RESUMO
Tomato interveinal chlorosis virus (ToICV; Begomovirus solanumintervenae, genus Begomovirus, family Geminiviridae) has been described infecting tomato (Solanum lycopersicum) and Macroptilium lathyroides in Northeastern (NE) Brazil for more than a decade (Albuquerque et al., 2012; Silva et al., 2012). During a survey in 2020, plants of the leguminous weed Rhynchosia minima exhibiting virus-like symptoms such as mosaic and interveinal chlorosis were observed in the state of Alagoas, NE Brazil. Symptomatic leaf samples of R. minima were randomly collected (n=15; supplementary figure 1). Total DNA from each sample was used as a template for PCR amplification of partial begomoviral DNA-A sequences using the degenerate primer pair PAL1v1978 and PAR1c496, universal for geminiviruses (Rojas et al., 1993). Amplicons of ~1.2 kbp were observed from 12 samples, although this should not be considered as incidence since only symptomatic plants were collected. To identify the begomovirus associated with R. minima, viral genomes were amplified from PCR-positive samples using rolling circle amplification (RCA) (Inoue-Nagata et al., 2004). The RCA products were digested with HindIII, cloned into the pBluescript II KS+ plasmid vector and bidirectionally Sanger-sequenced (Macrogen Inc., Seoul). BLASTn searches indicated that the clones (n=4) reported here corresponded to a begomovirus DNA-A component, and pairwise comparisons showed that they shared the highest identity with ToICV, at 92.4-94.7% nucleotide sequence identity. Based on the species demarcation criteria of ≥91% nucleotide identity for the genus Begomovirus (Brown et al., 2015), the begomoviruses obtained from R. minima are new isolates of ToICV. The new DNA-A sequences of 2,619-2,623 nt in length were deposited in GenBank under accession numbers PP639092 to PP639095. Multiple nucleotide sequence alignments were prepared using the MUSCLE algorithm implemented in MEGA v.11 (Kumar et al., 2018), and a maximum likelihood (ML) tree was reconstructed in RaxML-NG (Kozlov et al., 2019), assuming a general time reversible (GTR) nucleotide substitution model with a gamma (G) model of rate heterogeneity and 1,000 bootstrap replicates. The DNA-A-based tree showed that the ToICV sequences clustered into a monophyletic group, additionally supporting these isolates as members of the species Begomovirus solanumintervenae. At least two independent interspecies recombination events were predicted among the ToICV isolates, with breakpoints located in the Rep-encoding region and ToICV (GenBank Accession JF803253), tomato mottle leaf curl virus (JF803248) and soybean blistering mosaic virus (MN486865) detected as putative parents. To the best of our knowledge, this is the first report of ToICV infecting R. minima worldwide, expanding the host range of this begomovirus. Non-cultivated plants such as R. minima play a crucial role as reservoirs and sources of inoculum for begomoviruses (Paz-Carrasco et al., 2014), reinforcing their relevance to socioeconomically important crops.
RESUMO
A diversidade genética de catorze cultivares de cana-de-açúcar (Saccharum oficinarum) foi acessada por meio de marcadores moleculares ISSR. Objetivou-se caracterizar molecularmente as cultivares estudadas. Foram utilizados trinta e sete primers de ISSR, dos quais, oito foram eficientes na amplificação do DNA das amostras analisadas, sendo sete primers suficientes para distinguir todas as cultivares de cana-de-açúcar envolvidas nas análises. A faixa de amplicons variou de 300 a 2000 pb. As cultivares RB 92579 e RB 863129 apresentaram maiores coeficientes de similaridade (77 por cento) enquanto as cultivares RB 961 e RB 931611 formaram o grupo com menor similaridade (22 por cento). Os resultados indicam que os marcadores ISSR foram úteis na análise da diversidade genética e geração de padrões genéticos (fingerprint), em germoplasma de cana-de-açúcar. Marcadores ISSR cultivar-específico foram obtidos com o primer UBC 817 para as 14 cultivares testadas. Num próximo trabalho, mais primers ISSR serão utilizados para buscar mais polimorfismos dessas e de outras cultivares de cana-de-açúcar.
Genetic diversity of fourteen sugarcane cultivars was accessed by ISSR molecular markers. With the aim to characterizing and validating the efficiency of these markers in the fingerprint of studied cultivars, thirty seven ISSR primers were used, from which, eight were efficient for the DNA amplification. Seven primers were efficient to discriminate the fourteen studied sugarcane cultivars. The amplicons varied from 300 to 2000 bp. The cultivars RB 92579 and RB 863129 presented higher similarity coefficient (77 percent) while the cultivars RB 961 and RB 93611 formed the group with lower similarity (22 percent). The results suggested that ISSR markers were useful in the analysis of the genetic diversity and in the fingerprint in sugarcane germosplasm. In the next step more ISSR primers will be used in order to obtain more polymorphism from these varieties and to analyze more sugarcane cultivars.