A segment of the apospory-specific genomic region is highly microsyntenic not only between the apomicts Pennisetum squamulatum and buffelgrass, but also with a rice chromosome 11 centromeric-proximal genomic region

Bacterial artificial chromosome (BAC) clones from apomicts Pennisetum squamulatum and buffelgrass (Cenchrus ciliaris), isolated with the apospory-specific genomic region (ASGR) marker ugt197, were assembled into contigs that were extended by chromosome walking. Gene-like sequences from contigs were identified by shotgun sequencing and BLAST searches, and used to isolate orthologous rice contigs. Additional gene-like sequences in the apomicts' contigs were identified by bioinformatics using fully sequenced BACs from orthologous rice contigs as templates, as well as by interspecies, whole-contig cross-hybridizations. Hierarchical contig orthology was rapidly assessed by constructing detailed long-range contig molecular maps showing the distribution of gene-like sequences and markers, and searching for microsyntenic patterns of sequence identity and spatial distribution within and across species contigs. We found microsynteny between P. squamulatum and buffelgrass contigs. Importantly, this approach also enabled us to isolate from within the rice (Oryza sativa) genome contig Rice A, which shows the highest microsynteny and is most orthologous to the ugt197-containing C1C buffelgrass contig. Contig Rice A belongs to the rice genome database contig 77 (according to the current September 12, 2003, rice fingerprint contig build) that maps proximal to the chromosome 11 centromere, a feature that interestingly correlates with the mapping of ASGR-linked BACs proximal to the centromere or centromere-like sequences. Thus, relatedness between these two orthologous contigs is supported both by their molecular microstructure and by their centromeric-proximal location. Our discoveries promote the use of a microsynteny-based positional-cloning approach using the rice genome as a template to aid in constructing the ASGR toward the isolation of genes underlying apospory.     

金针菇基因组测序与组装策略分析

采用二代和三代测序技术分别对金针菇单核体菌株“6-3”进行测序,应用4种组装策略进行基因组的de novo组装,对比组装效果。基因组组装的参数方面,仅使用二代测序组装的效果最差,长度大于10kb的Contig全长只有24.6Mb,Contig N50只有23kb,组装率只有59.27%。采用三代组装二代校正的组装策略效果最好,长度大于10kb的Contig全长为38.3Mb,Contig N50为2.8Mb,组装率高达92.16%。保守单拷贝基因拼接效果方面,4种组装策略获得基因组序列与BUSCO数据库里的担子菌的保守单拷贝基因比对,基因完整性均大于94%。在组装准确性方面,经过PCR扩增、Sanger测序验证,三代组装二代校正的基因组序列完整并且连续,同时序列上碱基的SNP、InDel数量最少。综上所述,三代组装二代校正得到的基因组序列具有Contig N50值大、组装率高、碱基准确性高的特点,是食用菌基因组测序较为理想的方案。     

Construction of a BAC library and identification of Dmrt1 gene of the rice field eel, Monopterus albus

A bacterial artificial chromosome (BAC) library was constructed using nuclear DNA from the rice field eel (Monopterus albus). The BAC library consists of a total of 33,000 clones with an average insert size of 115 kb. Based on the rice field eel haploid genome size of 600 Mb, the BAC library is estimated to contain approximately 6.3 genome equivalents and represents 99.8% of the genome of the rice field eel. This is first BAC library constructed from this species. To estimate the possibility of isolating a specific clone, high-density colony hybridization-based library screening was performed using Dmrt1 cDNA of the rice field eel as a probe. Both library screening and PCR identification results revealed three positive BAC clones which were overlapped, and formed a contig covering the Dmrt1 gene of 195 kb. By sequence comparisons with the Dmrt1 cDNA and sequencing of first four intron-exon junctions, Dmrt1 gene of the rice field eel was predicted to contain four introns and five exons. The sizes of first and second intron are 1.5 and 2.6 kb, respectively, and the sizes of last two introns were predicted to be about 20 kb. The Dmrt1 gene structure was conserved in evolution. These results also indicate that the BAC library is a useful resource for BAC contig construction and molecular isolation of functional genes.     

