Development of a disposable pyruvate biosensor to determine pungency in onions (Allium cepa L.)

A disposable prototype pyruvate biosensor was constructed using pyruvate oxidase immobilised on mediated meldolas blue electrodes to determine pungency in onions (Allium cepa L.). The optimum operating potential was +150 mV (versus Ag/AgCl). A strong correlation between the biosensor response and untreated onion juice of known pyruvate concentration 2-12 micromol/g fresh weight (FW) was demonstrated. The biosensor was able to differentiate between low and high pungency onions. The detection limit using 1 unit of pyruvate oxidase was 1-2 micromol/g FW. Optimum concentrations of co-factors TPP, FAD and MgSO4 comprising the enzyme cocktail were determined as being 0.04, 0.1 and 30 mM, respectively.     

A PCR-based marker system monitoring CMS-(S), CMS-(T) and (N)-cytoplasm in the onion ( Allium cepa L.)

The chimerical mitochondrial CMS(1)-specific sequence in chives ( Allium schoenoprasum) was used to develop a PCR-marker that distinguishes both male-sterility inducing cytoplasms, CMS-(S) and CMS-(T), from the normal cytoplasm in onion ( Allium cepa). In combination with a previously described marker for CMS-(S), which anchors in the upstream region of the mitochondrial gene cob, all of the three known cytoplasms in the onion are distinguishable. The PCR-marker system was tested in 361 onion plants, which were selected from F(1)-hybrids and different open-pollinated varieties. The latter are mainly landraces from Turkey, in which all three cytoplasm types were detected.     

Development of a PCR-based marker to identify rice blast resistance gene,Pi-2(t), in a segregating population

The genomic clone RG64, which is tightly linked to the blast resistance gene Pi-2(t) in rice, provides means to perform marker-aided selection in a rice breeding program. The objective of this study was to investigate the possibility of generating a polymerase chain reaction (PCR)-based polymorphic marker that can distinguish the blast resistant gene, Pi-2(t), and susceptible genotypes within cultivated rice. RG64 was sequenced, and the sequence data was used to design pairs of specific primers for (PCR) amplification of genomic DNA from rice varieties differing in their blast disease responsiveness. The amplified products, known as sequenced-tagged-sites (STSs), were not polymorphic between the three varieties examined. However, cleavage of the amplified products with the restriction enzyme HaeIII generated a polymorphic fragment, known as specific amplicon polymorphism (SAP), between the resistant and the susceptible genotypes. To examine the power of the identified SAP marker in predicting the genotype of the Pi-2 (t) locus, we determined the genotypes of the F₂ individuals at this locus by performing progeny testing for the disease response in the F₃ generation. The results indicated an accuracy of more than 95% in identifying the resistant plants, which was similar to that using RG64 as the hybridization probe. The identification of the resistant homozygous plants increased to 100% when the markers flanking the genes were considered simultaneously. These results demonstrate the utility of SAP markers as simple and yet reliable landmarks for use in marker-assisted selection and breeding within cultivated rice.     

Trialling techniques in the breeding and prediction of recombinant inbred lines in onions (Allium cepa)

An outline is given of a novel breeding scheme for onions to produce highly vigorous, good quality inbred lines adapted for autumn-sowing in the UK. The scheme involves the production of advanced generation inbred lines in the absence of selection, by single seed descent, starting with 1500 F₂ plants from a Japanese × European cross. Concurrently a genetical analysis designed to predict the range of recombinant inbred lines derivable from this cross has been conducted using triple test cross (TTC) and basic generation (BG) families. Four trials are described, the results of which are used to define trialling techniques for the TTC and BG material which enable genetical predictions to be applied to direct-drilled onions at commercial spacings. The ranking of genotypes for important agronomic characters remains unchanged whether they are grown as transplanted spaced single plants or direct-drilled, however interactions may occur if pre-chitted seed is used.     

Prediction of the performance of inbred lines derived from a population cross in autumn-sown onions (Allium cepa L.)

Summary A design and model are presented to allow the prediction, in early generations, of the mean and distribution of recombinant inbred lines derived from a cross between two parental populations or partially inbred lines. The procedure has been tested in autumn-sown onions (in the UK) using a wide cross between the openpollinated Japanese cultivar, Senshyu, and a partially inbred line derived from the European cultivar, Rawska. The early generations used for prediction included the first self-pollinated generation of the two parental populations and the F₃ generation produced from the hybrid population. The predictions were tested by reference to the field performance of a random array of inbred lines, which were produced by single-seed descent (SSD) and had been selfed for three generations. The early generations, used for prediction, and a sample of SSD lines were raised alongside each other in each of two seasons. Within each season, good agreement was found between the predicted and observed performance of the recombinant inbred lines for three characters — yield, quality and maturity. This is used as evidence of the validity of the genetical model and the assumptions made. The effects of genotype x environment interactions prevented predictions made in one season being reliably applied to those made in the other and, therefore, reduce the attraction of this type of prediction study to the plant breeder.     

Differences between,and possible origins of,the cytoplasms found in fertile and male-sterile onions (Allium cepa L.)

Summary The DNA of the organellar genomes of Allium cepa has been examined to detect restriction fragment length polymorphisms. Differences can be shown between both the chloroplastal and mitochondrial genomes of the N and cms-S cytoplasms in their restriction fragment profiles. Southern blot analysis of the mtDNA profiles using probes containing defined mitochondrial genes also detected polymorphisms. No differences can be shown between the organellar genomes of the N and cms-T onions by either of these techniques. These data indicate different origins for the two sterility-conferring cytoplasms, suggesting autoplasmic and alloplasmic origins for the cms-T and cms-S cytoplasms, respectively. No evidence of the presence of virus-like particles was found in any of the cytoplasms.     

