Histogram score contributes for reliability of DNA content estimatives in Brachiaria spp

Ana Luiza De Oliveira Timbó,Roselaine Cristina Pereira,Lisete Chamma Davide,Vanderley Borges Dos Santos,Fausto Souza Sobrinho

doi:10.1590/s1413-70542012000600001

Abstract

Flow cytometry allows to estimate the DNA content of a large number of plants quickly. However, inadequate protocols can compromise the reliability of these estimates leading to variations in the values of DNA content the same species. The objective of this study was to propose an efficient protocol to estimate the DNA content of Brachiaria spp. genotypes with different ploidy levels using flow cytometry. We evaluated four genotypes (B. ruziziensis diploid and artificially tetraploidized; a tetraploid B. brizantha and a natural triploid hybrid), three buffer solutions (MgSO4, Galbraith and Tris-HCl) and three species as internal reference standards (Raphanus sativus, Solanum lycopersicum e Pisum sativum). The variables measured were: histogram score (1-5), coefficient of variation and estimation of DNA content. The best combination for the analysis of Brachiaria spp. DNA content was the use of MgSO4 buffer with R. sativus as a internal reference standard. Genome sizes expressed in picograms of DNA are presented for all genotypes and the importance of the histogram score on the results reliability of DNA content analyses were discussed.

Highlights

Brachiaria is used for feeding beef cattle, and is widely used for milk production in Brazil
Significant differences were observed for the general averages of the frequency and the coefficient of variation of the histogram in the estimates of the DNA content using different genotypes of Brachiaria spp, reference standards and nuclear isolation buffers (Table 3)
Considering the best average of the frequency of the histogram and lowest CV, the MgSO4 buffer and the reference standard R. sativus are indicated as the trustworthy estimate of the DNA content of the evaluated genotypes (Table 3)

Summary

Introduction

Brachiaria is used for feeding beef cattle, and is widely used for milk production in Brazil. In 2006, species from the Brachiaria genus represented 85% of the 120 million hectares of cultivated Brazilian pastures (MACEDO, 2006). The most cultivated species in Brazil are B. decumbens, B. brizantha, B. humidicola and B. ruziziensis, all of which present limitations such as being susceptible to leafhoppers or low tolerance to acidic, badly drained and poor soil. Leads to the necessity of polyploidy induction for the accomplishment of inter-specific crossings. The certification of the resultant plants whether by interspecific crossings, chromosomal duplication, from collections in pastures or in improved populations in open field can be carried out by flow cytometry

Objectives

Methods

Results