【目的】利用桑科的EST数据库进行桑树EST-SSR引物开发,为丰富桑树SSR数据库提供参考。【方法】下载NCBI中桑科的EST数据库,进行序列处理及拼接后,用SSRIT搜索其中的SSR位点,以Primer Premier 5.0设计引物,选用5个桑树品种各5株样品进...【目的】利用桑科的EST数据库进行桑树EST-SSR引物开发,为丰富桑树SSR数据库提供参考。【方法】下载NCBI中桑科的EST数据库,进行序列处理及拼接后,用SSRIT搜索其中的SSR位点,以Primer Premier 5.0设计引物,选用5个桑树品种各5株样品进行多态性引物筛选。【结果】下载的EST拼接后得到2008条拼接片段(contig),共搜索到831个SSR位点,分布在799个contig中。二、三核苷酸重复基元是主导类型,分别占总SSR的66.06%和32.37%;TC/AG和CT/GA是二核苷酸优势重复基元,分别占二核苷酸总数的28.64%和25.87%。设计合成77对EST-SSR引物,其中46对能够扩增出有效条带;经多态性筛选,26对引物可扩增出多态性条带。【结论】利用桑属近缘属的EST序列进行桑树EST-SSR引物的开发是可行的;开发出的26对多态性引物可用于桑树遗传多样性分析和种质鉴定等。展开更多
Amplified consensus genetic marker （ACGM） is a PCR-based marker technique that uses primers designed within conserved regions of coding sequences. After a comparison of Cryptomeria japonica and Arabidopsis ESTs to s...Amplified consensus genetic marker （ACGM） is a PCR-based marker technique that uses primers designed within conserved regions of coding sequences. After a comparison of Cryptomeria japonica and Arabidopsis ESTs to search for conserved sequences, 237 single e-PCR products were obtained. We randomly selected 110 candidate ACGM markers to test. Of the 110 candidate ACGM markers tested, 106 yielded stable and clear PCR products in C. japonica. We then tested the utility of these 106 primer pairs in 10 species, representing 7 genera of Taxodi- aceae. The number of specific amplification primer pairs among those 10 species varied from 49 to 103 （or 46.2±97.2%）. The 106 primer pairs （ACGM loci） were high transferable to Cryptomeria fortunei Hooibrenk （97.2%） but were low in Metasequoia glyptostroboides （46.2%）. The number of PCR bands per primer pair ranged from 1.06 to 1.15, which means that most of the ACGM primers can obtain a single band within these 10 Taxodiaceae species. In summary, our study shows that ACGM is a technique applicable for marker development even in species with limited sequence data.展开更多
Lentil(Lens culinaris Medik.), a diploid(2n = 14) with a genome size greater than 4000 Mbp, is an important cool season food legume grown worldwide. The availability of genomic resources is limited in this crop specie...Lentil(Lens culinaris Medik.), a diploid(2n = 14) with a genome size greater than 4000 Mbp, is an important cool season food legume grown worldwide. The availability of genomic resources is limited in this crop species. The objective of this study was to develop polymorphic markers in lentil using publicly available curated expressed sequence tag information(ESTs). In this study, 9513 ESTs were downloaded from the National Center for Biotechnology Information(NCBI) database to develop unigene-based simple sequence repeat(SSR) markers. The ESTs were assembled into 4053 unigenes and then analyzed to identify 374 SSRs using the MISA microsatellite identification tool. Among the 374 SSRs, 26 compound SSRs were observed.Primer pairs for these SSRs were designed using Primer3 version 1.14. To classify the functional annotation of ESTs and EST–SSRs, BLASTx searches(using E-value 1 × 10-5) against the public UniP rot(http://www.uniprot.org/) and NCBI(http://www.ncbi.nlh.nih.gov/) databases were performed. Further functional annotation was performed using PLAZA(version3.0) comparative genomics and GO annotation was summarized using the Plant GO slim category. Among the synthesized 312 primers, 219 successfully amplified Lens DNA. A diverse panel of 24 Lens genotypes was used to identify polymorphic markers. A polymorphic set of 57 markers successfully discriminated the test genotypes. This set of polymorphic markers with functional annotation data could be used as molecular tools in lentil breeding.展开更多
Expressed sequence tags（ESTs） are generated from single-pass sequencing of randomly picked cDNA clones and can be used for development of simple sequence repeat（SSR） markers or microsatellites.However,EST database...Expressed sequence tags（ESTs） are generated from single-pass sequencing of randomly picked cDNA clones and can be used for development of simple sequence repeat（SSR） markers or microsatellites.However,EST databases have been developed for only a small number of species.This paper provides a case study of the utility of freely available birch EST resources for the development of markers necessary for the genetic analysis of Betula luminifera.Based on birch EST data,primers for 80 EST-SSR candidate loci were developed and tested in birch.Of these,59 EST-SSR loci yielded single,stable and clear PCR products.We then tested the utility of those 59 markers in B.luminifera.The results showed 28（47.6%） yielded stable and clear PCR products for at least one B.luminifera genotype.In addition,this study describes a rapid and inexpensive alternative for the development of SSRs in species with scarce available sequence data.展开更多
生物信息学中,发现、鉴别新基因是承上启下的一步,它既承接了过往如“基因组测序”的工作,又是未来“后基因时代”研究的基石．“基因电脑克隆”是利用计算手段发现、鉴别新基因的方法,SiClone软件实现了“基因电脑克隆”功能．本文对Si...生物信息学中,发现、鉴别新基因是承上启下的一步,它既承接了过往如“基因组测序”的工作,又是未来“后基因时代”研究的基石．“基因电脑克隆”是利用计算手段发现、鉴别新基因的方法,SiClone软件实现了“基因电脑克隆”功能．本文对SiClone软件操作的数据库提出并行处理方案,并详述了基于MPI(message passing interface)平台实现的并行优化版本PSiClone．根据已得到的EST数据库,展示了软件并行版PSiClone的运行性能,试验数据库EST序列条数仅仅是NCBI(The National Center for Biotechnology Information)dbEST庞大数据库的很小部分,这也暗示我们软件的并行工作对于大数据库的比较和运算将更有应用前景．展开更多
