Temporal analysis of Arabidopsis genes activated by Eucalyptus grandis NAC transcription factors associated with xylem fibre and vessel development.

Secondary cell wall (SCW) deposition in Arabidopsis is regulated among others by NAC transcription factors, where SND1 chiefly initiates xylem fibre differentiation while VND6 controls metaxylem vessel SCW development, especially programmed cell death and wall patterning. The translational relevance of Arabidopsis SCW regulation theory and the utility of characterized transcription factors as modular synthetic biology tools for improving commercial fibre crops is unclear. We investigated inter-lineage gene activation dynamics for potential fibre and vessel differentiation regulators from the widely grown hardwood Eucalyptus grandis (Myrtales). EgrNAC26, a VND6 homolog, and EgrNAC61, an SND1 homolog, were transiently expressed in Arabidopsis mesophyll protoplasts in parallel to determine early and late (i.e. 7 and 14 hours post-transfection) gene targets. Surprisingly, across the time series EgrNAC26 activated only a subset of SCW-related transcription factors and biosynthetic genes activated by EgrNAC61, specializing instead in targeting vessel-specific wall pit and programmed cell death markers. Promoters of EgrNAC26 and EgrNAC61 both induced reporter gene expression in vessels of young Arabidopsis plants, with EgrNAC61 also conferring xylem- and cork cambium-preferential expression in Populus. Our results demonstrate partial conservation, with notable exceptions, of SND1 and VND6 homologs in Eucalyptus and a first report of cork cambium expression for EgrNAC61.

A time series transcriptome analysis of cassava (Manihot esculenta Crantz) varieties challenged with Ugandan cassava brown streak virus.

A time-course transcriptome analysis of two cassava varieties that are either resistant or susceptible to cassava brown streak disease (CBSD) was conducted using RNASeq, after graft inoculation with Ugandan cassava brown streak virus (UCBSV). From approximately 1.92 billion short reads, the largest number of differentially expressed genes (DEGs) was obtained in the resistant (Namikonga) variety at 2 days after grafting (dag) (3887 DEGs) and 5 dag (4911 DEGs). At the same time points, several defense response genes (encoding LRR-containing, NBARC-containing, pathogenesis-related, late embryogenesis abundant, selected transcription factors, chaperones, and heat shock proteins) were highly expressed in Namikonga. Also, defense-related GO terms of 'translational elongation', 'translation factor activity', 'ribosomal subunit' and 'phosphorelay signal transduction', were overrepresented in Namikonga at these time points. More reads corresponding to UCBSV sequences were recovered from the susceptible variety (Albert) (733 and 1660 read counts per million (cpm)) at 45 dag and 54 dag compared to Namikonga (10 and 117 cpm respectively). These findings suggest that Namikonga's resistance involves restriction of multiplication of UCBSV within the host. These findings can be used with other sources of evidence to identify candidate genes and biomarkers that would contribute substantially to knowledge-based resistance breeding.

QTL associated with resistance to cassava brown streak and cassava mosaic diseases in a bi-parental cross of two Tanzanian farmer varieties, Namikonga and Albert.

KEY MESSAGE: QTL consistent across seasons were detected for resistance to cassava brown streak disease induced root necrosis and foliar symptoms. The CMD2 locus was detected in an East African landrace, and comprised two QTL. Cassava production in Africa is compromised by cassava brown streak disease (CBSD) and cassava mosaic disease (CMD). To reduce costs and increase the precision of resistance breeding, a QTL study was conducted to identify molecular markers linked to resistance against these diseases. A bi-parental F1 mapping population was developed from a cross between the Tanzanian farmer varieties, Namikonga and Albert. A one-step genetic linkage map comprising 943 SNP markers and 18 linkage groups spanning 1776.2 cM was generated. Phenotypic data from 240 F1 progeny were obtained from two disease hotspots in Tanzania, over two successive seasons, 2013 and 2014. Two consistent QTLs linked to resistance to CBSD-induced root necrosis were identified in Namikonga on chromosomes II (qCBSDRNFc2Nm) and XI (qCBSDRNc11Nm) and a putative QTL on chromosome XVIII (qCBSDRNc18Nm). qCBSDRNFc2Nm was identified at Naliendele in both seasons. The same QTL was also associated with CBSD foliar resistance. qCBSDRNc11Nm was identified at Chambezi in both seasons, and was characterized by three peaks, spanning a distance of 253 kb. Twenty-seven genes were identified within this region including two LRR proteins and a signal recognition particle. In addition, two highly significant CMD resistance QTL (qCMDc12.1A and qCMDc12.2A) were detected in Albert, on chromosome 12. Both qCMDc12.1A and qCMDc12.2A lay within the range of markers reported earlier, defining the CMD2 locus. This is the first time that two loci have been identified within the CMD2 QTL, and in germplasm of apparent East African origin. Additional QTLs with minor effects on CBSD and CMD resistance were also identified.

Genetic diversity of African maize inbred lines revealed by SSR markers.

