共查询到20条相似文献,搜索用时 62 毫秒
1.
2.
BAC contig development by fingerprint analysis in soybean. 总被引:11,自引:0,他引:11
We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4-5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication. 相似文献
3.
Fine genetic mapping and BAC contig development for the citrus tristeza virus resistance gene locus in Poncirus trifoliata (Raf.) 总被引:1,自引:0,他引:1
Z. Deng S. Huang P. Ling C. Yu Q. Tao C. Chen M. Wendell H.-B. Zhang F. Gmitter Jr 《Molecular genetics and genomics : MGG》2001,265(4):739-747
A map-based cloning strategy has been employed to isolate Ctv, a single dominant gene from Poncirus trifoliata that confers resistance to citrus tristeza virus (CTV), the most important viral pathogen of citrus. Cloning of this gene will allow development of commercially acceptable, virus-resistant cultivars. A high-resolution genetic linkage map of the Ctv locus region was developed using a backcross population of 678 individuals. Three DNA markers that were closely linked or co-segregated with Ctv were identified and used to screen BAC libraries derived from an intergeneric hybrid of Poncirus and Citrus. Through chromosome walking and landing, two BAC contigs were developed: one encompassing the Ctv region, and the other spanning the allelic susceptibility gene region. The resistance gene contig consists of 20 BAC clones and is approximately 550 kb in length; the susceptibility gene contig consists of 16 BAC clones and extends about 450 kb. The Ctv locus was localized within a genomic region of approximately 180 kb by genetic mapping of BAC insert ends. The BAC contigs were integrated with the genetic map; variation in the ratio of genetic to physical distance was observed in the vicinity of Ctv. Southern hybridization data indicated that a few copies of NBS-LRR class sequences are distributed at or around the Ctv locus. Efforts are being made to assign the Ctv locus to a smaller genomic fragment whose function can be confirmed through genetic complementation of a CTV susceptible phenotype. These results indicate that map-based gene cloning is feasible in a woody perennial. 相似文献
4.
K.M. Boycott R.J. Zahorchak C.G. Summer N.P. Boycott V. Kotak C.G. Russell N.T. Bech-Hansen 《Genomics》1998,48(3):369
To generate sequence-ready templates for the gene-rich Xp11.23 region, we have constructed a 1.5-Mb bacterial artificial chromosome (BAC) contig spanning the interval between the DNA markers OATL1 and DXS255. The contig includes 28 BACs, ranging in size from 58 to 285 kb with an average size of 135 kb, which provide 2.5-fold coverage of the region. The BAC contig was constructed based entirely on the content of 40 DNA markers from a previously established YAC contig and 11 new markers developed from BAC-end DNA sequences, 4 of which were required to close gaps in the map. There was no evidence of rearrangement, instability, or chimerism in any of the BAC clones. The BAC cloning system appears to provide robust and total physical coverage of this gene-rich region with clones that are suitable for DNA sequencing. 相似文献
5.
AFLP-derived SCARs facilitate construction of a 1.1 Mb sequence-ready map of a region that spans the Vf locus in the apple genome 总被引:2,自引:0,他引:2
The availability of high-density anchored markers is a prerequisite for reliable construction of a deep coverage BAC contig, which leads to creation of a sequence-ready map in the target chromosomal region. Unfortunately, such markers are not available for most plant species, including woody perennial plants. Here, we report on an efficient approach to build a megabase-size sequence-ready map in the apple genome for the Vf region containing apple scab resistance gene(s) by targeting AFLP-derived SCAR markers to this specific genomic region. A total of 11 AFLP-derived SCAR markers, previously tagged to the Vf locus, along with three other Vf-linked SCAR markers have been used to screen two apple genome BAC libraries. A single BAC contig which spans the Vf region at a physical distance of approximately 1,100 kb has been constructed by assembling the recovered BAC clones, followed by closure of inter-contig gaps. The contig is 4 ×deep, and provides a minimal tiling path of 16 contiguous and overlapping BAC clones, thus generating a sequence-ready map. Within the Vf region, duplication events have occurred frequently, and the Vf locus is restricted to the ca. 290 kb region covered by a minimum of three overlapping BAC clones. 相似文献
6.
