真菌毒素生物测定方法研究概况：Ⅱ.细胞及细胞器水平的生物测定

本文从细胞和细胞器水平详细介绍了6种真菌毒素生物测定法,为研究毒素的定量测定和作用机制奠定基础。     

A rapid, seedling-based bioassay for identifying low cadmium-accumulating individuals of Durum wheat (Triticum turgidum L.).

We have developed a seedling-based bioassay that iscapable of identifying low Cd-accumulating phenotypes(homozygous and heterozygous) after 96–120 h ofexposure. Our experiments were conducted using¹⁰⁹Cd as a tracer at subtoxic concentrations tosimulate conditions that might be experienced in thefield. Supply of Cd (10^-11 M) to 4-d oldseedlings for 0–8 h resulted in no differences in rootand leaf Cd content between the low (TL05) and high(TL04) Cd-accumulating isolines. Increasing time ofexposure produced significant differences in leaf Cdaccumulation between isolines, with differencesbecoming most pronounced after the bulk of appliedcadmium ( 95%) was depleted from absorption solutions( 72 h). Similar results were obtained with8-d old seedlings, where differences between genotypeswere more pronounced in young leaves (2nd leaf) orshoot bases. Individuals from five low and highCd-accumulating near isogenic pairs (50individuals/isoline) were screened using Cdconcentration of shoot bases as the screeningcriterion. Mean scores within each isoline pair weresignificantly different, although overlap ofindividual scores was observed at intermediate foliarCd concentrations. The 2^nd leaf to root Cdcontent ratio, which reflects root to shoottranslocation, provided a better parameter todistinguish low from high Cd-accumulating isolines. Plants used for this bioassay could also be rescuedfor subsequent experimental crosses, providing a rapidand cost-effective tool for early detection of the lowCd-accumulating phenotype.     

Genic microsatellite markers for discrimination of spinach cultivars

A set of simple sequence repeat (SSR or microsatellite) markers was used to discriminate a collection of 33 Spinacia oleracea hybrid cultivars from seven different breeding stations all over the world. All SSR markers were genic microsatellite markers located in coding or non-coding regions of genes of known function. Cluster analysis based on 13 of the SSR markers showed that the spinach hybrids grouped into three clusters. The first two clusters consisted of European spinach types, which were well discriminated according to their origin from different breeding stations. The third cluster was a mixture of Asian as well as European types of spinach. Subclusters in this group did not reflect differences in morphology, earliness or company origin. The data show that genic microsatellites are a powerful tool for discrimination of spinach cultivars.     

Effect of Drechslera (Helminthosporium) sacchari Toxin upon the Permeability of Sugarcane Leaf and Callus Tissues

Sugarcane leaves and calli from highly susceptible and resistant varieties to eyespot disease were used to evaluate the Drechslera sacchari toxin effect at different concentrations and incubation times by measuring electrolyte leakage. This expression of disease resistance was observed not only in the leaf but also in the callus. Furthermore, the growth of resistant calli MS medium supplemented with DS toxin, was higher when compared to the susceptible ones. The possibility of obtaining resistant somaclones is confirmed.     

Molecular tagging of Asiatic soybean rust resistance in exotic genotype EC 241780 reveals complementation of two genes

Asian soybean rust (ASR) caused by Phakopsora pachyrhizi severely reduces seed yield in soybean. Molecular tagging of ASR resistance can help in the process of resistance breeding. In this study, an F₂ population of cross (susceptible cultivar ‘NRC 7’ × resistant exotic genotype EC 241780) was used for bulked segregant analysis (BSA) with 25 SSR (simple sequence repeat) primers linked with six Rpp genes. Among them, five polymorphic SSR markers, viz., Sct 187, SSR 1859, Satt 191 (Rpp1b like loci) and Satt 215, Sat_361 (Rpp2 loci) distinguished the ASR resistant and susceptible bulks and individuals. In combined marker analysis, the markers Satt 191 (Rpp1b like loci) and Satt 215 (Rpp2 loci) were linked with ASR severity score and were also confirmed in individual 110 F₂ segregants. Hence, these markers could be utilized in the marker assisted rust resistance breeding of Rpp1b like and Rpp2 genes. In silico candidate gene analysis for hypersensitive response revealed that Satt 191 linked region was rich in genes encoding apoptotic ATPase having leucine‐rich repeat (LRR) domain.     