Genomic analysis and marker development for the Tsn1 locus in wheat using bin-mapped ESTs and flanking BAC contigs

The wheat Tsn1 gene confers sensitivity to the host-selective toxin Ptr ToxA produced by the tan spot fungus (Pyrenophora tritici-repentis). The long-term goal of this research is to isolate Tsn1 using a positional cloning approach. Here, we evaluated 54 ESTs (expressed sequence tags) physically mapped to deletion bin 5BL 0.75–0.76, which is a gene-rich region containing Tsn1. Twenty-three EST loci were mapped as either PCR-based single-stranded conformational polymorphism or RFLP markers in a low-resolution wheat population. The genetic map corresponding to the 5BL 0.75–0.76 deletion bin spans 18.5 cM and contains 37 markers for a density of 2 markers/cM. The EST-based genetic map will be useful for tagging other genes, establishing colinearity with rice, and anchoring sequence ready BAC contigs of the 5BL 0.75–0.76 deletion bin. High-resolution mapping showed that EST-derived markers together with previously developed AFLP-derived markers delineated Tsn1 to a 0.8 cM interval. Flanking markers were used to screen the Langdon durum BAC library and contigs of 205 and 228 kb flanking Tsn1 were assembled, sequenced, and anchored to the genetic map. Recombination frequency averaged 760 kb/cM across the 228 kb contig, but no recombination was observed across the 205 kb contig resulting in an expected recombination frequency of more than 10 Mb/cM. Therefore, chromosome walking within the Tsn1 region may be difficult. However, the sequenced BACs allowed the identification of one microsatellite in each contig for which markers were developed and shown to be highly suitable for marker-assisted selection of Tsn1.     

通过染色体步查建立水稻G200座位的重叠群

分别以G200及其旁侧区的Y2587L、Y4073L和L688片段为探针筛选水稻基因组YAC文库,共得到4个阳性克隆,均与G200、Y2587L和Y4073L座位重叠,插入片段的大小为240～650kb。用反向PCR法分离YAC克隆插入片段的末端序列,并利用这些末端序列确定克隆的方向以及进行染色体步查,共筛选到7个YAC克隆,建立了一个8厘摩(cM)的G200 YAC重叠群。     

The centromere composition of multiple repetitive sequences on rice chromosome 5

The large-scale primary structure of the centromeric region of rice chromosome 5 was analyzed, the first example in a cereal species. The yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) contigs aligned on the centromere of rice chromosome 5 (CEN5) covered a distance of more than 670 kb. Strong suppression of genetic recombination, one of the features of a functional centromere, occurred along the contig region. The most remarkable feature of CEN5 is the composition of the multiple repetitive elements. Oryza-specific RCS2 short tandem repeats were clustered along less than 100 kb at one end of the contig. At least 15 copies of the conserved domain of the 1.9 kb RCE1 centromeric repeats, which are similar to the long terminal repeats (LTRs) of gypsy-type retrotransposon RIRE7, were dispersed mainly in 320 kb stretches next to RCS2 tandem clusters. Many copies of the LTR-like sequences of RIRE3 and RIRE8, another gypsy-type retrotransposon, were also found throughout the contig. On the other hand, the gagpol region was less conserved in the contig. These results indicate that the rice centromere is composed of multiple repetitive sequences with the RCS2 tandem cluster probably being situated as the core of a functional centromere of some hundreds of kilobases to megabases in length.     

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 lanes by correlation of adjacent bads.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 mapping.The number of contigs with 600 kb in length on average was 464.of contigs with 1000kb in length on average was 107; of contigs with 1500 kb in length on average was Construction of Oryza Sativa genome contigs.23.Therefor,all the contigs we have obtained ampunted 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.     

