Random amplified polymorphic DNA (RAPD) markers were used to identify the male parents of the half-sib progeny from a single clone within a Japanese black pine (Pinus thunbergii Parl.) clonal seed orchard consisting of 16 pinewood-nematode [Bursaphelenchus xylophilus (Steiner et Buhrer) Nickle] resistant clones. Twenty-one primers, with 28 polymorphic and reproducible fragments, were selected from 320 primers examined. The genotypes of orchard clones at each fragment were fingerprinted by RAPD analyses of both diploid needles and haploid tissues of megagametophytes from each clone. We used all 28 RAPD markers to identify the male parents of the half-sib progeny of a single orchard clone, Tanabe-(t)54. By comparing the genotype of orchard clones with the phenotype in the seedling at each RAPD locus, we were able to identify the male parents of 82 out of 85 seedlings. Pollen contamination and natural selfing occurred at rates of only 2.4% and 1.2%, respectively. Mating with the clone deviated significantly from panmixia. More than half of the progeny of the clone were produced through fertilization by only two clones. Seven clones did not contribute at all to fertilization as the male parent. Deviations from panmixia have been caused by a difference in the distance between clones, variation in the amounts of pollen, and phenological synchrony between clones. The possibility and limitations of RAPD markers for paternity analysis in conifer seed orchards are discussed.
To assess the genetic diversity and genetic structure of East Asian wild radish (Raphanus sativus var. hortensis f. raphanistroides), 13 natural populations from Japan and Korea were analyzed for amplified fragment length polymorphism (AFLP). On the average, 77.4% of the AFLP markers generated by eight primer pairs were polymorphic. Both Japanese and Korean populations of wild radish showed a high within population variation (66.3% polymorphic markers, Shannon’s information index HO = 3.486, and genetic diversity HEP = 0.128). The majority of the genetic variation of wild radish (96.7%) was observed within populations. Although no appreciable local differentiation of AFLP markers was detected, AFLP markers were more effective than allozymes in classifying natural populations of East Asian wild radish. AFLP variation showed a very close genetic relationship between R. raphanistrum and R. sativus, particularly Kazakhstan R. sativus, confirming the assumption that R. raphanistrum might be involved in the origin of R. sativus.
The inheritance of citrus leaf shape was investigated by analyzing a diallel set of crosses of five citrus varieties/selections. Leaf contours were extracted by image processing, and described by elliptic Fourier descriptors. Mathematically independent shape characteristics were then identified by principal component analysis of the descriptors. The 1st, 2nd, 3rd and 4th components accounted for 51%, 20%, 10% and 6% of the total shape variation, and were good measures of the ratio of length to width, the position of the centroid, the curvature and the leaf wing size, respectively. Diallel analyses indicated that these components differed substantially in their mode of inheritance. The broad sense heritability was fairly high (>0.90) in the 2nd and 4th components and high (>0.70) in the 1st component, whereas the narrow sense heritability was high only in the 2nd component. There was no significant genetic effect in the 3rd component. The 1st, 2nd and 4th components showed over-, incomplete- and complete- dominance, respectively. The results indicate that elliptic Fourier descriptors can be successfully applied to the quantitative genetic analysis of citrus leaf shape.
Effects of the dwarfing gene sd1 originating from the Taiwanese rice variety ‘Dee-geo-woo-gen’ on yield, yield components and other traits were investigated using two isogenic lines, Td and Sd, and their respective parental varieties, ‘Taichung 65’ (T65) and ‘Shiokari’. At Kochi University, Td and T65 were grown at three fertilizer levels in 1998, and at one fertilizer level in 1995. Sd and ‘Shiokari’ were grown at one fertilizer level at Hokkaido University in 1996. Yields of Td and T65 increased as the fertilizer level was increased in 1998. The yield of Td was lower than that of T65 under all four growing conditions. Similarly, the yield of Sd was lower than that of ‘Shiokari’. In spikelet number per panicle, Td and Sd were significantly smaller than their respective parental varieties under all growing conditions. In panicle number per m2, differences between Td and T65 were nonsignificant under all growing conditions. In this trait, Sd was larger than ‘Shiokari’. In ripened-grain percentage, Td was higher than T65. In 1000 grain weight, on the other hand, Td was smaller than T65 under all growing conditions except that in 1995. In these two traits, however, Sd and ‘Shiokari’ were similar to each other. Td and T65 were similar to each other in length and width of spikelet. To sum up, the lower yields of Td and Sd were mainly due to their small numbers of spikelets per panicle in comparison with their respective parental varieties. In spikelet number per m2 and in sink size, Td and Sd were smaller than their respective parental varieties, indicating that sd1 decreases sink size. In LAI and leaf weight per m2 at the 80%-heading stage, Td and T65 were similar to each other, indicating that sd1 has almost no effect on the total amount of photosynthetic organ.
