M.G.K. Jones

Production of somatic hybrids by electrofusion in Solanum.

SummaryConditions are described for large scale electrofusion of mesophyll protoplasts of dihaploid S. tuberosum with those of diploid S. brevidens. Overall fusion frequencies of 20%–30% were achieved, and following fusion, large numbers of protoplast-derived calli were obtained. Putative somatic hybrid plants were selected from the regenerated shoots by examining their morphological characteristics. Twenty-one somatic hybrids were confirmed by isoenzyme analysis and six somatic hybrids were further confirmed by Southern hybridization. Tetraploid hybrids were obtained, but cytogenetic studies indicated that more of the regenerated hybrids were hexaploid than had previously been found following chemical fusion of the same partners. Some advantages of electrofusion over chemical fusion are discussed.

Validation of molecular markers for wheat breeding

Five sets of markers were assessed for their usefulness in breeding, two linked to wheat stem rust gene Sr2, several markers linked to a chromosome segment conferring Yr17/Lr37/Sr38 resistance, two reported markers for the linked genes Lr35 andSr39, one for Lr28, and one linked to flour colour. The gene for Sr2 confers adult plant resistance to stem rust (Puccinia graminis f.sp. tritici) and was originally transferred to bread wheat from the tetraploid emmer (‘Yaroslav’) to the cultivars Hope and H-44. The gene is located on the short arm of chromosome 3B and confers a durable adult plant resistance to stem rust usually expressed only in the field. The chromosome segment carrying the Lr37, Sr38, Yr17 resistance genes is located on 2AS and was originally introduced into wheat through an Aegilops ventricosa Triticum persicum cross, followed by a cross to the cultivar Marne (VPM1). The flour colour quantitative trait locus was originally described in a Yarralinka Schomburg cross and is located on chromosome 7A. The primers as originally developed required optimisation for more routine use in a breeding program.

A polymerase chain reaction assay for cucumber mosaic virus in lupin seeds

Seed is the main source of infection of narrow-leafed lupin (Lupinus angustifolius) crops by cucumber mosaic virus (CMV). The ELISA procedure is currently used for large-scale, routine testing of lupin seed samples, but a more sensitive, reliable and labour-saving assay is needed which detects levels of seed infection as low as 0.1%. A Polymerase Chain Reaction (PCR) using ground dry seed samples was developed for this purpose. Primers based on concensus sequences of eight published CMV coat protein cDNAs (RNA3) of CMV subgroups 1 and 2 were used. The assay involved (1) a reverse transcription step for cDNA synthesis and (2) amplification of a specific fragment (482-501 bp depending on the strain) by PCR. Two methods of extracting virus from infected lupin material were used: (i) a rapid procedure which was effective for samples with higher levels of infection, e.g. infected leaves and 20.5% infected seed; (ii) a phenol-chloroform procedure, which led to greater sensitivity, enabling reliable detection of 0.1% seed infection. It detected CMV in 16 commercial seed samples (0.1-8% seed infection) belonging to seven cultivars from 12 different localities. Both methods were suitable for routine testing of the flour derived from grinding dry seed. On dissection of infected seeds, CMV was detected in the cotyledons and embryo and usually in or on the testa. The PCR assay detected virus from both CMV subgroups, but only subgroup 2 was found in lupin seed samples. The two CMV subgroups can be distinguished by digestion of amplified DNA with the restriction enzyme EcoRI; only CMV strains of subgroup 2 are digested to yield two fragments of size 330 and 170 bp.

Transient gene expression in electroporated protoplasts and intact cells of sugar beet.

Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.

Molecular, cytogenetic and morphological characterization of somatic hybrids of dihaploid Solanum tuberosum and diploid S. brevidens.