Genetic and physical mapping of xa13, a recessive bacterial blight resistance gene in rice 总被引:6,自引:0,他引:6
A. C. Sanchez L. L. Ilag D. Yang D. S. Brar F. Ausubel G. S. Khush M. Yano T. Sasaki Z. Li N. Huang 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1999,98(6-7):1022-1028
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 相似文献
7.
Game L Close J Stephens P Mitchell J Best S Rochette J Louis-dit-Sully C Riley J See CG Sanseau P Kearney L Bethel G Humphray S Dunham I Mungall A Thein SL 《Genomics》2000,64(3):264-276
Genetic studies have previously assigned a quantitative trait locus (QTL) for hemoglobin F and F cells to a region of approximately 4 Mb between the markers D6S408 and D6S292 on chromosome 6q23. An initial yeast artificial chromosome contig of 13 clones spanning this region was generated. Further linkage analysis of an extended kindred refined the candidate interval to 1-2 cM, and key recombination events now place the QTL within a region of <800 kb. We describe a high-resolution bacterial clone contig spanning 3 Mb covering this critical region. The map consists of 223 bacterial artificial chromosome (BAC) and 100 P1 artificial chromosome (PAC) clones ordered by sequence-tagged site (STS) content and restriction fragment fingerprinting with a minimum tiling path of 22 BACs and 1 PAC. A total of 194 STSs map to this interval of 3 Mb, giving an average marker resolution of approximately one per 15 kb. About half of the markers were novel and were isolated in the present study, including three CA repeats and 13 single nucleotide polymorphisms. Altogether 24 expressed sequence tags, 6 of which are unique genes, have been mapped to the contig. 相似文献
8.
9.
Hohmann U Jacobs G Telgmann A Gaafar RM Alam S Jung C 《Molecular genetics and genomics : MGG》2003,269(1):126-136
In sugar beet (Beta vulgaris L.), early bolting is caused by a single dominant gene, designated B. Twenty AFLP markers selected from a 7.8-cM segment of the B region on chromosome 2 were used to screen a YAC library, and a first-generation physical map including the B gene, made up of 11 YACs, was established. Because the genome coverage of the YAC library was low, a BAC library was constructed in the vector pBeloBAC11. This library consists of 57,600 clones with an average insert size of 116 kb, corresponding to 8.8 genome equivalents. Screening of the BAC library with chloroplast and mitochondrial DNA probes indicated that less than 0.1% of the clones contained organelle-derived DNA. To fill the gaps in the physical map around the B gene, the BAC library was screened with four AFLP markers and 10 YAC-derived probes. In total, 54 different BACs were identified. Overlaps between BACs were detected by using BAC termini amplified by PCR as probes, and by RFLP fingerprinting. In this way, a minimal tiling path of the central 4.6-cM region was constructed, which consists of 14 BACs. The B locus was localized to a 360-kb contig, a size which makes positional cloning of the gene feasible. 相似文献
10.