Protein composition of central region of white-core grains and non-white-core grains in the same rice cultivar for sake brewery was analyzed to detect the proteins specific to the white-core by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). We divided grains of two cultivars for sake brewery, Yamadanishiki and Gohyakumangoku into two groups, that is, grains with white-core and grains without white-core. We made a comparison between 2D-PAGE protein profiles of the central region of the white-core grains and that of non-white-core grains. The results showed that storage proteins, i.e., glutelin and prolamin, were contained in the white-core region as well as the central region of non-white-core grains. We found a protein spot in the 2D-PAGE profile specific to the white-core, which has an isoelectric point value of 4.5 and molecular weight size of 60 kDa. The protein spot was detected in the profiles of white-core region of two cultivars cultivated in 1998 and 2000, but not in those of non-white-core grains. The protein is considered to be associated with the expression of white-core in rice grains.
A classification based on RFLP markers and morphological trait index was conducted in four photoperiod- and/or thermo-sensitive genic male sterile rice (PGMS or TGMS) lines and 49 rice varieties of nine different ecotypic origins. The results indicated that the combination of the two classification methods enabled to classify clearly and similarly the materials into two groups, indica and japonica. The 49 varieties were test-crossed to the four PGMS or TGMS lines used as female parents. Grain yield of the test-crossed F1s of diverse ecotypes exhibited remarkable differences, especially in three parameters, i.e. number of combinations over a check, extent of standard heterosis and heterobeltiosis. In general, the first two showed higher values in indica-japonica crosses than in crosses between two different ecotypes within the same subspecies. For each female parent except for LS2S, there were corresponding optimum ecotypes of male parents. In terms of grain yield advantages and above-mentioned parameters, patterns of heterotic combinations were as follows: Peiai64S in combination with japonica varieties of northeastern China, restorers of japonica rice hybrids, or japonica varieties of North China; N422S in combination with indica varieties of China, indica varieties of Korea; 108S with American new varieties, japonica varieties of northeastern China, and indica varieties of China. LS2S showed a lower general combining ability.
Sixty Asian pear accessions from 6 Pyrus species were genetically identified by 9 SSR markers with a total of 133 putative alleles. Among them, 58 varieties could be successfully differentiated except for 2 pairs of synonymous or clonal varieties. All the SSR markers produced 1 or 2 discrete amplified fragments for all the diploid accessions, whereas a triploid variety showed 3 fragments with some SSRs. The number of putative alleles ranged from 7 to 20, with an average value of 14.8. The observed heterozygosity and the power of discrimination were 0.63 and 0.91, respectively. A phenogram based on the SSR genotypes was obtained, showing 3 major groups corresponding to the Japanese, Chinese and European groups. The SSR markers were highly polymorphic and could be utilized as a reliable tool for cultivar identification in Asian pears.
Interspecific hybrids between cultivated rice (Oryza sativa, 2n = 24, AA) and two wild species (O. minuta, 2n = 48, BBCC and O. officinalis, 2n = 24, CC) have been produced by cross pollination. The embryo development in these interspecific hybrids was observed to study the deterioration features in detail. All hybrids showed either abnormal embryo development with degenerating endosperm or embryo retardation itself. Abnormally developed embryos were observed by the end of 7 days after pollination and the percentage of abnormally developed embryos increased from 7 to 14 DAP. The embryos were excised at 7 to 14 DAP and cultured on MS medium supplemented with 1, 3 and 5 mg/l benzylaminopurine, 1 g/l casein hydrolysate, 0.8% agar and 3% sucrose. The optimal time for rescue turned out to be 11-14 DAP. There was a significant difference (P = 0.05) in the regeneration efficiency rate with rescue time but no significant difference with of culture medium in these cross combinations. The F1 hybrids were morphologically intermediate between their parents. All F1 hybrid plants thus obtained were completely sterile. Chromosome doubling was attempted by treating F1 hybrid tillers with colchicine to overcome their sterility. The morphology of the colchicine-treated F1 plants closely resembled the female parent and fertility.
