysis of the genetic diversity of mango (Mangifera indica) germplasm in Brazil.

We evaluated genetic variability of mango (Mangifera indica) accessions maintained in the Active Germplasm Bank of Embrapa Meio-Norte in Teresina, Piauí, Brazil, using RAPDs. Among these accessions, 35 originated from plantings in Brazil, six from the USA and one from India. Genomic DNA, extracted from leaf material using a commercial purification kit, was subjected to PCR with the primers A01, A09, G03, G10, N05, and M16. Fifty-five polymorphic loci were identified, with mean of 9.16 ± 3.31 bands per primer and 100% polymorphism. Application of unweighted pair group method using arithmetic average cluster analysis demonstrated five genotypic groups among the accessions examined. The genotypes Rosa 41, Rosa 48 and Rosa 49 were highly similar (94% similarity), whereas genotypes Sensation and Rosa 18 were the most divergent (only 7% similarity). The mango accessions were found to have considerable genetic variability, demonstrating the importance of analyzing each genotype in a collection in order to efficiently maintain the germplasm collection.