To perform rapid amplified fragment length polymorphism (AFLP) analysis and efficiently clone AFLP fragments with non-radioactive primers, we modified the current AFLP protocol. When the modified procedure was applied to the rice genome, 30 to 80 AFLP fragments (∼40 to 400 bp in length) could be detected in a single PCR reaction. To assess the suitability of the technique for the analysis of the rice genome, we first applied it to quantify genetic diversity in a sample of 55 predominantly Japanese modern and domestic cultivars. In this analysis, more than 75% of the Japanese rices were differentiated by the combination of six prinrer pairs. Then we used the technique to identify and clone AFLP fragments unique to the Japanese variety Kinuhikari compared to those found in the closely related cultivar Koshihikari. In this cloning, we obtained sequences derived from chromosomal segments of an Indica genome, which had introgressed into the Kinuhikari genome. These two studies demonstrated that the modified AFLP technique is suitable for the genome analysis in rice.
Genetic analysis of narrow- sense earliness in wheat was carried out, by using four segregating populations derived from the crosses between five cultivars with a large variation in narrow-sense earliness. Narrow-sense earliness measured at 15 °C, exhibited a large variation among five parental cultivars ranging from 29.8 to 51.2 days. It was earlier in F1 hybrids than in the mid-parent and later by a few days than in the early parent in all cross combinations, indicating the partial dominance of earliness. The polygenic nature of narrow-sense earliness was indicated by continuous variation in F2 populations, and heritability in a broad sense ranged from 0.90 to 0.99 among four F2 populations. In randomly selected progenies, the parent-offspring correlation coefficient between parents (F2 plants) and F3 lines proved to be statistically significant (r=0.608-0.742, P<0.01). These results indicated that narrow-sense earliness is a highly heritable character, inspite of its quantitative nature. Artificial selection in F2 populations resulted in significant improvement in early and late selections as compared with randomly selected F3 lines, indicating the effectiveness of artificial selection for optimizing narrow-sense earliness.
The components of gene effects for protein content were studied following a six parameter model of Hayman and three parameter model of Jinks and Jones, using the means of the six basic populations (P1, P2, F1, F2, BC1P1 and BC1P2) from four crosses derived from four diverse genotypes of long bean. The scaling tests indicated the presence of non-allelic interactions for pod protein and seed protein in two crosses. Additive effects are more important than dominance effects for pod protein. The interaction effects contributed more than the main genetic component for seed protein. Among the digenic epistatic interactions, dominance × dominance (1) contributed the most followed by additive × additive (i) and additive × dorninance (h) effects. Pedigree, bulk or single seed descent methods are suggested for developing elite populations. Duplicate epistasis was noticed for the two interacting crosses.
Regenerated plants were obtained by culturing leaf explants of Akame and Aome strains of horseradish (Armoracia rusticana Gaertn.) on MS agar-solidified medium supplemented with 1 mg/l NAA + 1 mg/l TDZ. Extracted DNA from leaves of mother plants was amplified using random and simple sequence repeat (SSR) primers. The SSR primers generated a larger nwnber of scorable bands than the random ones, although inter-and intra-strain polymorphism in the banding pattern was detected by both SSR and random primers at alnrost the same level. An SSR primer, (CAGA)4, was selected for detecting somaclonal variation among regenerants from leaf-derived calli. Somaclonal variation among the regenerants was observed in the PCR products as well as in the leaf shape and color. Seventy one percent of the regenerants exhibited different banding patterns from those of their mother plant, whereas variation in the leaf shape and color was observed in 5 % of the regenerants. These findings suggest that inter-SSR PCR is an efficient tool for detecting somaclonal variation in horseradish. The frequency of the regenerants exhibiting different banding patterns from those of their mother plants and the number of variant bands per regenerant were higher in the Akame than in the Aome strain, suggesting that somaclonal variation in horseradish was affected by the genotype. Fifty percent and 60% of the missing and newly acquired bands, respectively, for the same fragment size were common to the regenerants originating from different leaf-derived calli, suggesting that there were mutable sites in the horseradish genome during the tissue culture process.
