Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyn...Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.展开更多
A bacterial artificial chromosome （BAC） library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based clon...A bacterial artificial chromosome （BAC） library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA Inserts Is needed. We have developed a BAC library of the restoring line 0-613-2R for Isolating the fertility restorer （Rf1） gene and genomic research in cotton （Gossypium hirsutum L.）. The BAC library contains 97 825 clones stored In 255 pieces of a 384-well mlcrotiter plate. Random samples of BACs digested with the Notl enzyme Indicated that the average Insert size Is approximately 130 kb, with a range of 80-275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hlndiii enzymes. Thus, the atabiiity of a single BAC clone can be sustained at iesat for 100 generations. Eight simple sequence repeat （SSR） markers flanking the Rf; gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.展开更多
A bacterial artificial chromosome （BAC） library was constructed for Gossypium hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert ...A bacterial artificial chromosome （BAC） library was constructed for Gossypium hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carried out by four-round poiymerase chain reactions using 23 simple sequence repeats （SSR） markers, three sequence-related amplified polymorphism markers and one pair of primers for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 （A12） and 26 （D12） according to the genetic map, these BAC clones are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with important agronomic traits.展开更多
A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for co...A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds.展开更多
Acyl-coenzyme A （Acyl-CoA） desaturases play a key role in the biosynthesis of female moth sex pheromones. Desaturase genes are encoded by a large multigene family, and they have been divided into five subgroups on t...Acyl-coenzyme A （Acyl-CoA） desaturases play a key role in the biosynthesis of female moth sex pheromones. Desaturase genes are encoded by a large multigene family, and they have been divided into five subgroups on the basis of biochemical functionality and phylogenetic affinity. In this study both copy numbers and transcriptional levels of desaturase genes in the European corn borer （ECB）, Ostrinia nubilalis, were investigated. The results from genome-wide screening of ECB bacterial artificial chromosome （BAC） library indicated there are many copies of some desaturase genes in the genome. An open reading frame （ORF） has been isolated for the novel desaturase gene ECB ezi-A11~ from ECB gland complementary DNA and its functionality has been analyzed by two yeast expression systems. No functional activities have been detected for it. The expression levels of the four desaturase genes both in the pheromone gland and fat body of ECB and Asian corn borer （ACB）, O. furnacalis, were determined by real-time polymerase chain reaction. In the ECB gland, All is the most abundant, although the amount of A14 is also considerable. In the ACB gland, A14 is the most abundant and is 100 times more abundant than all the other three combined. The results from the analysis of evolution of desaturase gene transcription in the ECB, ACB and other moths indicate that the pattern of A 11 gene transcription is significantly different from the transcriptional patterns of other desaturase genes and this difference is tied to the underlying nucleotide composition bias of the genome.展开更多
基金supported by the National High-Tech R&D Program in China (2013AA102603)the Natural Science Foundation of Shandong Province,China (ZR2014YL014)+3 种基金the Youth Scientific Research Foundation of Shandong Academy of Agricultural Sciences,China (2014QNZ03)the Taishan Scholars Program of Shandong Province,China (2016-2020)the National Natural Science Foundation of China (31101425)Prof. Alain Palloxin,French National Institute for Agricultural Research (INRA),for kindly providing the pepper genotype HDA149
文摘Pepper （Capsicum annuum. L.） is a widely cultivated vegetable crop worldwide and has the second largest planting area and the first largest vegetable output and value in China. Pepper root-knot nematode （Meloidogyne spp.） is one of the most serious pests of pepper, which caused huge losses every year. Previous studies showed that the Me3 gene is resistant to a wide range of Meloidogyne species, including M. arenaria, M. javanica, and M. incognita. HDA149, a double haploid pepper genotype, harboring the root-knot nematode resistance gene Me3, was used to construct bacterial artificial chro- mosome library （BAC） via the vector of CopyControFM pCC1 in this study. The library consists of 210 200 BAC clones and is equivalent to 5.3 pepper genomes. The average insert size is 95 kb, and most of them are 90-120 kb; but the empty clones are less than 3%. In order to screen the BAC library easily, 550 super pools with 384 BAC clones of each pool were further developed in this study. Specific primers from Me3 gene locus were used for BAC library screening, and more than 20 positive BAC clones were obtained. Then the selected positive BAC clones were analyzed by restriction enzyme digestion, BAC-end sequencing, marker development, and new positive BAC clones exploration, respectively. Finally, the contig with total length of about 300 kb linked to the Me3 locus was constructed based on chromosome walking strategy, which made a solid foundation for the cloning of the important root-knot nematode resistance gene Me3.