Construction of a bacterial artificial chromosome (BAC) library and identification of overlapping BAC clones with chromosome 4-specific RFLP markers in rice

 To facilitate construction of physical map of the rice genome, a bacterial artificial chromosome (BAC) library of IR64 genomic DNA was constructed. It consists of 18 432 clones and contains 3.28 rice genomic equivalents. The insert size ranged from 37 to 364 kb with an average of 107 kb. We used 31 RFLP markers on chromosome 4 to screen the library by colony hybridization. Sixty eight positive clones were identified with 2.2 positive clones per RFLP marker. The positive clones were analyzed to generate 29 contigs whose sizes ranged from 50 to 384 kb with an average of 145.6 kb. Chromosome walking was initiated for ten contigs linked to resistance genes. Thirty eight BAC clones were obtained and two contigs were integrated. Altogether, they covered 5.65 Mb (15.1%) of chromosome 4. These contigs may be used as landmarks for physical mapping of chromosome 4, and as starting points for chromosome walking towards the map-based cloning of disease resistance genes which were located nearby. Received: 15 November 1996 / Accepted: 24 January 1997     

Assembly and analysis of cosmid contigs in the CEA-gene family region of human chromosome 19.

The carcinoembryonic antigen (CEA)-like genes are members of a large gene family which is part of the immunoglobulin superfamily. The CEA family is divided into two major subgroups, the CEA-subgroup and the pregnancy-specific glycoprotein (PSG)-subgroup. In the course of an effort to develop a set of overlapping cosmids spanning human chromosome 19, we identified 245 cosmids in a human chromosome 19 cosmid library (6-7X redundant) by hybridization with an IgC-like domain fragment of the CEA gene. A fluorescence-based restriction enzyme digest fingerprinting strategy was used to assemble 212 probe-positive cosmids, along with 115 additional cosmids from a collection of approximately 8,000 randomly selected cosmids, into five contigs. Two of the contigs contain CEA-subgroup genes while the remaining three contigs contain PSG-subgroup genes. These five contigs range in size from 100 kb to over 300 kb and span an estimated 1 Mb. The CEA-like gene family was determined by fluorescence in situ hybridization to map in the q13.1-q13.2 region of human chromosome 19. Analysis of the two CEA-subgroup contigs provided verification of the contig assembly strategy and insight into the organization of 9 CEA-subgroup genes.     

Estimates of conserved microsynteny among the genomes of <Emphasis Type="Italic">Glycine max</Emphasis>, <Emphasis Type="Italic">Medicago truncatula</Emphasis> and <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A growing body of research indicates that microsynteny is common among dicot genomes. However, most studies focus on just one or a few genomic regions, so the extent of microsynteny across entire genomes remains poorly characterized. To estimate the level of microsynteny between Medicago truncatula (Mt) and Glycine max (soybean), and also among homoeologous segments of soybean, we used a hybridization strategy involving bacterial artificial chromosome (BAC) contigs. A Mt BAC library consisting of 30,720 clones was screened with a total of 187 soybean BAC subclones and restriction fragment length polymorphism (RFLP) probes. These probes came from 50 soybean contig groups, defined as one or more related BAC contigs anchored by the same low-copy probe. In addition, 92 whole soybean BAC clones were hybridized to filters of HindIII-digested Mt BAC DNA to identify additional cases of cross-hybridization after removal of those soybean BACs found to be repetitive in Mt. Microsynteny was inferred when at least two low-copy probes from a single soybean contig hybridized to the same Mt BAC or when a soybean BAC clone hybridized to three or more low-copy fragments from a single Mt BAC. Of the 50 soybean contig groups examined, 54% showed microsynteny to Mt. The degree of conservation among 37 groups of soybean contigs was also investigated. The results indicated substantial conservation among soybean contigs in the same group, with 86.5% of the groups showing at least some level of microsynteny. One contig group was examined in detail by a combination of physical mapping and comparative sequencing of homoeologous segments. A TBLASTX similarity search was performed between 1,085 soybean sequences on the 50 BAC contig groups and the entire Arabidopsis genome. Based on a criterion of sequence homologues <100 kb apart, each with an expected value of < or =1e-07, seven of the 50 soybean contig groups (14%) exhibited microsynteny with Arabidopsis.     

