Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas ...Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production. In this study, on the basis of detection and verification of the genotypes of 22 rice varieties har- boring known blast resistance genes （Pi-ta and Pi-b） and blast susceptibility genes （pi-ta and pi-b）, two multiple PCR systems for these genes were established by us- ing the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta and pi-b. In addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resis- tance in rice breeding.展开更多
A total of 144 F2 individuals were obtained from the crossing between 1047 (susceptible) and A21 (resistant). Two RAPD markers were screened out in 200 random primers using BSA(Bulked Segregant Analysis). Two RAPD mar...A total of 144 F2 individuals were obtained from the crossing between 1047 (susceptible) and A21 (resistant). Two RAPD markers were screened out in 200 random primers using BSA(Bulked Segregant Analysis). Two RAPD markers, designated as AG13/2000 and U16/660, were 7.7 cM and 8.38 cM apart from the TuMV resistant gene, respectively. The two RAPD fragments were converted to SCAR markers. SCAR markers were confined in germplasm.展开更多
The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular ...The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2× 10^7 cells mL-1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS 188. The construct was transferred into the protoplasts through electroporation （1 kV, 15 μS）. The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL-t zeoein. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.展开更多
S. gesnerioides （Willd） Vatke is a major biological constraint to cowpea production in the dry savanna of sub-Saharan Africa. Yield losses caused by S. gesnerioides in these regions are estimated in millions of tons...S. gesnerioides （Willd） Vatke is a major biological constraint to cowpea production in the dry savanna of sub-Saharan Africa. Yield losses caused by S. gesnerioides in these regions are estimated in millions of tons annually, and prevalence of Striga soil infestation is steadily increasing. The availability of molecular markers tightly linked to S. gesnerioides resistance genes opens up the possibility of applying Marker-Assisted Selection （MAS） to cowpea and would fast track the process of developing resistance varieties to the parasite. In the present study, we report the use of Fast Technology for Analysis （FTA） also known as PlantSaver Cards （Whatman~ FTA）, developed by Flinder Technology associate to retrieve DNA from plant tissue for molecular analysis. A total of 100 F2 individual plants derived from two crosses were validated for SG3 resistance using two different SCAR markers （MahSe2 and C42B） linked to Striga race 3 （SG3） and 5 （SG5） resistance in other segregating populations. Genomic DNA was successfully recovered from leaf tissues of cowpea pressed onto FTA classic card and the DNA obtained from the FTA papers was found to be suitable for molecular analysis by PCR-based techniques. The marker efficiency of SCAR MahSe2 and C42B in detecting SG3 resistance was 98.5% and 93% respectively. This result revealed the utility of SCAR markers in cowpea breeding programme. Therefore, the application of MAS using FTA technology has the potential to increase efficiency of selection and for molecular characterization of cowpea lines for Striga resistance..展开更多
基金Supported by Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province[CX(12)1003]Science and Technology Support Program of Jiangsu Province(BE2013301)Special Fund for the Construction of Modern Agriculture Industry System of China(CARS-01-47)~~
文摘Rice blast is one of the important diseases in major rice producing areas of China. The main blast resistance genes Pi-ta and Pi-b showed broad-spectrum and durable resistance to rice blast in many rice growing areas of China, which have been widely utilized in rice breeding and commercial production. In this study, on the basis of detection and verification of the genotypes of 22 rice varieties har- boring known blast resistance genes （Pi-ta and Pi-b） and blast susceptibility genes （pi-ta and pi-b）, two multiple PCR systems for these genes were established by us- ing the functional markers of blast resistance genes Pi-ta and Pi-b as well as blast susceptibility genes pi-ta and pi-b, respectively. Specifically, multiple PCR system I could simultaneously detect blast resistance genes Pi-ta and Pi-b, while system II could detect simultaneously blast susceptibility genes pi-ta and pi-b. In addition, the genotypes of 336 high generation breeding materials were detected with these two multiple PCR systems. The results were highly consistent with those of conventional single mark detection, indicating that these two multiplex PCR systems were stable, reliable and time-saving. The established multiplex PCR systems may serve as a rapid and efficient method to identify and screen rice germplasm resources and can be applied in marker-assisted selection to polymerize multiple genes for blast resis- tance in rice breeding.
文摘A total of 144 F2 individuals were obtained from the crossing between 1047 (susceptible) and A21 (resistant). Two RAPD markers were screened out in 200 random primers using BSA(Bulked Segregant Analysis). Two RAPD markers, designated as AG13/2000 and U16/660, were 7.7 cM and 8.38 cM apart from the TuMV resistant gene, respectively. The two RAPD fragments were converted to SCAR markers. SCAR markers were confined in germplasm.
基金supported by the Provincial Natural Science Foundation of Shandong Province,China (2013ZRB14055)and the National Natural Science Foundation of China (Grant No.31372518)
文摘The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker. It can control the phleomycin resistance, and significantly increase the tolerance of hosts to zeocin. The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin. We selected ble as the selective marker for the genetic transformation of N. oculata. After the algal cells at a density of 2× 10^7 cells mL-1 was digested with 4% hemicellulase and 2% driselase for 1 h, the protoplasts accounted for 90% of the total. The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii, yielding a recombinant expression construct pMS 188. The construct was transferred into the protoplasts through electroporation （1 kV, 15 μS）. The transformed protoplasts were cultured in fresh f/2 liquid medium, and selected on solid f/2 medium supplemented with 500 ng mL-t zeoein. The PCR result proved that ble existed in the transformants. Three transformants had been cultured for at least 5 generations without losing ble. Southern blotting analysis showed that the ble has been integrated into the genome of N. oculata. The ble will serve as a new dominant selective marker in genetic engineering N. oculata.
文摘S. gesnerioides （Willd） Vatke is a major biological constraint to cowpea production in the dry savanna of sub-Saharan Africa. Yield losses caused by S. gesnerioides in these regions are estimated in millions of tons annually, and prevalence of Striga soil infestation is steadily increasing. The availability of molecular markers tightly linked to S. gesnerioides resistance genes opens up the possibility of applying Marker-Assisted Selection （MAS） to cowpea and would fast track the process of developing resistance varieties to the parasite. In the present study, we report the use of Fast Technology for Analysis （FTA） also known as PlantSaver Cards （Whatman~ FTA）, developed by Flinder Technology associate to retrieve DNA from plant tissue for molecular analysis. A total of 100 F2 individual plants derived from two crosses were validated for SG3 resistance using two different SCAR markers （MahSe2 and C42B） linked to Striga race 3 （SG3） and 5 （SG5） resistance in other segregating populations. Genomic DNA was successfully recovered from leaf tissues of cowpea pressed onto FTA classic card and the DNA obtained from the FTA papers was found to be suitable for molecular analysis by PCR-based techniques. The marker efficiency of SCAR MahSe2 and C42B in detecting SG3 resistance was 98.5% and 93% respectively. This result revealed the utility of SCAR markers in cowpea breeding programme. Therefore, the application of MAS using FTA technology has the potential to increase efficiency of selection and for molecular characterization of cowpea lines for Striga resistance..