SummaryFifty-eight somatic hybrid plants, produced both by chemical (11) and electrical fusion (47) of protoplasts of dihaploid Solanum tuberosum and S. brevidens, have been analysed by molecular, cytological and morphological methods. The potentially useful euploid plants constituted 34% of the total, of which 20% were tetraploid and 14% hexaploid; the remainder were aneuploid at the tetraploid, hexaploid and octoploid levels. Analysis of chloroplast DNA showed that 55% of hybrids contained chloroplasts from S. brevidens and 45% from S. tuberosum. Hexaploids, the products of three protoplasts fusing together, were analyzed with specific DNA probes, and this revealed that nuclear genome dosages could be either 2∶1 S. tuberosum∶S. brevidens, or vice-versa. Chloroplast types of hexaploids were not influenced by nuclear genome dosage, and all six possible combinations of genome dosage and chloroplast types were found amongst tetraploids and hexaploids. To examine the morphology of the hybrid population and its possible relation to the chromosome number and chloroplast DNA type, 18 morphological characteristics were measured on greenhouse-grown plants and analyzed by principal component and canonical variate analyses. Both analyses showed that nuclear ploidy has the most prominent influence on the overall morphology of the hybrids. Differential parental genome expression in the morphology of the hybrids is discussed. These results provide useful data on the range of genetic combinations that can be expected to occur amongst somatic hybrid plants.

Mapping and validation of the genes for resistance to Pyrenophora teres f. teres in barley (Hordeum vulgare L.)

Identification and deployment of disease resistance genes are key objectives of Australian barley breeding programs. Two doubled haploid (DH) populations derived from Tallon × Kaputar (TK) and VB9524 × ND11231 (VN) crosses were used to identify markers for net type net blotch (NTNB) (Pyrenophora teres f. teres). The maps included 263 and 250 markers for TK and VN populations, respectively. The TK population was screened with 5 pathotypes and the VN population with 1 pathotype of NTNB as seedlings in the glasshouse. In addition, the TK population was subjected to natural infection in the field at Hermitage Research Station, Qld. Analyses of the markers were performed using the software packages MapManager and Qgene. One region on chromosome 6H was strongly associated with resistance to NTNB in both populations (R2 = 83% for TK and 66% for VN). In the TK population, 2 more quantitative trait loci (QTLs) were identified on chromosomes 2H and 3H, with R2 values of 30% and 31%, respectively. These associations were consistent over all pathotypes studied during the seedling stage. The same QTL on chromosome 6H was also found to be highly significantly associated (R2 = 65%) with the adult plant (field) response in the TK population. There are several very closely linked markers showing strong associations in these regions. Association of the 4 markers on chromosome 6H QTL with resistance to the NTNB has been validated in 2 other DH populations derived from barley crosses Pompadour × Stirling and WPG8412 × Stirling. These markers present an opportunity for marker assisted selection of lines resistant to NTNB in barley breeding programs.

Using laser capture microdissection to study gene expression in early stages of giant cells induced by root‐knot nematodes

SUMMARY Root-knot nematodes (Meloidogyne spp.) are economically important plant parasites that induce specific feeding cells called giant cells in host roots. Study of molecular events involved in induction and differentiation of giant cells has been limited because it is difficult to obtain pure cytoplasm specifically from the highly specialized cells. In this work, laser capture microdissection (LCM) was used to collect cytoplasmic contents from paraffin-embedded sections of 4 day post-inoculation giant cells in tomato roots. Total RNA was isolated from the sections, and used in RT-PCR to investigate expression of cell cycle genes in giant cells. Two D-type cyclin genes, LeCycD3;2 and LeCycD3;3, were expressed at higher levels in giant cells compared with other cell-cycle-related cyclin genes, suggesting that the induction of the G1 phase of the cell cycle may be triggered in response to stimulation by the infecting nematode. LCM provides a powerful new tool to study the molecular basis of host-pathogen interactions at the cellular or subcellular level.

Chromosome variation in dividing protoplasts and cell suspensions of wheat.