Genetic and physical mapping of a rice blast resistance locus,Pi-CO39(t), that corresponds to the avirulence gene AVR1-CO39 of Magnaporthe grisea

We have identified, genetically mapped and physically delineated the chromosomal location of a new rice blast resistance locus, designated Pi-CO39(t). This locus confers resistance to Magnaporthe grisea isolates carrying the AVR1-CO39 avirulence locus. The AVR1-CO39 locus is conserved in non-rice (cereals and grasses)-infecting isolates of M. grisea, making Pi-CO39(t) useful for engineering M. grisea resistance in rice and other cereals. The resistance in the rice line CO39 was inherited as a single dominant locus in segregating populations derived from F(2) and F(3) crosses between disease-resistant (CO39) and susceptible (51583) rice genotypes. Microsatellite, RFLP and resistance gene analog (RGA) markers were used to map the Pi-CO39(t) locus to a 1.2-cM interval between the probenazole-responsive ( RPR1) gene (0.2 cM) and RFLP marker S2712 (1.0 cM) on the short arm of rice chromosome 11. RFLP markers G320 and F5003, and resistance gene analogs RGA8, RGA38 and RGACO39 were tightly linked to the Pi-CO39(t) locus (no recombination detected in a sample of ~2400 gametes). A large-insert genomic library of CO39 was constructed in the binary plant transformation vector pCLD04541. A library screen using RGA8, RGA38 and probes derived from the ends of CO39 clones, as well as BAC end probes from the corresponding locus in the rice cv. Nipponbare, resulted in the assembly of three CO39 contigs of 180 kb, 110 kb and 145 kb linked to the Pi-CO39(t) locus. A 650-kb contig was also constructed representing the susceptible locus, pi-CO39(t), in the Nipponbare genome. The two genomes are highly divergent with respect to additions, deletions and translocations at the Pi-CO39(t) locus, as revealed by the presence or absence of mapping markers.     

One large-insert plant-transformation-competent BIBAC library and three BAC libraries of Japonica rice for genome research in rice and other grasses

We report one large-insert BIBAC library and three BAC libraries for japonica rice cv Nipponbare. The BIBAC library was constructed in the HindIII site of a plant-transformation-competent binary vector (pCLD04541) and the three BAC libraries were constructed in the BamHI, HindIII and EcoRI sites of a BAC vector (pECBAC1), respectively. Each library contains 23,040 clones, has an average insert size of 130 kb, 170 kb, 150 kb and 156 kb, and covers 6.7x, 8.7x, 7.7x and 8.0 x rice haploid genomes, respectively. The combined libraries contain 92,160 clones in total, covering 31.1 x rice haploid genomes. To demonstrate their utility, we screened the libraries with 55 DNA markers mapped to chromosome 8 of the rice genetic maps and analyzed a number of clones by the restriction fingerprinting and contig assembly method. The results indicate that the libraries completely cover the rice genome and, thus, are well-suited for genome research in rice and other gramineous crops. The BIBAC library represents the first plant-transformation-competent large-insert DNA library for rice, which will streamline map-based cloning, functional analysis of the rice genome sequence and molecular breeding in rice and other grass species. These libraries are being used in the development of a whole-genome, BAC/BIBAC-based, integrated physical, genetic and sequence map of rice and in the research of genome-wide comparative genomics of grass species.     

Genetic and physical mapping of xa13, a recessive bacterial blight resistance gene in rice

