家蚕cDNA文库构建及大规模EST测序

EST(expressedsequencetag,表达序列标签 )测序分析技术广泛应用于基因功能和表达模式的分析研究。以家蚕品种“大造”为材料 ,采用非均一化的Oligo dT引物定向克隆技术构建了 13个组织的cDNA文库 ,进行了cDNA克隆 5′端测序 ,共获得 84 791万条EST序列 ,初步拼接得到 2 76 93个非重复序列 ,其中包括 10 4 33个Contigs,172 6 0个Singletons。

关于基因重组中OLC算法的改进研究

针对基因组组装问题,从数据预处理,利用KMP算法在O(m+n)的时间上快速确定某两个碱基片段的最大重复度,将读长序列依据Overlap图连成Contigs链以及Contigs N50的确定4个环节,改进现有的OLC拼接技术,并给出优化后的模型和算法,较好地解决了基因组组装问题.

对基因组拼接算法的深入分析和探讨

研究基因组拼接算法,进而更高效地实现全基因组拼接、获取生物体遗传信息,对于生命科学研究具有重要的意义。拼接之前对数据进行一定的预处理。拼接采用经典的DBG算法,分析了DBG算法的实现步骤以及需要注意的问题。根据read切割K-mer,然后采用位运算压缩存入Hash表。以有效的K-mer为顶点,相邻的K-mer之间连有向边,建立DBG图,然后在该图中寻找Euler路径。每一条Euler路径对应一条contig。最后进行参数组合实验,并将结果与专业软件CLC的结果进行对比。两者效率相近,说明了算法的高效性。

AutoSeqMan:batch assembly of contigs for Sanger sequences

With the wide application of DNA sequencing technology, DNA sequences are still increasingly generated through the Sanger sequencing platform. SeqMan （in the LaserGene package） is an excellent program with an easy-to-use graphical user interface （GUI） employed to assemble Sanger sequences into contigs. However, with increasing data size, larger sample sets and more sequenced loci make contig assemble complicated due to the considerable number of manual operations required to run SeqMan. Here, we present the ＇autoSeqMan＇ software program, which can automatedly assemble contigs using SeqMan scripting language. There are two main modules available, namely, ＇Classification＇ and ＇Assembly＇. Classification first undertakes preprocessing work, whereas Assembly generates a SeqMan script to consecutively assemble contigs for the classified files. Through comparison with manual operation, we showed that autoSeqMan saved substantial time in the preprocessing and assembly of Sanger sequences. We hope this tool will be useful for those with large sample sets to analyze, but with little programming experience. It is freely available at https：//github.com/ Sun-Yanbo/autoSeqMan.

Construction of Oryza sativa genome contigs by fingerprint strategy

We described the construction of BAC contigs of the genome of a indica variety of Oryza sativa, Guang Lu Ai 4.An entire representative (sixfold coverage of rice chromosomes) and genetically stable BAC library of rice genome constructed in this lab has been systematically analysed by restriction enzyme fragmentation and polyacrylamide gel electrophoresis. And all the images thus obtained were subject to image-processing, which consisted of preliminary location of bands, cooperative tracking of lanesby correlation of adjacent bands, a precise densitometric pass, alignment at the marker bands with the standard,optional interactive editing, and normalization of the accepted bands. The contigs were generated based on the Computer Software specially designed for genome map ping. The number of contigs with 600 kb in length on average was 464; of contigs with 1000 kb in length on average was 107; of contigs with 1500 kb in length on average was 23. Therefore, all the contigs we have obtained amounted up to 420 megabases in length. Considering the size of rice genome (430 megabased), the contigs generated in this lab have covered nearly 98% of the rice genome. We are now in the process of mapping the contigs to chromosomes.

高通量测序中拼接问题的研究现状

近年来,随着第二代测序技术的普及和第三代测序技术的逐步发展,高通量测序技术在实际研究中的应用越来越广泛。高速率、高性价比是其主要优点。相对于传统的桑格(Sanger)法测序来言,高通量测序得到的片段长度较为短小,故如何拼接得到完整的序列一直是炙手可热的研究方向。本文总结了现阶段高通量测序中拼接问题的研究结果,针对现在流行的各种算法进行了简单介绍。

Construction of the Primary Physical Map of Rice Chromosome 12

A primary physical map of rice chromosome 12 was constructed using marker-based chromosome landing and chromosome walking. A BAC library from IR64 was screened using 84 RFLP markers, 4 STS markers and 6 microsatellite markers on chromosome 12 by colony hybridization and polymerase chain reaction (PCR) amplification. A total of 59 contigs consisting of 419 BAC clones including 5 single-clones were physically aligned on rice chromosome 12 with the largest BAC contig covering 855 kb. The whole physical map had a size of ～16 Mb and covered about 52% of rice chromosome 12. This physical map will be certainly helpful for map-based gene cloning of agronomically and biological important genes and understanding the genome structure of the chromosome.

