Molecular phytogeny of conifers using RFLP analysis of PCR-amplified specific chloroplast genes

We investigated the molecular phylogeny of conifers using restriction endonuclease fragment length polymorphism of six polymerase chain reaction-amplified chloroplast genes — frxC, rbcL, psbA, psbD, trnK, and 16S. We detected 227 total site changes among species, representing 23, 26, 38, 48, 67, and 25 site changes in frxC, psbA, psbD, rbcL, trnK and 16S, respectively. The mean nucleotide substitution was 10.75% (SD 0.573) among species in five families. Forty maximally parsimonious trees were obtained using the Wagner parsimony method, and a 50% majority-rule consensus tree was obtained from them. Data analysis produced similar basic patterns when both the Wagner parsimony and the neighbor-joining methods were applied, and the main lineages were clearly separated. Taxaceae and Cephalotaxaceae species were used as the out-groups when applying Wagner parsimony methods. With the Wagner method, the consistency index was 0.510, the retention index was 0.879, and tree length was 435 steps. Our results indicated that Cupressaceae and Taxodiaceae are closely related families and that Sciadopitys verticillata is the basal lineage of Cupressaceae and Taxodiaceae. The neighbor-joining tree is similar to the 50% majority-rule consensus of the 40 Wagner parsimony trees except for the position of Keteleeria daversifolia, the Picea and Cedrus group, and the divergence within Cupressaceae.     

Conversion of a RAPD-generated PCR product,containing a novel dispersed repetitive element,into a fast and robust assay for the presence of rye chromatin in wheat

Bulk segregant analysis was used to obtain a random amplified polymorphic DNA (RAPD) marker specific for the rye chromosome arm of the 1BL.1RS translocation, which is common in many high-yielding bread wheat varieties. The RAPD-generated band was cloned and end-sequenced to allow the construction of a pair of oligonucleotide primers that PCR-amplify a DNA sequence only in the presence of rye chromatin. The amplified sequence shares a low level of homology to wheat and barley, as judged by the low strength of hybridization of the sequence to restriction digests of genomic DNA. Genetic analysis showed that the amplified sequence was present on every rye chromosome and not restricted to either the proximal or distal part of the 1RS arm. In situ hybridization studies using the amplified product as probe also showed that the sequence was dispersed throughout the rye genome, but that the copy number was greatly reduced, or the sequence was absent at both the centromere and the major sites of heterochromatin (telomere and nucleolar organizing region). The probe, using both Southern blot and in situ hybridization analyses, hybridized at a low level to wheat chromosomes, and no hybridizing restriction fragments could be located to individual wheat chromosomes from the restriction fragment length polymorphism (RFLP) profiles of wheat aneuploids. The disomic addition lines of rye chromosomes to wheat shared a similar RFLP profile to one another. The amplified sequence does not contain the RIS 1 sequence and therefore represents an as yet undescribed dispersed repetitive sequence. The specificity of the amplification primers is such that they will provide a useful tool for the rapid detection of rye chromatin in a wheat background. Additionally, the relatively low level of cross-hybridization to wheat chromatin should allow the sequence to be used to analyse the organization of rye euchromatin in interphase nuclei of wheat lines carrying chromosomes, chromosome segments or whole genomes derived from rye.     

Identification of the most represented repeated motifs in Arabidopsis thaliana microsatellite loci

The major simple sequence repeats present in the Arabidopsis genome were identified by Southern hybridizations with 49 oligonucleotide probes matching all the possible combinations of motifs up to 4 nucleotides long. The method used allowed us to perform all the hybridizations under the same temperature conditions. A good correlation was observed with the data obtained from database analysis, indicating that the method can be useful for identifying the major classes of microsatellite loci in species for which few or no sequence data are available. AG/CT, AAG/CTT, ATG/CAT and GTG/CAC are the major motifs present in the Arabidopsis genome that can be used as convenient probes to isolate microsatellite loci by screening libraries. AAG/CTT is the more frequent of these motifs, and its relative frequency in Arabidopsis is much higher than averagely found in the plant kingdom. About 8% of the cDNA clones from an immature silique library contains AG/CT, AAG/CTT or ATG/CAT microsatellite loci. Several microsatellite loci were isolated by screening genomic and cDNA libraries. Twenty-six tri-nucleotide loci were PCR amplified from four different ecotypes, and polymorphism was observed for 12 of them; 10 loci showing two alleles and 2 loci showing three alleles.     