 The recessive gene, xa13, confers resistance to Philippine race 6 (PXO99) of the bacterial blight pathogen Xanthomonas oryzae pv oryzae. Fine genetic mapping and physical mapping were conducted as initial steps in an effort to isolate the gene. Using nine selected DNA markers and two F₂ populations of 132 and 230 plants, xa13 was fine-mapped to a genomic region <4 cM on the long arm of rice chromosome 8, flanked by two RFLP markers, RG136 and R2027. Four DNA markers, RG136, R2027, S14003, and G1149, in the target region were used to identify bacterial artificial chromosome (BAC) clones potentially harboring the xa13 locus from a rice BAC library. A total of 11 BACs were identified, forming four separate contigs including a single-clone contig, 29I3, associated with the RG136 STS marker, the S14003 contig consisting of four clones (44F8, 41O2, 12A16, and 12F20), the G1149 contig with two clones, 23D11 and 21H18, and the R2027 contig consisting of four overlapping clones, 42C23, 30B5, 6B7 and 21H14. Genetic mapping indicated that the xa13 locus was contained in the R2027 contig. Chromosomal walking on the R2027 contig resulted in two more clones, 33C7 and 14L3. DNA fingerprinting showed that the six clones of the R2027 contig were overlapping. Clone 44F8 hybridized with a single fragment from the clone 14L3, integrating the R2027 and S14003 contigs into a single contig consisting of ten BAC clones with a total size of approximately 330 kb. The physical presence of the xa13 locus in the contig was determined by mapping the ends of the BAC inserts generated by TAIL-PCR. In an F₂ population of 230 plants, the BAC-end markers 42C23R and 6B7F flanked the xa13 locus. The probes 21H14F and 21H14R derived from BAC clone 21H14 were found to flank xa13 at a distance of 0.5 cM on either side, using a second F₂ population of 132 plants. Thus, genetic mapping indicated that the contig and the 96-kb clone, 21H14, contained the xa13 locus. Received: 15 August 1998 / Accepted: 29 September 1998     

A BAC-based physical map of the channel catfish genome

Catfish is the major aquaculture species in the United States. To enhance its genome studies involving genetic linkage and comparative mapping, a bacterial artificial chromosome (BAC) contig-based physical map of the channel catfish (Ictalurus punctatus) genome was generated using four-color fluorescence-based fingerprints. Fingerprints of 34,580 BAC clones (5.6x genome coverage) were generated for the FPC assembly of the BAC contigs. A total of 3307 contigs were assembled using a cutoff value of 1x10(-20). Each contig contains an average of 9.25 clones with an average size of 292 kb. The combined contig size for all contigs was 0.965 Gb, approximately the genome size of the channel catfish. The reliability of the contig assembly was assessed by both hybridization of gene probes to BAC clones contained in the fingerprinted assembly and validation of randomly selected contigs using overgo probes designed from BAC end sequences. The presented physical map should greatly enhance genome research in the catfish, particularly aiding in the identification of genomic regions containing genes underlying important performance traits.     

A physical map with yeast artificial chromosome (YAC) clones covering 63% of the 12 rice chromosomes.

A new YAC (yeast artificial chromosome) physical map of the 12 rice chromosomes was constructed utilizing the latest molecular linkage map. The 1439 DNA markers on the rice genetic map selected a total of 1892 YACs from a YAC library. A total of 675 distinct YACs were assigned to specific chromosomal locations. In all chromosomes, 297 YAC contigs and 142 YAC islands were formed. The total physical length of these contigs and islands was estimated to 270 Mb which corresponds to approximately 63% of the entire rice genome (430 Mb). Because the physical length of each YAC contig has been measured, we could then estimate the physical distance between genetic markers more precisely than previously. In the course of constructing the new physical map, the DNA markers mapped at 0.0-cM intervals were ordered accurately and the presence of potentially duplicated regions among the chromosomes was detected. The physical map combined with the genetic map will form the basis for elucidation of the rice genome structure, map-based cloning of agronomically important genes, and genome sequencing.     

An approach to cloning of Pi-b rice blast resistance gene

A contig of clones from BAC rice genomic library encompassing blast resistance gene Pi-b was constructed. On an average eight clones (8 ± 2.6) were picked up by each marker, which was expected basing on the BAC library size (Nakamura et al. 1997). The 2.4 cM distance between flanking RFLP markers G 1234 and RZ 213 (Miyamoto et al. 1996) was spanned with 4 steps of contig including 25 clones. The physical distance of 370 kb between flanking markers corresponds to a small ratio of physical and genetical distances (155 kb/cM) due to a probable structure of the gene locus near the telomeric end of the chromosome. Markers cosegregating with blast resistance against Magnoporthe grisea were localized in a 2 kb restriction fragment. A new border marker was found on the telomeric side of the Pi-b gene, less than 10 kb from cosegregating markers. No clear marker for the centromeric side of the gene was found but the position of Pi-b rice blast resistant gene was narrowed to within at least 50 kb, which is to our knowledge the most precised estimation of the position of this gene.     