Cabbage (Brassica oleracea L. var. capitata) has been reported to show a lower regenerative ability in microspore culture than broccoli (B. oleracea var. italica) or some other Brassica crops. Since the genotype is considered to be the most important factor affecting the embryogenesis of Brassica species, varietal differences in embryogenic and plant regenerative abilities from isolated microspores of cabbage accessions were examined to identify more responsive genotypes. Significant differences (<1%) were observed among the accessions in both the total number of embryos per 1 × l0ˆ5 microspores (embryo yield) and regenerative embryo yield. ‘Matsu-nanri', an F1 hybrid cultivar, showed the highest embryo yield and the highest efficiency of plant regeneration from microspores among the 38 accessions examined. The embryogenic ability of this cultivar was five to ten times higher than that previously reported for other cabbage cultivars. Although ‘M05', a breeding line, showed the second highest embryo yield, the plant regeneration ability was found to be very low. Enrbryos of‘M05' often developed abnormally and produced calli. No correlation was observed between the total embryo yield and the percentage of regenerative embryos among all the accessions studied and in the accessions without abnormal embryogenesis. These findings suggest that plant regeneration from the embryo is controlled by genetic factors other than those controlling the embryogenesis itself. Forty doubled haploid (DH) Iines derived from microspores of‘Matsunami' were examined for efficiency in embryo yield. Significant differences (< 1%) in total enrbryo yield were observed among these DH lines. Though most of the DH Iines showed a lower embryo yield than ‘Matsunami' itself, four lines showed a higher yield than ‘Matsunanri'. These four DH Iines are considered to be suitable for use in the haploid method of breeding in cabbage.
We analyzed 124 markers for linkage in 138 F2 plants derived from a cross between 2 inbred lines, cabbage and kale (both Brassica oleracea). Ninety-nine RAPDs, 21 RFLPs, 2 isozyme genes, and 2 morphological characters were used to construct a genetic map spanning 9 linkage groups covering 823.6 cM of the B. oleracea genome. The number of mapped loci for each linkage group ranged from 9 to 23. The average interval between 2 Ioci was 7.16 cM. The maximum distance between two loci in the linkage groups was 28.3 cM. Most of the markers segregated in a normal Mendelian ratio. However, biased segregation was observed at 10 loci. Most of the loci were clustered in a particular area of the genome, and showed an over-abundance of the male parent, indicating the presence of a lethal allele in the neighborhood of these markers or a quantitative selection advantage for one or nrore alleles. One QTL for resistance to clubroot disease was identified. QTL nrapping confirmed the identification of the probable location and effect of a QTL on linkage group 3. The QTL was separated from the nearest marker by a distance of 1.0 cM, and its R2 value was 0.300 indicating phenotypic variation.
Products of disease resistance genes cloned from several plant species share structural motifs such as a nucleotide binding site (NBS) and leucine-rich repeats. The presence of conserved domains in resistance genes anabled the cloning of nunrerous additional resistance gene analogs from rice using a polymerase chain reaction (PCR) with degenerate oligonucleotide primers to the NBS motifs. Ten resistance gene analogs (RGAs) were amplified from rice varieties Aichiasahi and Toride-1. The sequences coded open reading frames with additional sequences characteristic of NBSs. A hounology search through a database revealed significant similarities to known resistance genes. Southern blot analysis of eight rice varieties probed with RGAS demonstrated that most of them were single copy genes with diverse restriction fragment length polymorphisms (RFLPs). RFLPs Were observed between japonica and indica type rice in some RGAs and within bothjaponica and indica varieties. Some of their map positions were linked to known true resistance loci and some to field resistance (quantitative trait) Ioci. The transcription for RGAs could be detected by RT-PCR; however, their transcription levels were extremely low and varied with the variety.
Seeds of the common bean (Phaseolus vuggaris L.) contain proteinous α-amylase inhibitor (αAI) that inhibits insect and mamnalian α-amylase enzymes. Based on the specific inhibition of α-amylase enzylnes, common beans were classified into four different αAI types, αAI-1, 2, 3, and null type. This trait is controlled by a single locus, and alleles encoding the three αAI variants, αAI-1, 2, and -3, are codominant. Two different inhibitor proteins were purified from a common bean cultivar containing αAI-3. The two proteins are encoded at the same locus and designated αAI-3a and αAI-3b. αAI-3a inhibited the activity of porcine pancreatic α-amylase, while αAI-3b inhibited the activity of Mexican bean weevil larvae. N-terminal sequences and the specificity of αAI-3a and αAI-3b were very sinrilar to αAI-1 and αAI-2, respectively, indicating that the common bean classified als αAI-3 type contains both analogs of αAI-1 and a αAI-2.