基金Supported by the National Natural Science Foundation of China （30270848）.
文摘A bacterial artificial chromosome （BAC） library containing a large genomlc DNA insert is an important tool for genome physical mapping, map-based cloning, and genome sequencing. To Isolate genes via a map-based cloning strategy and to perform physical mapping of the cotton genome, a high-quality BAC library containing large cotton DNA Inserts Is needed. We have developed a BAC library of the restoring line 0-613-2R for Isolating the fertility restorer （Rf1） gene and genomic research in cotton （Gossypium hirsutum L.）. The BAC library contains 97 825 clones stored In 255 pieces of a 384-well mlcrotiter plate. Random samples of BACs digested with the Notl enzyme Indicated that the average Insert size Is approximately 130 kb, with a range of 80-275 kb, and 95.7% of the BAC clones in the library have an average insert size larger than 100 kb. Based on a cotton genome size of 2 250 Mb, library coverage is 5.7 × haploid genome equivalents. Four clones were selected randomly from the library to determine the stability of the BAC clones. There were no different fingerprints for 0 and 100 generations of each clone digested with Notl and Hlndiii enzymes. Thus, the atabiiity of a single BAC clone can be sustained at iesat for 100 generations. Eight simple sequence repeat （SSR） markers flanking the Rf; gene were chosen to screen the BAC library by pool using PCR method and 25 positive clones were identified with 3.1 positive clones per SSR marker.
基金Supported in part by the National Natural Science Foundation of China(30730067)111 Project (B08025).
文摘A bacterial artificial chromosome （BAC） library was constructed for Gossypium hirsutum acc. TM-1, a genetic and genomic standard line for Upland cotton. The library consists of 147 456 clones with an average insert size of 122.8 kb ranging from 97 to 240 kb. About 96.0% of the clones have inserts over 100 kb. Therefore, this library represents theoretically 7.4 haploid genome equivalents based on an AD genome size of 2 425 Mb. Clones were stored in 384 384- well plates and arrayed into multiplex pools for rapid and reliable library screening. BAC screening was carried out by four-round poiymerase chain reactions using 23 simple sequence repeats （SSR） markers, three sequence-related amplified polymorphism markers and one pair of primers for a gene associated with fiber development to test the quality of the library. Correspondingly, in total 92 positive BAC clones were identified with an average four positive clones per SSR marker, ranging from one to eight hits. Additionally, since these SSR markers have been localized to chromosome 12 （A12） and 26 （D12） according to the genetic map, these BAC clones are expected to serve as seeds for the physical mapping of these two homologous chromosomes, sequentially map-based cloning of quantitative trait loci or genes associated with important agronomic traits.
文摘A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds.
文摘Acyl-coenzyme A （Acyl-CoA） desaturases play a key role in the biosynthesis of female moth sex pheromones. Desaturase genes are encoded by a large multigene family, and they have been divided into five subgroups on the basis of biochemical functionality and phylogenetic affinity. In this study both copy numbers and transcriptional levels of desaturase genes in the European corn borer （ECB）, Ostrinia nubilalis, were investigated. The results from genome-wide screening of ECB bacterial artificial chromosome （BAC） library indicated there are many copies of some desaturase genes in the genome. An open reading frame （ORF） has been isolated for the novel desaturase gene ECB ezi-A11~ from ECB gland complementary DNA and its functionality has been analyzed by two yeast expression systems. No functional activities have been detected for it. The expression levels of the four desaturase genes both in the pheromone gland and fat body of ECB and Asian corn borer （ACB）, O. furnacalis, were determined by real-time polymerase chain reaction. In the ECB gland, All is the most abundant, although the amount of A14 is also considerable. In the ACB gland, A14 is the most abundant and is 100 times more abundant than all the other three combined. The results from the analysis of evolution of desaturase gene transcription in the ECB, ACB and other moths indicate that the pattern of A 11 gene transcription is significantly different from the transcriptional patterns of other desaturase genes and this difference is tied to the underlying nucleotide composition bias of the genome.