Bacterial artificial chromosome library construction of root-knot nematode resistant pepper genotype HDA149 and identification of clones linked to Me3 resistant locus

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.

The YAC Contig Construction for Human X Chromosome Xp21. 3—11. 3

The YAC contig construction has been done for the Human X chromosome short armXp21.3—p11.3, a region which contains several genetic disease gene loci and is of highlybiomedical importance. Using known probes(OTC, DXS166, DMDcDNA) and STS markersof this region, YAC screenings are performed by both YAC colony in situ hybridization andPCR methods. Totally 55 YACs are obtained from the YAC libraries of CEPH, ICRF andthe Institute. The size determination, the analysis of 26 pairs of microsatelite STS, the single copy probe hybridization and the Alu-PCR fingerprinting are performed for these YACs.The mapping of these YACs is performed, and finally, 6 YAC contigs in Xp21.3—11.3 are obtained, covering about 15 Mb. This work will greatly facilitate the positional cloning of disease genes or the genome sequencing in this important region.

Construction of a Contig Encompassing Ef(t) Gene Locus Using a Rice BAC Library

A major gene for heading date in rice, Ef(t), was mapped on the same position as one RFLP marker C1369 on chromosome 10, which was located between two RFLP markers C234 and G37 at 1.0 cM interval. Initially, these three RFLP markers were used to screen a rice BAC library and seven independent clones with the size ranged from 70kb to 180kb were identified. By the comparisons of Hind III restriction fragment in each clone, the relative location of these clones were determined and two primary contigs, contig C1369 and contig G37, were obtained. Chromosome walking was performed with one outmost BAC end of the primary BAC contig C1369, then two contigs were integrated into one. The resultant contig encompassing Ef(t) gene locus which consisted of 7 BAC clones was developed. It will facilitate the isolation of Ef(t) gene using map based cloning approach.

CONSTRUCTION OF A CONTIG MAP OF THE RICE GENOME

In August 1992, the State S&T Commission(SSTC) of China announced that China would launch a Rice Genome Program, a counterpart to the US Program of Human Genome, in a bid to decipher the genetic code of rice at the molecular level and then apply the obtained results to the cultivation of improved strains of the crop.

Fine mapping of a semidwarf gene sd-g in indica rice (Oryza sativa L.)

The semidwarf gene sd-g which has been used in indica rice breeding in southern China is a new one, non-allelic to sd-1. To map sd-g, an F2 population derived from the cross between Xinguiaishuangai and 02428 was con-structed. The sd-g was roughly mapped between two mi-crosatellite markers RM440 and RM163, with genetic dis-tances of 0.5 and 2.5 cM, respectively. Then nine new poly-morphic microsatellite markers were developed in this region. The sd-g was further mapped between two microsatellite markers SSR5-1 and SSR5-51, with genetic distances of 0.1 and 0.3 cM, respectively, while cosegregated with SSR418. A BAC contig was found to span the sd-g locus, the region be-ing delimited to 85 kb. This result was very useful for cloning of the sd-g gene.

Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project

As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome （BAC） by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones. Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa.

Genetic mapping of a new semi-dwarf gene,sd-t(t),in indica rice and estimating of the physical distance of the mapping region

Application and functional study of dwarf and semi-dwarf genes are of great importance to both crop breeding and molecular biology. A new semi-dwarf gene, sd-t(t), non-allelic to sd-1,had been identified in an indica rice variety, Aitaiyin 2. In this study the gene was genetically mapped by using an F2 population, which consisted of 474 individuals developed from a cross between Aitaiyin 2 and B30. The sd-t(t) gene was located between the RFLP markers R514 and R1408B with a distance of 1.1 cM to R514, and 4.5 cM to R1408B on chromosome 4. A physical contig covering the sd-t(t) mapping region was further constructed by screening a BAC library with R514 and R1408B as probes, and the physical distance between R514 and R1408B was estimated at approximately 147 kb. This result will facilitate map-based cloning of the sd-t(t) gene.