Construction of a BAC contig containing the xa5 locus in rice

 The recessive gene xa5 confers resistance to bacterial blight in rice. To generate a physical map of the xa5 locus, three RFLP markers RG556, RG207 and RZ390, closely linked to xa5, were used to screen a rice bacterial artificial chromosome (BAC) library. The identified overlapping BAC clones formed two small contigs which were extended to both sides by chromosome walking. The final physical map consisted of 14 BAC clones and covered 550 kb. Genetic analysis with an F₂ population showed that two RFLP markers 28N22R and 40F20R, derived from the BAC clones in the contig, flanked the xa5 locus. To further delimit the location of the xa5 locus, RFLP markers RG556 and RG207 were converted to sequence tagged sites and used to perform genetic analysis. The results indicated that the xa5 locus was most likely located between RG207 and RG556. Among the BAC clones in the contig, one clone, 44B4, hybridized to both RG207 and RG556. This suggests that BAC clone 44B4 carried the xa5 locus. Received: 12 January 1998 / Accepted: 27 May 1998     

A BAC clone-based physical map of ovine major histocompatibility complex

An ovine bacterial artificial chromosome (BAC) library containing 190,000 BAC clones was constructed and subsequently screened to construct a BAC-based physical map for the ovine major histocompatibility complex (MHC). Two hundred thirty-three BAC clones were selected by 84 overgo probes designed on human, mouse, and swine MHC sequence homologies. Ninety-four clones were ordered by DNA fingerprinting to form contigs I, II, and III that correspond to ovine MHC class I-class III, class IIa, and class IIb. The minimum tiling paths of contigs I, II, and III are 15, 4, and 4 BAC clones, spanning approximately 1900, 400, and 300 kb, respectively. The order and orientation of most BAC clones in each contig were confirmed by BAC-end sequencing. An open gap exists between class IIa and class III. This work helps to provide a foundation for detailed study of ovine MHC genes and of evolution of MHCs in mammals.     

Local de novo assembly of RAD paired-end contigs using short sequencing reads

Despite the power of massively parallel sequencing platforms, a drawback is the short length of the sequence reads produced. We demonstrate that short reads can be locally assembled into longer contigs using paired-end sequencing of restriction-site associated DNA (RAD-PE) fragments. We use this RAD-PE contig approach to identify single nucleotide polymorphisms (SNPs) and determine haplotype structure in threespine stickleback and to sequence E. coli and stickleback genomic DNA with overlapping contigs of several hundred nucleotides. We also demonstrate that adding a circularization step allows the local assembly of contigs up to 5 kilobases (kb) in length. The ease of assembly and accuracy of the individual contigs produced from each RAD site sequence suggests RAD-PE sequencing is a useful way to convert genome-wide short reads into individually-assembled sequences hundreds or thousands of nucleotides long.     

Fluorescence in situ hybridization mapping of human chromosome 19: mapping and verification of cosmid contigs formed by random restriction enzyme fingerprinting.

Automated restriction enzyme fingerprinting of 7900 cosmids from chromosome 19 and calculation of the likelihood of their overlap based on shared fragments have resulted in the assembly of 743 sets of overlapping cosmids (contigs). We have mapped 22% of the formed contigs (n = 165) and all of the contigs with minimal tiling paths exceeding 6 members (n = 50) to chromosomal bands by fluorescence in situ hybridization using DNA from at least one member cosmid. The estimated average size of the formed contigs is 60-70 kb. Thus, members of a correctly formed contig are expected to lie close to each other in metaphase and interphase chromatin. Therefore, we tested the contig assembly process by comparing the band assignment of two or more members selected from each of 97 contigs. Forty-two of these contigs were further characterized for valid assembly by determining the proximity of members in interphase chromatin. Using these tests, we surveyed a total of 431 joins counted along the minimal tiling path (280 in interphase as well as metaphase) and found 6 erroneous joins, one in each of 6 contigs (6% of tested).