Marker assisted accelerated introgression of null allele of kunitz trypsin inhibitor in soybean

Development of kunitz trypsin inhibitor (KTI)-free soybean is crucial for soy-food industry as the heat inactivation employed to inactivate the anti-nutritional factor in regular soybean incurs extra cost and affects protein solubility. In the presented work, a null allele of KTI from PI542044 was introgressed into cultivar ‘JS97-52’ (recurrent parent) through marker assisted backcrossing. Foreground selection in BC₁F₂, BC₂F₂ and BC₃F₂ was carried out using the null allele-specific marker in tandem with SSR marker Satt228, tightly linked with a trypsin inhibitor Ti locus. Background selection in null allele-carrying plants through 106 polymorphic SSR markers across the genome led to the identification of 9 KTI-free lines exhibiting 98.6% average recurrent parent genome content (RPGC) after three backcrosses, which otherwise had required 5–6 backcrosses through conventional method. Introgressed lines (ILs) were free from KTI and yielded at par with recurrent parent. Reduction of 68.8–83.5% in trypsin inhibitor content (TIC) in ILs compared to the recurrent parent (‘JS97-52’) was attributed to the elimination of KTI.     

Development of a sequence-specific PCR marker linked to the Ku gene which removes the vernalization requirement in narrow-leafed lupin

Wild types of Lupinus angustifolius require vernalization to promote flowering. Modern domesticated cultivars carry the early-flowering gene Ku which removes this requirement. A microsatellite-anchored fragment length polymorphism marker was identified as co-segregating with the Ku gene in a recombinant inbred line (RIL) population derived from a domesticated × wild-type cross. DNA sequencing showed that the marker contained a 7 bp insertion/deletion polymorphism, as well as a single nucleotide polymorphism. A pair of sequence-specific primers was designed and successfully converted the size polymorphism into a simple polymerase chain reaction based co-dominant marker. This marker is closely linked to the Ku gene, as it co-segregates with the Ku phenotyping in a population consisting of 106 RILs.     

Segregation distortion in doubled haploid lines of barley (Hordeum vulgare L.) detected by simple sequence repeat (SSR) markers

A total of 147 simple sequence repeat (SSR) markers (including86 barley and 61 wheat microsatellite markers) were tested for their segregation in a doubled haploid (DH) and an F₂ population of barley. The DH population consisted of 71 doubled haploid lines, developed from F₁ plants of a cross between Tadmor and WI2291using isolated microspore culture technique. A genetic linkage map consisting of 43 microsatellite markers was constructed using the DH population. Particularly on chromosome 4H microsatellite markers showed distorted segregation ratios. Segregation of DH lines based on molecular markers were compared with segregation of 92 F₂ lines from the same cross. The proportion of loci deviating from the expected monogenic segregation ratios in the DH population was significantly higher (19/43loci, 44%) than in the F₂ population (7/43 loci, 16%). The deviation was biased towards the WI2291 parent alleles. In line with this observation, WI2291 was found to perform better than Tadmor in regenerating green plantlets with the isolated microspore-culture technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

DNA fingerprinting in soybean (Glycine max (L.) Merrill) with oligonucleotide probes for simple repetitive sequences

Summary Soybean DNA fingerprints were analyzed by digoxigenin-labeled oligonucleotide probes complementary to simple repetitive sequences. The clearest and most polymorphic patterns were obtained with (AAT)₆ as a probe, with which all 47 soybean cultivars tested could be distinguished. However, DNA fingerprints of individuals within cultivars showed the same pattern. Using (CT)₈, (GAA)₅ or (AAGG)₄ as probes, clear polymorphic patterns among cultivars and accessions in the subgenus Soja (Glycine max and Glycine soja) were not observed, while quite different patterns were found in accessions in the subgenus Glycine. The results suggest that G. max and G. soja are closer in their genome structure. DNA fingerprints of reciprocal crosses between cultivars and accessions in the subgenus Soja were similar, and contained bands of both parents. In an F₂ population from these crosses, such bands segregated in a Mendelian fashion.