Combined genetic and physiological analysis of a locus contributing to quantitative variation

The natural variation of many traits is controlled by multiple genes, individually referred to as quantitative trait loci (QTL), that interact with the environment to determine the ultimate phenotype of any individual. A QTL has yet to be described molecularly, in part because strategies to systematically identify them are underdeveloped and because the subtle nature of QTLs prevents the application of standard methods of gene identification. Therefore, it will be necessary to develop a systematic approach(es) for the identification of QTLs based upon the numerous positional data now being accumulated through molecular marker analyses. We have characterized a QTL by the following three-step approach: (1) identification of a QTL in complex populations, (2) isolation and genetic mapping of this QTL in near-isogenic lines, and (3) identification of a candidate gene using map position and physiological criteria. Using this approach we have characterized a plant height QTL in maize that maps to chromosome 9 near the centromere. Both map position and physiological criteria suggest the gibberillin biosynthesis gene dwarf3 as a candidate gene for this QTL.     

Localization of genes for bacterial canker resistance in Lycopersicon peruvianum using RFLPs

A backcross population of the L. peruvianum accession LA 2157, which is resistant to bacterial canker caused by Clavibacter michiganensis ssp. michiganensis, with the susceptible L. peruvianum accession LA 2172 was evaluated for the segregation of C. michiganenis resistance and of RFLP markers in order to map the loci involved in this resistance. The development of symptoms of the disease was scored using an ordinal scale. The mapping of the disease resistance was hampered by distorted segregation ratios of a large number of markers and unexpected quantitative inheritance of the resistance. By means of the Kruskal-Wallis rank-sum test, five regions on chromosomes 1, 6, 7, 8 and 10 were identified that may be involved in C. michiganensis resistance.     

Differential accumulation of the transcripts of 22 novel protein kinase genes in Arabidopsis thaliana

22 novel members of the Arabidopsis thaliana protein kinase family (AKs) were identified by using degenerate oligonucleotide primers directed to highly conserved amino acid sequences of the protein kinase (PK) catalytic domain. Of these 22 genes, 16 turned out to carry intron sequences. Homologies of AK sequences were detected to S-locus receptor protein kinases (SRKs) from Brassica spp., to SRK-like PKs from maize and A. thaliana and to several other receptor PKs from A. thaliana. Sequence similarity was also detected to Ca²⁺-dependent PKs (CDPKs) from rape and soybean, to SNF1 and to CDC2 homologues. The genomic organization and the accumulation of the mRNAs from these 22 AK genes were investigated.     

Repair mechanisms of UV-induced DNA damage in soybean chloroplasts

In order to better understand the biochemical mechanisms of DNA metabolism in chloroplasts, repair of UV induced plastome damage in vivo was determined by exposure of soybean suspension cells to UV light and subsequent quantitation of the damage remaining in nuclear and chloroplast encoded genes with time by quantitative polymerase chain reaction (QPCR). The kinetics of damage rapir in the nuclear rbcS gene suggest that photoreactivation and dark mechanisms are active, while for the plastome encoded psbA gene only a light-dependent repair process was detected which is considerably slower than would be expected for photolyase-mediated photoreactivation.     

小鼠造血干细胞增殖和分化的分子生物学证据

本文采用Ｙ染色体特异的性别决定基因（Ｓｒｙ）作为新的细胞遗传标志,通过ＰＣＲ技术来追踪观察造血干细胞的增殖与分化性能。该方法具有简便、灵敏和特异等优点。雌性受体小鼠输注雄鼠骨髓细胞和１３天脾结节（ＣＦＵ－Ｓ１３）细胞后,Ｓｒｙ ＰＣＲ测试受体小鼠的ＣＦＵ－Ｓ结果表明,它们均为供体来源的ＸＹ细胞。用Ｓｒｙ ＰＣＲ骨髓细胞和骨髓中脾结节生成细胞（ＣＰＵ－Ｓ）的长期重建造血能力,结果表明,在存活雌性小鼠