Knowledge of genetic diversity (GD) and relationships among maize inbred lines is indispensable in a breeding program. Our objectives were to (1) investigate the level of genetic diversity among maize inbred lines and (2) assess their genetic structures by applying simple sequence repeat (SSR) markers. Fifty-six highland and mid-altitude maize inbred lines obtained from CIMMYT programs in Ethiopia and Zimbabwe were genotyped using 27 SSR loci. All of the genotypes studied could unequivocally be distinguished with the combination of the SSRs used. In total, 104 SSR alleles were identified, with a mean of 3.85 alleles per locus. The average polymorphism information content (PIC) was 0.58. GD expressed as Euclidean distance, varied from 0.28 to 0.73 with an average of 0.59. Cluster analysis using unweighted pair group method with arithmetic average (UPGMA) suggested five groups among the inbred lines. Most of the inbred lines adapted to the highlands and the mid-altitudes were positioned in different clusters with a few discrepancies. The pattern of groupings of the inbred lines was mostly consistent with available pedigree information. The variability detected using SSR markers could potentially contribute towards effective utilization of the inbred lines for the exploitation of heterosis and formation of genetically diverse source populations in Ethiopian maize improvement programs.

Complete genetic linkage maps from an interspecific cross between Fusarium circinatum and Fusarium subglutinans.

The Gibberella fujikuroi complex includes many plant pathogens of agricultural crops and trees, all of which have anamorphs assigned to the genus Fusarium. In this study, an interspecific hybrid cross between Gibberella circinata and Gibberella subglutinans was used to compile a genetic linkage map. A framework map was constructed using a total of 578 AFLP markers together with the mating type (MAT-1 and MAT-2) genes and the histone (H3) gene. Twelve major linkage groups were identified (n=12). Fifty percent of the markers showed significant deviation from the expected 1:1 transmission ratio in a haploid F(1) cross (P <0.05). The transmission of the markers on the linkage map was biased towards alleles of the G. subglutinans parent, with an estimated 60% of the genome of F(1) individuals contributed by this parent. This map will serve as a powerful tool to study the genetic architecture of interspecific differentiation and pathogenicity in the two parental genomes.

Development and assessment of microarray-based DNA fingerprinting in Eucalyptus grandis.

Development of improved Eucalyptus genotypes involves the routine identification of breeding stock and superior clones. Currently, microsatellites and random amplified polymorphic DNA markers are the most widely used DNA-based techniques for fingerprinting of these trees. While these techniques have provided rapid and powerful fingerprinting assays, they are constrained by their reliance on gel or capillary electrophoresis, and therefore, relatively low throughput of fragment analysis. In contrast, recently developed microarray technology holds the promise of parallel analysis of thousands of markers in plant genomes. The aim of this study was to develop a DNA fingerprinting chip for Eucalyptus grandis and to investigate its usefulness for fingerprinting of eucalypt trees. A prototype chip was prepared using a partial genomic library from total genomic DNA of 23 E. grandis trees, of which 22 were full siblings. A total of 384 cloned genomic fragments were individually amplified and arrayed onto glass slides. DNA fingerprints were obtained for 17 individuals by hybridizing labeled genome representations of the individual trees to the 384-element chip. Polymorphic DNA fragments were identified by evaluating the binary distribution of their background-corrected signal intensities across full-sib individuals. Among 384 DNA fragments on the chip, 104 (27%) were found to be polymorphic. Hybridization of these polymorphic fragments was highly repeatable (R2>0.91) within the E. grandis individuals, and they allowed us to identify all 17 full-sib individuals. Our results suggest that DNA microarrays can be used to effectively fingerprint large numbers of closely related Eucalyptus trees.

Comparative genetic linkage maps of Eucalyptus grandis, Eucalyptus globulus and their F1 hybrid based on a double pseudo-backcross mapping approach.

Comparative genetic mapping in interspecific pedigrees presents a powerful approach to study genetic differentiation, genome evolution and reproductive isolation in diverging species. We used this approach for genetic analysis of an F(1) hybrid of two Eucalyptus tree species, Eucalyptus grandis (W. Hill ex Maiden.) and Eucalyptus globulus (Labill.). This wide interspecific cross is characterized by hybrid inviability and hybrid abnormality. Approximately 20% of loci in the genome of the F(1) hybrid are expected to be hemizygous due to a difference in genome size between E. grandis (640 Mbp) and E. globulus (530 Mbp). We investigated the extent of colinearity between the two genomes and the distribution of hemizygous loci in the F(1) hybrid using high-throughput, semi-automated AFLP marker analysis. Two pseudo-backcross families (backcrosses of an F(1) individual to non-parental individuals of the parental species) were each genotyped with more than 800 AFLP markers. This allowed construction of de novo comparative genetic linkage maps of the F(1) hybrid and the two backcross parents. All shared AFLP marker loci in the three single-tree parental maps were found to be colinear and little evidence was found for gross chromosomal rearrangements. Our results suggest that hemizygous AFLP loci are dispersed throughout the E. grandis chromosomes of the F(1) hybrid.

High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer.

Amplified fragment length polymorphism (AFLP) analysis is currently the most powerful and efficient technique for the generation of large numbers of anonymous DNA markers in plant and animal genomes. We have developed a protocol for high-throughput AFLP analysis that allows up to 70,000 polymorphic marker genotype determinations per week on a single automated DNA sequencer. This throughput is based on multiplexed PCR amplification of AFLP fragments using two different infrared dyelabeled primer combinations. The multiplexed AFLPs are resolved on a two-dye, model 4200 LI-COR automated DNA sequencer, and the digital images are scored using semi-automated scoring software specifically designed for complex AFLP banding patterns (AFLP-Quantar). Throughput is enhanced by using high-quality genomic DNA templates obtained by a 96-well DNA isolation procedure.