Structural studies of microsporogenesis in fertile and male-sterile onions (Allium cepa L.) containing the cms-S cytoplasm

Summary The structure of anther tissues has been studied during microsporogenesis in male-sterile and -fertile onions. Three types of abnormal tapetal behaviour have been observed within the single line II/3ms containing the cms-S cytoplasm: type 1, the premature breakdown of the tapetum at the tetrad stage, type 2, the hypertrophy of the tapetum after the diad stage followed by its premature autolysis and, type 3, in which the tapetum remains in good condition but for an abnormally long period of time. Tapetal autolysis proceeds in the same manner in both male-steriles and -fertiles with only the stage at which it occurs differing between the types of plants. Mitochondria were prominent in the tapetal tissue of all onion types throughout all stages of microsporogenesis and were still visible during the last stages of tapetal autolysis. In a detailed study of type 2 behaviour, no differences in mitochondrial volumes were found until the tapetum hypertrophied.     

Triple test cross predictions of the performance of recombinant inbred lines from a wide cross in onions (Allium cepa)

In conjunction with an onion breeding scheme to produce recombinant inbred lines from a wide cross genetical experiments were carried out to predict the likely outcome. Results are given of genetical predictions, obtained from two triple test cross experiments, of the expected performance of inbred lines derivable by single seed descent from a Japanese × Polish cross. For bulb maturity, c. 12% of the inbred lines are expected to be earlier than the earlier parent; for bulb hardness over 50% of the inbred lines are expected to exceed the better parent. For bulb weight, predictions from the basic model suggest 5–25% of the inbred lines may exceed the better parent, but when this model is modified to take account of the likely inbreeding depression in the open-pollinated Japanese parent, only 0–2% of such high yielding inbred lines are expected. Other potential sources of bias which may affect the predictions are discussed.     

Identification of a novel chimeric gene, orf725, and its use in development of a molecular marker for distinguishing among three cytoplasm types in onion (Allium cepa L.)

A novel chimeric gene with a 5′ end containing the nearly complete sequence of the coxI gene and a 3′ end showing homology with chive orfA501 was isolated by genome walking from two cytoplasm types: CMS-S and CMS-T, both of which induce male-sterility in onion (Allium cepa L.). In addition, the normal active and variant inactive coxI genes were also isolated from onions containing the normal and CMS-S cytoplasms, respectively. The chimeric gene, designated as orf725, was nearly undetectable in normal cytoplasm, and the copy number of the normal coxI gene was significantly reduced in CMS-S cytoplasm. RT-PCR results showed that orf725 was not transcribed in normal cytoplasm. Meanwhile, the normal coxI gene, which is essential for normal mitochondrial function, was not expressed in CMS-S cytoplasm. However, both orf725 and coxI were transcribed in CMS-T cytoplasm. The expression of orf725, a putative male-sterility-inducing gene, was not affected by the presence of nuclear restorer-of-fertility gene(s) in male-fertility segregating populations originating from the cross between a male-sterile plant containing either CMS-T or CMS-S and a male-fertile plant whose genotypes of nuclear restorer gene(s) might be heterozygous. The specific stoichiometry of orf725 and coxI in the mtDNA of the three cytoplasm types was consistent among diverse germplasm. Therefore, a molecular marker based on the relative copy numbers of orf725 and coxI was designed for distinguishing among the three cytoplasm types by one simple PCR. The reliability and applicability of the molecular marker was shown by testing diverse onion germplasm.     

Development of a PCR-based SNP marker system for effective selection of kernel length and kernel elongation in rice

Kernel length in rice (Oryza sativa L.) is controlled by various quantitative trait loci of which GS3 is the most important, being responsible for 80–90% of the variation in kernel length. A mutation in the second exon of this gene has been reported to be associated with maximum variations in the kernel length. We have developed a simple PCR-based marker system named DRR-GL which targets the functional nucleotide polymorphism at GS3. This marker system has the advantages that it is easy to use, saves time and cost, and is amenable for large-scale marker-assisted selection for the trait of kernel length. Validation of this marker in a segregating population and 152 rice varieties, which includes 30 elite basmati varieties, reveals its effective co-segregation and association with the traits of kernel length as well as kernel elongation after cooking. We recommend utilization of this simple, low-cost marker system in breeding programs targeted at improvement of key rice grain quality traits, kernel length and kernel elongation.     

Mutation loci and intragenic selection marker of the granule-bound starch synthase gene in waxy maize

Four pairs of specific PCR primers have been designed on the basis of the sequence of the granule-bound starch synthase gene (GBSS; dominant non-waxy gene Wx) and used to amplify its homologous sequence from thirteen waxy and two non-waxy inbred lines. Results from electrophoresis indicated that the recessive waxy gene was wx, derived from the dominant non-waxy gene Wx by mutation at its 3′ end. The sequence of the mutated 3′ end was amplified by the TAIL-PCR technique. Sequence alignment showed that the mutation of the wx gene was caused by transposition of the aldehyde dehydrogenase gene rf2. Two pairs of specific primers were designed on the basis of the sequence difference between the dominant gene Wx and its mutated recessive allele wx and used as intragenic selection markers to identify individual plants of genotypes WxWx, Wxwx, and wxwx by PCR amplification from the segregating population of the F₂ generation crossed between waxy and non-waxy inbred lines. Iodine solution staining and starch component assay showed that all the 35 F₂ plants identified as genotype WxWx produced non-waxy kernels of the F₃ generation and that all 33 F₂ plants identified as genotype wxwx produced waxy kernels of the F₃ generation. This result can be used to improve the selection efficiency of waxy maize breeding and for selection of other single genes and major polygenes.