SummaryThe cytology of bread wheat (Triticum aestivum) suspension lines, recycled lines (selected for high division frequency) and their dividing protoplasts, have been examined. Extensive numerical and structural chromosome variation was present in all the lines. The most frequently observed chromosome numbers were around 2n=32, indicating that considerable chromosome loss from the normal wheat complement (2n=6x=42) had occurred during selection of the lines. Chromosome aberrations also indicated loss of chromosome arms and chromosome segments. The implications of this variation for studies on transformation and for the potential regeneration of whole plants from protoplasts of bread wheat are discussed.

Species-specific sequences in the genus Solanum: identification, characterization, and application to study somatic hybrids of S. brevidens and S. tuberosum

SummaryTo aid in the identification and analysis of somatic hybrids between potato (Solanum tuberosum, dihaploid line PDH 40) and the non tuber-bearing wild species S. brevidens, a series of species-specific repetitive DNA sequences have been isolated. This was accomplished by making libraries of HaeIII-digested total DNA of S. tuberosum and S. brevidens, by cloning fragments into the SmaI site of plasmid pUC18 and transforming them into E. coli (JM83). The S. brevidens library consisted of 1,000 recombinant clones, and that of S. tuberosum, 700. Nitrocellulose filters with recombinant clones were hybridised to nick-translated total DNA of S. brevidens and also S. tuberosum, and, following autoradiography, clones that hybridised strongly to the DNA of only one of the species were chosen. Two highly repeated S. brevidens clones (pSB1, 400 bp and pSB7,210 bp), one highcopy-number s. tuberosum clone (pST10, 200 bp) and one low-copy-number sequence of S. tuberosum (pST3, 1.5 kbp) were selected for further analysis by Southern hybridisation to digested total DNA. Clone pSB7 gave a ladder pattern on hybridisation to EcoR1-digested total DNA of S. brevidens, with signals at multiples of 200 bp DNA. Using these probes it was possible to verify the hybridity of putative hybrids of dihaploid S. tuberosum and S.brevidens, and to confirm by Southern analysis and by slot blots the parental genome dosage of hexaploid hybrids (two s. brevidens: one S. tuberosum, and vice-versa). The S. tuberosum-specific probe, pSTIO, hybridised with DNA of three other tuber-bearing wild species (S. hjertingii, S. capsicibaccatum and S. berthaultii). A squash-blot procedure was developed using the probes that would allow early identification of somatic hybrid callus. There are a number of useful applications of such species-specific probes in the identification and analysis of somatic hybrids.

Genotyping single nucleotide polymorphisms for selection of barley β-amylase alleles

A high-throughput single nucleotide polymorphism (SNP) genotyping system was developed and used to select barley seedlings carrying superior alleles of β-amylase. In the malting process, β-amylase is a key enzyme involved in the degradation of starch. Four allelic forms of the enzyme are found in barley, each exhibiting a different rate of thermal inactivation, or thermostability. The level of thermostability influences starch degradation, which determines the yield of fermentable sugars for alcohol production during brewing. Control of the fermentability level is important for barley breeding programs to allow targeting quality profiles of new varieties to suit end-user requirements. Alignment of the cDNA sequences encoding the 4 enzyme forms revealed 6 SNPs (cSNPs). The 4 alleles could be identified unambiguously by codominantly genotyping 2 of the cSNPs using a duplex single nucleotide primer extension (SNuPE) assay. Two genotyping primers with their 3′ ends directly flanking the selected SNPs were annealed to the amplified target sequences and extended by single dideoxynucleotides complementary to the polymorphic nucleotides. Extended primers were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Making use of the inherent molecular weight difference between DNA bases, incorporated nucleotides were identified by the increase in mass of the extended primers. A cleaved amplified polymorphic sequence (CAPS) assay enabling broader classification of the alleles was also developed to facilitate the transfer of this marker to other laboratories. Plants carrying alternative β-amylase alleles were selected at the seedling stage for barley breeding.