Suppressed recombination around the MXC3 locus, a major gene for resistance to poplar leaf rust 总被引:8,自引:0,他引:8
B. Stirling G. Newcombe J. Vrebalov I. Bosdet H.D. Bradshaw Jr. 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2001,103(8):1129-1137
A positional cloning strategy is being implemented in Populus for the isolation of the dominant MXC3 allele, which confers resistance to poplar leaf rust caused by Melampsora×columbiana (pathotype 3). AFLP markers were used to saturate the chromosomal region around the MXC3 locus in a large (n=1,902) Populus trichocarpa×P. deltoides (T×D) mapping pedigree segregating 1:1 for rust resistance and susceptibility. The high-resolution linkage map developed
around the MXC3 locus contains 19 AFLP markers and spans a genetic distance of 2.73 cM. Of the 19 AFLP markers, seven were found to co-segregate
with the locus. One co-segregating AFLP marker, CCG.GCT_01, was converted to an STS marker (BVS1) and used to identify a physical contig of overlapping BAC clones from the MXC3 region. Genetic and physical mapping of markers isolated from the BAC contig failed to delimit the MXC3 locus within a 300-kb interval defined by the overlapping BAC clones. This result indicates a >25-fold reduction in recombination
frequency in the MXC3 region compared to the average rate of recombination for the Populus genome.
Received: 8 December 2000 / Accepted: 1 March 2001 相似文献
11.
The citrus tristeza virus resistance gene (Ctv) is a single dominant gene in Poncirus trifoliata, a sexually compatible relative of citrus. To clone this gene, a bacterial artificial chromosome (BAC) library has been constructed from an individual plant that was homozygous for Ctv. This library contains 45,696 clones with an average insert size of 80 kb, corresponding to 9.6 genome equivalents. Screening of the BAC library with five chloroplast DNA probes indicated that 0.58% of the BAC clones contained chloroplast-derived inserts. The chromosome walk across the Ctv locus was initiated using three closely linked genetic markers: C19, AD8, and Z16. The walk has been completed and a contig of ca. 1.2 Mb was constructed. Based on new data, the genetic map in the Ctv region was revised, with Ctv being located between AD8-Z16 and C19 at distances of 1.2 and 0.6 cM, respectively. Utilizing DNA fragments isolated from the contig as RFLP markers, the Ctv locus was further mapped to a region of ca. 300 kb. This contig contains several putative disease-resistance genes similar to the rice Xa21 gene, the tomato Cf-2 gene, and the Arabidopsis thaliana RPS2 gene. This library will therefore allow cloning of Ctv and other putative disease-resistance genes. 相似文献
12.
Chromosome landing at the bacterial blight resistance gene<Emphasis Type="Italic"> Xa4</Emphasis> locus using a deep coverage rice BAC library 总被引:6,自引:0,他引:6
Wang W Zhai W Luo M Jiang G Chen X Li X Wing RA Zhu L 《Molecular genetics and genomics : MGG》2001,265(1):118-125
Xa4 is a dominantly inherited rice gene that confers resistance to Philippine race 1 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae in rice. In order to isolate the gene by positional cloning, a bacterial artificial chromosome (BAC) library was constructed from genomic DNA isolated from an Xa4-harboring accession, IRBB56. The library contains 55,296 clones with an average insert size of 132 kb, providing 14 rice genome equivalents. Three DNA markers closely linked to Xa4 were used to screen the library. The marker RS13, a resistance gene analogue that co-segregates with Xa4, identified 18 clones, of which four and six, respectively, were simultaneously detected by the other two markers, G181 and L1044. Fingerprinting and Southern analysis indicated that these clones overlapped and define an interval spanning 420 kb. In an F2 population derived from an indica variety, IR24, and its Xa4-containing near isogenic line (NIL), IRBB4, the susceptible plants were screened in order to map the Xa4 gene genetically and physically. Out of 24 insert ends isolated from the BACs in the contig, three revealed polymorphisms between IR24 and IRBB4. Two insert ends, 56M22F and 26D24R, flanked Xa4 on each side. Based on the overlap of the BACs, six overlapping clones were considered to include the Xa4 allele, one of which, 106P13, was chosen for further investigation. 相似文献
13.
