Resistance gene analogues in sugarcane and sorghum and their association with quantitative trait loci for rust resistance.

Fifty-four different sugarcane resistance gene analogue (RGA) sequences were isolated, characterized, and used to identify molecular markers linked to major disease-resistance loci in sugarcane. Ten RGAs were identified from a sugarcane stem expressed sequence tag (EST) library; the remaining 44 were isolated from sugarcane stem, leaf, and root tissue using primers designed to conserved RGA motifs. The map location of 31 of the RGAs was determined in sugarcane and compared with the location of quantitative trait loci (QTL) for brown rust resistance. After 2 years of phenotyping, 3 RGAs were shown to generate markers that were significantly associated with resistance to this disease. To assist in the understanding of the complex genetic structure of sugarcane, 17 of the 31 RGAs were also mapped in sorghum. Comparative mapping between sugarcane and sorghum revealed syntenic localization of several RGA clusters. The 3 brown rust associated RGAs were shown to map to the same linkage group (LG) in sorghum with 2 mapping to one region and the third to a region previously shown to contain a major rust-resistance QTL in sorghum. These results illustrate the value of using RGAs for the identification of markers linked to disease resistance loci and the value of simultaneous mapping in sugarcane and sorghum.

Markers associated with stalk number and suckering in sugarcane colocate with tillering and rhizomatousness QTLs in sorghum.

Two important factors influencing sugar yield, the primary focus of sugarcane plant breeding programs, are stalk number and suckering. Molecular markers linked to both of these traits are sought to assist in the identification of high sugar yield, high stalk number, low-suckering sugarcane clones. In this preliminary mapping study, 108 progeny from a biparental cross involving two elite Australian sugarcane clones were evaluated at two sites for two years for both stalk number and suckering. A total of 258 DNA markers, including both restriction fragment length polymorphisms (RFLPs) and radio-labelled amplified fragments (RAFs), were scored and evaluated using single-factor analysis. Sixteen (7 RFLPs and 9 RAFs) and 14 (6 RFLPs and 8 RAFs) markers were identified that were significantly associated (P < 0.01) with stalk number and suckering, respectively, across both years and sites. The seven and six RFLP markers associated with stalk number and suckering, respectively, were generated by eight different RFLP probes, of which seven had been mapped in sorghum and (or) sugarcane. Of significant interest was the observation that all seven RFLP probes could be shown to be located within or near QTLs associated with tillering and rhizomatousness in sorghum. This observation highlights the usefulness of comparative mapping between sorghum and sugarcane and suggests that the identification of useful markers for stalk number and suckering in sugarcane would be facilitated by focussing on sorghum QTLs associated with related traits.

Homologues of the maize rust resistance gene Rp1-D are genetically associated with a major rust resistance QTL in sorghum.

As part of a comparative mapping study between sugarcane and sorghum, a sugarcane cDNA clone with homology to the maize Rp1-D rust resistance gene was mapped in sorghum. The cDNA probe hybridised to multiple loci, including one on sorghum linkage group (LG) E in a region where a major rust resistance QTL had been previously mapped. Partial sorghum Rp1-D homologues were isolated from genomic DNA of rust-resistant and -susceptible progeny selected from a sorghum mapping population. Sequencing of the Rp1-D homologues revealed five discrete sequence classes: three from resistant progeny and two from susceptible progeny. PCR primers specific to each sequence class were used to amplify products from the progeny and confirmed that the five sequence classes mapped to the same locus on LG E. Cluster analysis of these sorghum sequences and available sugarcane, maize and sorghum Rp1-D homologue sequences showed that the maize Rp1-D sequence and the partial sugarcane Rp1-D homologue were clustered with one of the sorghum resistant progeny sequence classes, while previously published sorghum Rp1-D homologue sequences clustered with the susceptible progeny sequence classes. Full-length sequence information was obtained for one member of a resistant progeny sequence class ( Rp1-SO) and compared with the maize Rp1-D sequence and a previously identified sorghum Rp1 homologue ( Rph1-2). There was considerable similarity between the two sorghum sequences and less similarity between the sorghum and maize sequences. These results suggest a conservation of function and gene sequence homology at the Rp1 loci of maize and sorghum and provide a basis for convenient PCR-based screening tools for putative rust resistance alleles in sorghum.

The major excretory/secretory protease from Lucilia cuprina larvae is also a gut digestive protease.

