Sugarcane bacilliform viruses (SCBV; genus Badnavirus; family Caulimoviridae) are a threat to the global exchange of sugarcane germplasm. To investigate the prevalence and genetic diversity of SCBV across major Chinese sugarcane-growing areas, a total of 280 sugarcane leaf tissue samples collected from six provinces in China, 25 from three states in the USA and five from Queensland, Australia, were tested for the presence of SCBV by polymerase chain reaction (PCR) using newly designed degenerate primers targeting a 720-base pair (bp) fragment of the reverse transcriptase/ribonuclease H (RT/RNase H) genomic region. PCR-amplified fragments from 94 SCBV-positive samples were then cloned, sequenced and analysed for their genetic diversity. The results revealed a considerable haplotype diversity within individual SCBV isolates. Recombination analyses showed weak signatures of recombination among some of the SCBV sequences. Phylogenetic analysis revealed the segregation of global SCBV isolates into three major monophyletic clades encompassing 18 subgroups, including five previously undescribed subgroups named as SCBV-N to -R. Population genetic analysis data indicated that relatively low levels of genetic exchange have occurred between SCBV populations from different sugarcane production regions of the world. Together with the new set of degenerate SCBV-specific primers designed in this study, our results will advance the understanding of SCBV population structure in a semi-perennial host plant and aid the screening of global sugarcane germplasm to minimize the spread of genetic variants of the virus via contaminated plant materials.

A total of 332 sugarcane leaf samples were collected from seven provinces in China during 2010 to 2013. RT-PCR was performed using the YLS111/YLS462 primers to detect the causal pathogen Sugarcane yellow leaf virus (SCYLV) causing yellow leaf disease in sugarcane. The incidence of SCYLV infection ranged from 24 to 38 % depending on geographic areas. A new primer pair (P0-R3/P0-F2) was designed to prime the amplification of a 968-nucleotide (nt) fragment from SCYLV-positive samples. The fragment contained a partial 5′ untranslated region, the complete ORF0 encoding P0, and a partial ORF1. A phylogenetic analysis of 141 P0 fragment sequences (937 nt) worldwide showed that all SCYLV isolates were clustered into eight genotypes. The 107 SCYLV isolates collected in this study were classified into three genotypes (BRA, HAW, and CHN3), of which the BRA genotype (102/107) was the most prevalent and was detected in all evaluated locations. One isolate (1/107) of genotype HAW was found in Hainan province. Four isolates (4/107) of the genotype CHN3 were observed in Guangxi, Guizhou and Hainan provinces. These findings help understand the genotypic diversity and distribution of SCYLV isolates in China.

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Background Saccharum species such as sugarcane and energy cane are key players in the expanding bioeconomy for sugars, bioenergy, and production of high-value proteins. Genomic tools such as culm-regulated promoters would be of great value in terms of improving biomass characteristics through enhanced carbon metabolism for sugar accumulation and/or fiber content for biofuel feedstock. Unlike the situation in dicots, monocot promoters currently used are limited and mostly derived from highly expressed constitutive plant genes and viruses. In this study, a novel promoter region of Sugarcane bacilliform virus (SCBV; genus Badnavirus, family Caulimoviridae), SCBV21 was cloned and mapped by deletion analysis and functionally characterized transiently in monocot and dicot species and stably in sugarcane.ResultsIn silico analysis of SCBV21 [1816 base pair (bp)] identified two putative promoter regions (PPR1 and PPR2) with transcription start sites (TSS1 and TSS2) and two TATA-boxes (TATAAAT and ATATAA), and several vascular-specific and regulatory elements. Deletion analysis revealed that the 710 bp region spanning PPR2 (with TSS2 and ATATAA) at the 3′ end of SCBV21 retained the full promoter activity in both dicots and monocots, as shown by transient expression of the enhanced yellow fluorescent protein (EYFP) gene. In sugarcane young leaf segments, SCBV21 directed a 1.8- and 2.4-fold higher transient EYFP expression than the common maize ubiquitin 1 (Ubi1) and Cauliflower mosaic virus 35S promoters, respectively. In transgenic sugarcane, SCBV21 conferred a preferential expression of the β-glucuronidase (GUS) gene in leaves and culms and specifically in the culm storage parenchyma surrounding the vascular bundle and in vascular phloem cells. Among the transgenic events and tissues characterized in this study, the SCBV21 promoter frequently produced higher GUS activity than the Ubi1 or 35S promoters in a manner that was not obviously correlated with the transgene copy number.ConclusionsThe newly developed plant viral SCBV21 promoter is distinct from the few existing SCBV promoters in its sequence and expression pattern. The potential of SCBV21 as a tissue-regulated promoter with a strong activity in the culm vascular bundle and its storage parenchyma makes it useful in sugarcane engineering for improved carbon metabolism, increased bioenergy production, and enhanced stress tolerance.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0850-9) contains supplementary material, which is available to authorized users.

Sugarcane-infecting badnaviruses (sugarcane bacilliform viruses, SCBVs) represent a genetically heterogeneous species complex, posing a serious threat to the yield and quality of sugarcane in all major producing regions. SCBVs are commonly transmitted across regions by the exchange of sugarcane germplasm. In this study, we develop two quick, sensitive, and reliable protocols for real-time quantitative PCR (qPCR) of Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV) using two sets of TaqMan probes and primers targeting the reverse transcriptase/ribonuclease H (RT/RNase H) region. The two assays had a detection limit of 100 copies of plasmid DNA and were 100 times more sensitive than conventional PCR. High specificity of the two assays was observed with respect to SCBIMV and SCBMOV. A total of 176 sugarcane leaf tissue samples from Fujian and Yunnan provinces were collected and analyzed in parallel by conventional PCR, SCBIMV-qPCR, and SCBMOV-qPCR. The SCBIMV-qPCR and SCBMOV-qPCR assays indicated that 50% (88/176) and 47% (83/176) samples tested positive, respectively, whereas only 29% (51/176) tested positive with conventional PCR with the primer pairs SCBV-F and SCBV-R. We demonstrate for the first time that SCBIMV and SCBMOV occur in China and reveal coinfection of both Badnavirus species in 29% (51/176) of tested leaf samples. Our findings supply sensitive and reliable qPCR assays for the detection and quantitation of SCBV in sugarcane quarantine programs.

The Tibetan Mastiff is one of the most archaic, ferocious and the largest dogs in the world. The Kunming dog is the chief working-dog breed in China. In this study, ten microsatellite loci were used to assess the genetic diversity and evolutionary relationships in six dog breeds, including Tibetan Mastiff, Kunming dog, Belgian Malinois, Labrador Retriever, English Springer Spaniel, and German Shepherd. The highest genetic diversity was exhibited by the Tibetan Mastiff, indicating useful protection and little inbreeding in the modern Tibetan Mastiff. Higher genetic diversity was observed in European breeds, supporting the hypotheses that breeders outcross their pure breed dogs occasionally to avoid deleterious effects in Europe. Evolutionary relationships showed that English Springer Spaniel and Labrador Retriever were clustered together, then with the Tibetan Mastiff, consistent with previous cluster results. German Shepherd and Kunming dog were grouped together, coinciding with the breeding history of Kunming dog. It is the first time that Tibetan Mastiff and Kunming dog have been analyzed with microsatellites.