The sunn mutation of Medicago truncatula is a single-gene mutation that confers a novel supernodulation phenotype in response to inoculation with Sinorhizobium meliloti. We took advantage of the publicly available codominant PCR markers, the high-density genetic map, and a linked cytogenetic map to define the physical and genetic region containing sunn. We determined that sunn is located at the bottom of linkage group 4, where a fine-structure genetic map was used to place the locus within a approximately 400-kb contig of bacterial artificial chromosome (BAC) clones. Genetic analyses of the sunn contig, as well as of a second, closely linked BAC contig designated NUM1, indicate that the physical to genetic distance within this chromosome region is in the range of 1000 -1100 kb.cM-1. The ratio of genetic to cytogenetic distance determined across the entire region is 0.3 cM.microm(-1). These estimates are in good agreement with the empirically determined value of approximately 300 kb.microm(-1) measured for the NUM1 contig. The assignment of sunn to a defined physical interval should provide a basis for sequencing and ultimately cloning the responsible gene. 相似文献
14.
C Y Wang J D Shi Y Q Huang P E Cruz B Ochoa B Hawkins-Lee A Davoodi-Semiromi J X She 《Genomics》1999,60(1):12-19
Urofacial (Ochoa) syndrome is an autosomal recessive disease characterized by distorted facial expression and urinary abnormalities. Previously, we mapped the UFS gene to chromosome 10q23-q24 and narrowed the interval to one YAC clone of 1410 kb. Here, we have constructed a BAC/PAC contig of the 1-Mb region using STS content mapping with 42 BAC/PAC-end sequences, 9 previously reported and 16 newly identified microsatellite markers, and 14 EST markers. A total of 26 polymorphic microsatellite markers were genotyped for 31 UFS patients from Colombia and 2 patients from the United States. Haplotype analyses suggest that the UFS gene is located within two overlapping BAC clones, a region of <360 kb of DNA sequence. We tested 42 EST markers previously mapped to the D10S1709-D10S603 interval against the BAC/PAC contig and identified 11 ESTs located in the 1-Mb region. Four of the 11 ESTs mapped to the 360-kb UFS critical region. Shotgun sequencing of the two BAC clones and BLASTN search of the EST databases revealed 3 other ESTs contained in the UFS critical region. These results will facilitate the cloning and identification of the UFS gene. 相似文献
15.
A bacterial artificial chromosome contig spanning the major domestication locus Q in wheat and identification of a candidate gene 总被引:6,自引:0,他引:6
The Q locus played a major role in the domestication of wheat because it confers the free-threshing character and influences many other agronomically important traits. We constructed a physical contig spanning the Q locus using a Triticum monococcum BAC library. Three chromosome walking steps were performed by complete sequencing of BACs and identification of low-copy markers through similarity searches of database sequences. The BAC contig spans a physical distance of approximately 300 kb corresponding to a genetic distance of 0.9 cM. The physical map of T. monococcum had perfect colinearity with the genetic map of wheat chromosome arm 5AL. Recombination data in conjunction with analysis of fast neutron deletions confirmed that the contig spanned the Q locus. The Q gene was narrowed to a 100-kb segment, which contains an APETALA2 (AP2)-like gene that cosegregates with Q. AP2 is known to play a major role in controlling floral homeotic gene expression and thus is an excellent candidate for Q. 相似文献
16.
PCR Sampling of disease resistance-like sequences from a disease resistance gene cluster in soybean 总被引:2,自引:0,他引:2
Graham A Marek F Shoemaker C 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2002,105(1):50-57
Clusters of Resistance-like genes (RLGs) have been identified from a variety of plant species. In soybean, RLG-specific primers and BAC-fingerprinting were used to develop a contig of overlapping BACs for a cluster of RLGs on soybean linkage group J. The resistance genes Rps2 (Phytophthora stem and root rot) and Rmd-c (powdery mildew) and the ineffective nodulation gene Rj2 were previously mapped to this region of linkage group J. PCR hybridization was used to place two TIR/NBD/LRR cDNAs on overlapping BACs from this contig. Both of the cDNAs were present on BAC 34P7. Fingerprinting of this BAC suggested as many as twelve different RLGs were present. Given the high nucleotide identity shared between cDNAs LM6 and MG13 (>90%), direct sequencing of this region would be difficult. More sequence information was needed about the RLGs within this region before sequencing could be undertaken. By comparing the genomic sequences of cDNAs LM6 and MG13 we identified conserved regions from which oligonucleotide primers specific to BAC 34P7 RLGs could be designed. The nine primer pairs spanned the genomic sequence of LM6 and produced overlapping RLG products upon amplification of BAC 34P7. Amplification products from 12 different RLGs were identified. On average, nucleotide identity between RLG sequences was greater than 95%. Examination of RLG sequences also revealed evidence of additions, deletions and duplications within targeted regions of these genes. Using previously mapped cDNAs we were able to quickly and inexpensively access multiple RLGs within a single specific cluster. 相似文献
17.