In this paper, we characterized the Wx-mq gene for low amylose content in a rice variety, Milky Queen, at the molecular level. The Wx-mq gene was cloned by RT-PCR, and a nearly full-length cDNA sequence of the gene was determined. Sequence comparison between the Wx-mq gene and the wild type allele (Wx-b), cloned from cv. Koshihikari, revealed that two base changes existed within the coding region; a G to A base change at nucleotide position 497 and a T to C base change at nucleotide position 595. Each nucleotide substitution should generate a missense base change (an Arg-158 to His-158 change in exon4, and a Tyr-191 to His-191 change in exon5). However, it is not known which missense mutation is essential for the activity of the WX protein. To identify rice varieties and lines, which harbored the Wx-mq gene, PCR primers were designed at the gene level. These primers were able to amplify the Wx-mq specific 741 bp band in Milky Queen, and in other rice variety and lines, Milky Princess, Joiku 436 and Etsunan 190, all of which have the same pedigree as that of Milky Queen. On the other hand, no 741 bp band was amplified with the primers in Koshihikari which harbored the wild type allele (Wx-b), and the other low-amylose content variety and line, Snow Pearl and NM391, which do not have the pedigree. Thus, it is possible to detect the Wx-mq gene by PCR.
Non-viable hybrid seedlings were produced from a cross between Nicotiana tabacum (2n = 48; SSTT genome) and N. suaveolens (2n = 32). Our objective was to identify a N. tabacum chromosome that carried gene(s) inducing hybrid lethality in this cross. We crossed 10 monosomic lines (2n = 47; Haplo-M to Z, except for Haplo-p and Haplo-V) of N. tabacum to N. suaveolens and produced hybrids by test-tube pollination and ovule culture using synthetic media. We pollinated 178 placentae (13 to 62 per cross), cultured 5215 ovules (136 to 840 per cross), and produced a total of 85 seedlings. Of the 85 seedlings, 81 died before the cotyledonary and leaf-expansion stages of growth, while 4 from a cross to Haplo-Q produced viable hybrids. Three plants grew to maturity and flowered but one plant was infected by fungi and died at the young stage. Chromosome counting, RAPD and LSC analyses revealed that 3 viable plants were true F1 hybrids. These results suggested that the Q chromosome of the S genome harbored a gene or genes that induced non-viable seedlings from a cross between N. tabacum and N. suaveolens.
A rice semi-dwarf variety, IR8, known as “miracle rice” enabled dramatic increases rice production and its widespread adoption averted predicted food shortages in Asia during the 1960s to 1990s. This remarkable achievement was referred to as “green revolution”. The short stature of IR8 was derived from the semi-dwarf gene, sd1, and the sd1 gene contributed significantly to the rice “green revolution”. In this paper, we described the physiological, molecular genetic and biochemical characterization of the sd1 gene. The sd1 mutant contained lower gibberellin (GA) levels than wild-type plants but responded sensitively to exogenous GA. Cloning and sequence analyses revealed that the SD1 gene encoded a GA biosynthetic enzyme, GA20 oxidase. In all of the sd1 mutants tested, nucleotide deletions or substitutions were observed in the GA20 oxidase gene (GA20ox-2), which induced an internal stop codon or single amino acid substitutions, respectively. The sd1 plants, which the wild-type GA20ox-2 gene was introduced showed the normal height. A recombinant GA20ox-2 protein produced from the cDNA clone in E. coli catalyzed the conversion of GA53 to GA20. These results confirmed that SD1 encodes an active GA20 oxidase. The expression of GA20ox-2 was down-regulated by GA in a similar manner to that of some GA20oxs in other plants. The rice genome carried at least two GA 20-oxidase genes (GA20ox-1 and GA20ox-2) and SD1 corresponded to GA20ox-2, which is highly expressed in the leaves and flowers, whereas GA20ox-1 is preferentially expressed in the flowers. The reduced plant height associated with the sd1 alleles was due to the low amount of active GA in leaves, which was caused by a mutation of the GA20ox-2 gene. On the basis of these results, we discussed the importance of GA in the regulation of plant height in crop breeding.