The larvae of the fly Lucilia cuprina excrete or secrete a chymotrypsia (LCTb) onto the skin of sheep to facilitate the establishment of the larval infestation. A combination of immunoblotting and RT-PCR approaches has established that this protease is also a gut digestive protease. LCTb is synthesized primarily in the cardia, a small highly specialized organ located at the anterior end of the midgut and by midgut cells. There is also some expression by the hindgut but no expression by salivary glands. Excretion of LCTb with waste products or regurgitation of the gut contents of the larvae may explain how this protease is transferred from the larval gut onto ovine skin. LCTb is first expressed in eggs and constitutively expressed throughout each larval instar, but is not expressed in pupae or adult flies. It is concluded that LCTb could be involved in the establishment of larvae on sheep skin as well as acting as a general gut digestive enzyme.

Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA.

Two chymotrypsin-like proteases were purified from the secretory and excretory material of first-instar larvae of Lucilia cuprina. The hydrolysis of N-succinyl-L-phenylalanine-nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor-Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease (Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis-causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.

Isolation of a trypsin-like serine protease gene family from the sheep blowfly Lucilia cuprina.

Various protease inhibitors active against both trypsin- and chymotrypsin-like serine proteases were used to characterize gut proteases from Lucilia cuprina by in vitro feeding assays. Significant larval growth retardation was observed on feeding first-instar larvae with trypsin inhibitors, particularly soybean trypsin inhibitor. Feeding of chymostatin, a specific chymotrypsin inhibitor, resulted in no significant growth retardation. This information suggests that trypsin-like serine proteases are probably the major gut digestive enzymes. A DNA fragment obtained by PCR which coded for part of a putative trypsin gene from L. cuprina was used to isolate a four-member multigene family of trypsins. The full nucleotide sequence of one of the genes and partial sequence from the other three genes were determined. Transcription of at least one of the genes has been confirmed. All four of the genes appear to have arisen by two separate gene duplication events.

Characterisation of a family of multi-copy genes encoding rhoptry protein homologues in Babesia bovis, Babesia ovis and Babesia canis.

A monoclonal antibody that had been raised against a protease-containing fraction of Babesia bovis, and shown to bind to a protein located in the rhoptries, was used to screen a B. bovis cDNA expression library. The sequence of the protein encoded by a positive clone was almost identical to the equivalent region of a previously described B. bovis 60-kDa rhoptry protein (Bv60). A tandem repeat of the gene encoding Bv60 was identified in all Australian isolates of B. bovis examined. Genes encoding homologous of Bv60 were cloned from Babesia ovis and Babesia canis. In B. ovis, 5 closely linked genes were identified. Four of these genes appeared to encode very similar proteins (Bo60.1-4). The protein (Bo60.5) encoded by the fifth B. ovis gene had 72% amino acid identity to Bo60.1-4 in the amino-terminal 306 amino acids, but no significant similarities in the carboxy-terminal region. In B. canis one gene (Bc60.2) was sequenced and a second closely linked gene was identified. A further member of the family, p58, has also been described previously from Babesia bigemina. Tandemly repeated genes subject to extensive gene conversion appear to be a feature of this family of babesial rhoptry protein homologous. No proteins significantly related to any members of the gene family were identified in a search of translated DNA and protein sequence databases. Thus the function of this family of proteins remains a matter for speculation.

Inversion polymorphism in populations of Drosophila sulphurigaster albostrigata and Drosophila nasuta albomicans from Phuket, Thailand.

Two species of the Drosophila nasuta subgroup of the Drosophila immigrans group, D. sulphurigaster albostrigata and D. nasuta albomicans were investigated in this study. Collections of both species were made from Phuket, Thailand. Both species have similar salivary chromosomes, with four autosomal arms and one sex chromosome arm, and both are highly polymorphic for paracentric inversions. D.s. albostrigata accounted for the majority of the isolines collected and exhibited the greater number of inversions. One inversion, C14 was common to both species, indicating common ancestry. A non-random distribution of inversions was observed on the proximal end of chromosome III in both D.s. albostrigata and D. n. albomicans. An inter-collection comparison revealed that both rigid and flexible chromosomal polymorphism were operating in the two species, with a seasonal variation noted for one inversion in D. s. albostrigata. A non-random association of two inversions was observed in D. n. albomicans. Based on a comparison of the indices of crossing over, both D. s. albostrigata and D. n. albomicans were found to be more heterozygous than in previous studies, with D. n. albomicans appearing to have evolved further than D. s. albostrigata.