RESUMEN
Previous studies of gene-flow in agriculture have used a range of physical and biochemical markers, including transgenes. However, physical and biochemical markers are not available for all commercial varieties, and transgenes are difficult to use when trying to estimate gene flow in the field where the use of transgenes is often restricted. Here, we demonstrate the use of simple sequence repeat microsatellite markers (SSRs) to study gene flow in maize. Developing the first quantitative analysis of pooled SSR samples resulted in a high sampling efficiency which minimised the use of resources and greatly enhanced the possibility of hybrid detection. We were able to quantitatively distinguish hybrids in pools of ten samples from non-hybrid parental lines in all of the 24 pair-wise combinations of commercial varieties tested. The technique was used to determine gene flow in field studies, from which a simple model describing gene flow in maize was developed.
Asunto(s)
Cruzamiento/métodos , Flujo Génico/genética , Técnicas Genéticas , Repeticiones de Microsatélite/genética , Zea mays/crecimiento & desarrollo , Zea mays/genética , Análisis de Varianza , Calibración , Genética de Población , Modelos Genéticos , Polen/genéticaRESUMEN
Chlorella protothecoides cultures grown in a nitrogen-free bleaching medium (BM-N) in the dark rapidly degraded chlorophyll (Chl) to red catabolites. This degreening process was investigated under different growth conditions. Supply of nitrogen to the culture medium (BM+N) inhibited bleaching and the synthesis of catabolites as did the addition to BM-N of cycloheximide or a chelator, 2,2'-bipyridyl. In contrast, chloramphenicol or the protease inhibitor E64 had no effect. During bleaching, Chl breakdown was accompanied by the degradation of cellular proteins such as light-harvesting complex II, cytochrome f and protochlorophyllide oxido-reductase. During growth in BM-N, protease activity increased and proteins immunologically detectable with an antibody against a senescence-enhanced cysteine protease accumulated. cDNAs from BM-N and BM+N cells were used for differential and subtractive screening to isolate cDNAs representing genes with degreening-enhanced expression (dee) in C. protothecoides. Several different dees were identified with different patterns of expression during Chlorella growth but which were all expressed at higher levels during bleaching. Among these, dee4 was most abundant and its expression was exclusive in BM-N cultures. Analysis of the dee sequences showed that they encode different proteins including a novel amino acid carrier (dee4), ferritin, ATP-dependent citrate lyase, a Ca2+-binding protein, MO25, ubiquinone-cytochrome c-reductase and several new proteins.
Asunto(s)
Chlorella/genética , Clorofila/metabolismo , Proteínas Algáceas/efectos de los fármacos , Proteínas Algáceas/genética , Proteínas Algáceas/metabolismo , Secuencia de Aminoácidos , Chlorella/efectos de los fármacos , Chlorella/metabolismo , Clonación Molecular , Medios de Cultivo/química , Medios de Cultivo/farmacología , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Regulación de la Expresión Génica/efectos de los fármacos , Datos de Secuencia Molecular , Nitrógeno/farmacología , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
We combined single pollen typing with laser-mediated manipulation. After drilling a hole in the wall of a pollen grain from a dioecious plant (Silene latifolia) with a UV-laser microbeam, the single pollen grain was recovered directly in the cap of a PCR tube, using a non-contact method called laser pressure catapulting. The entire genome of the single pollen grain was then amplified with improved primer-extension-preamplification PCR (I-PEP PCR). Nested PCR with sequence tagged site (STS)-specific primers was used to analyze several loci in the haploid genome. The single copy gene MROS1 was detected in most of the single pollen grains analyzed. Bgl10, which is localized on the Y chromosome, was detected in approximately half of the pollen grains. MROS3 is reported to be localized on the X chromosome. Using inverse PCR, we isolated two genomic clones of MROS3: MROS3A and MROS3B. The single pollen analysis using nested PCR showed that MROS3A and MROS3B are derived from different loci that are not located on the X chromosome. Single pollen typing not only reveals sex chromosome-linkage within the haploid genome, but can also discriminate between alleles and different loci. This method should also be useful for measuring recombination frequencies without genetic crossover analysis.