文摘【目的】利用桑科的EST数据库进行桑树EST-SSR引物开发,为丰富桑树SSR数据库提供参考。【方法】下载NCBI中桑科的EST数据库,进行序列处理及拼接后,用SSRIT搜索其中的SSR位点,以Primer Premier 5.0设计引物,选用5个桑树品种各5株样品进行多态性引物筛选。【结果】下载的EST拼接后得到2008条拼接片段(contig),共搜索到831个SSR位点,分布在799个contig中。二、三核苷酸重复基元是主导类型,分别占总SSR的66.06%和32.37%;TC/AG和CT/GA是二核苷酸优势重复基元,分别占二核苷酸总数的28.64%和25.87%。设计合成77对EST-SSR引物,其中46对能够扩增出有效条带;经多态性筛选,26对引物可扩增出多态性条带。【结论】利用桑属近缘属的EST序列进行桑树EST-SSR引物的开发是可行的;开发出的26对多态性引物可用于桑树遗传多样性分析和种质鉴定等。
基金funded by the Natural Science Foundation of China (30800879)project 2009R50035 supported by Forest Seedling Industry Innovative Team of Zhejiang province in China
文摘Amplified consensus genetic marker （ACGM） is a PCR-based marker technique that uses primers designed within conserved regions of coding sequences. After a comparison of Cryptomeria japonica and Arabidopsis ESTs to search for conserved sequences, 237 single e-PCR products were obtained. We randomly selected 110 candidate ACGM markers to test. Of the 110 candidate ACGM markers tested, 106 yielded stable and clear PCR products in C. japonica. We then tested the utility of these 106 primer pairs in 10 species, representing 7 genera of Taxodi- aceae. The number of specific amplification primer pairs among those 10 species varied from 49 to 103 （or 46.2±97.2%）. The 106 primer pairs （ACGM loci） were high transferable to Cryptomeria fortunei Hooibrenk （97.2%） but were low in Metasequoia glyptostroboides （46.2%）. The number of PCR bands per primer pair ranged from 1.06 to 1.15, which means that most of the ACGM primers can obtain a single band within these 10 Taxodiaceae species. In summary, our study shows that ACGM is a technique applicable for marker development even in species with limited sequence data.
基金Financial assistance from ICARDA, Morocco, in the form of a brief projectgrant support from the Northern Pulse Growers Association and the USA Dry Pea and Lentil Council are gratefully acknowledged
文摘Lentil(Lens culinaris Medik.), a diploid(2n = 14) with a genome size greater than 4000 Mbp, is an important cool season food legume grown worldwide. The availability of genomic resources is limited in this crop species. The objective of this study was to develop polymorphic markers in lentil using publicly available curated expressed sequence tag information(ESTs). In this study, 9513 ESTs were downloaded from the National Center for Biotechnology Information(NCBI) database to develop unigene-based simple sequence repeat(SSR) markers. The ESTs were assembled into 4053 unigenes and then analyzed to identify 374 SSRs using the MISA microsatellite identification tool. Among the 374 SSRs, 26 compound SSRs were observed.Primer pairs for these SSRs were designed using Primer3 version 1.14. To classify the functional annotation of ESTs and EST–SSRs, BLASTx searches(using E-value 1 × 10-5) against the public UniP rot(http://www.uniprot.org/) and NCBI(http://www.ncbi.nlh.nih.gov/) databases were performed. Further functional annotation was performed using PLAZA(version3.0) comparative genomics and GO annotation was summarized using the Plant GO slim category. Among the synthesized 312 primers, 219 successfully amplified Lens DNA. A diverse panel of 24 Lens genotypes was used to identify polymorphic markers. A polymorphic set of 57 markers successfully discriminated the test genotypes. This set of polymorphic markers with functional annotation data could be used as molecular tools in lentil breeding.
基金funded by the Provincial Natural Science Foundation of Zhejiang in China (Y307465)
文摘Expressed sequence tags（ESTs） are generated from single-pass sequencing of randomly picked cDNA clones and can be used for development of simple sequence repeat（SSR） markers or microsatellites.However,EST databases have been developed for only a small number of species.This paper provides a case study of the utility of freely available birch EST resources for the development of markers necessary for the genetic analysis of Betula luminifera.Based on birch EST data,primers for 80 EST-SSR candidate loci were developed and tested in birch.Of these,59 EST-SSR loci yielded single,stable and clear PCR products.We then tested the utility of those 59 markers in B.luminifera.The results showed 28（47.6%） yielded stable and clear PCR products for at least one B.luminifera genotype.In addition,this study describes a rapid and inexpensive alternative for the development of SSRs in species with scarce available sequence data.
文摘生物信息学中,发现、鉴别新基因是承上启下的一步,它既承接了过往如“基因组测序”的工作,又是未来“后基因时代”研究的基石．“基因电脑克隆”是利用计算手段发现、鉴别新基因的方法,SiClone软件实现了“基因电脑克隆”功能．本文对SiClone软件操作的数据库提出并行处理方案,并详述了基于MPI(message passing interface)平台实现的并行优化版本PSiClone．根据已得到的EST数据库,展示了软件并行版PSiClone的运行性能,试验数据库EST序列条数仅仅是NCBI(The National Center for Biotechnology Information)dbEST庞大数据库的很小部分,这也暗示我们软件的并行工作对于大数据库的比较和运算将更有应用前景．