Construction of a rice bacterial artificial chromosome library and identification of clones linked to the Xa-21 disease resistance locus 总被引:21,自引:0,他引:21
Guo-Liang Wang Thomas E. Holsten Wen-Yuan Song He-Ping Wang Pamela C. Ronald 《The Plant journal : for cell and molecular biology》1995,7(3):525-533
A bacterial artificial chromosome (BAC) library consisting of 11 000 clones with an average DNA insert size of 125 kb was constructed from rice nuclear DNA. The BAC clones were stable in E. coli after 100 generations of serial growth. Transformation of the BAC clones by electroporation into E. coli was highly efficient and increased with decreasing size of the DNA inserts. The library was evaluated for the presence of organellar, repeated, and telomeric sequences. A very low percentage (<0.3%) of the library consisted of chloroplast and mitochondrial clones. Eighteen BACs were identified that hybridized with an Arabidopsis telomere repeat. Sixteen BACs hybridized with the AA genome-specific repetitive sequence pOs48. Twelve clones were isolated that hybridized with three DNA markers linked to the Xa-21 disease resistance locus. The results indicate that the BAC system can be used to clone and manipulate large pieces of plant DNA efficiently. 相似文献
18.
The interaction between soybean and the phytopathogenic oomycete Phytophthora sojae is controlled by host resistance (Rps) genes and pathogen avirulence (Avr) genes. We have mapped the Avr1a locus in F(2) populations derived from four different P. sojae races. Four RAPD and nine AFLP markers linked to Avr1a were initially identified. Nine markers were used to compare genetic linkage maps of the Avr1a locus in two distinct F(2) populations. Distorted segregation ratios favoring homozygous genotypes were noted in both crosses. Segregation analysis of all the markers in one F(2) population of 90 progeny generated a map of 113.2 cM encompassing Avr1a, with one marker cosegregating with the gene. The cosegregating DNA marker was used to isolate P. sojae BAC clones and construct a physical map covering 170 kb, from which additional DNA markers were developed. Three markers occurring within the BAC contig were mapped in an enlarged population of 486 F(2) progeny. Avr1a was localized to a 114-kb interval, and an average physical to genetic distance ratio of 391 kb/cM was calculated for this region. This work provides a basis for the positional cloning of Avr1a. 相似文献
19.
20.
Chinese alligator (Alligator sinensis) is a rare and endangered species endemic to China. To better understand genetic details of the Chinese alligator genomic structure, a highly redundant bacterial artificial chromosome (BAC) library was constructed. This library consists of 216,238 clones with an average insert size of about 90kb, indicating that the library contains 6.8-fold genome equivalents. Subsequently, we constructed a 516kb contig map for the Chinese alligator olfactory receptor (OR) genes, which spans nine BAC clones, and subjected the BACs to full sequencing. The sequence analysis revealed that this contig contained 16 OR functional genes and meanwhile demonstrated that the nine BACs, which constituted the contig, overlapped correctly, proving the usability of this genome library. As a result, this BAC library could provide a useful platform for physical mapping, genome sequencing or complex analysis of targeted genomic regions for this rare species. 相似文献