Integration of physical and genetic maps in apple confirms whole-genome and segmental duplications in the apple genome

A total of 355 simple sequence repeat (SSR) markers were developed, based on expressed sequence tag (EST) and bacterial artificial chromosome (BAC)-end sequence databases, and successfully used to construct an SSR-based genetic linkage map of the apple. The consensus linkage map spanned 1143 cM, with an average density of 2.5 cM per marker. Newly developed SSR markers along with 279 SSR markers previously published by the HiDRAS project were further used to integrate physical and genetic maps of the apple using a PCR-based BAC library screening approach. A total of 470 contigs were unambiguously anchored onto all 17 linkage groups of the apple genome, and 158 contigs contained two or more molecular markers. The genetically mapped contigs spanned ～421 Mb in cumulative physical length, representing 60.0% of the genome. The sizes of anchored contigs ranged from 97 kb to 4.0 Mb, with an average of 995 kb. The average physical length of anchored contigs on each linkage group was ～24.8 Mb, ranging from 17.0 Mb to 37.73 Mb. Using BAC DNA as templates, PCR screening of the BAC library amplified fragments of highly homologous sequences from homoeologous chromosomes. Upon integrating physical and genetic maps of the apple, the presence of not only homoeologous chromosome pairs, but also of multiple locus markers mapped to adjacent sites on the same chromosome was detected. These findings demonstrated the presence of both genome-wide and segmental duplications in the apple genome and provided further insights into the complex polyploid ancestral origin of the apple.     

Construction and characterization of BAC libraries from major grapevine cultivars

Genome projects were initiated on grapevine (Vitis vinifera L., 2n=38, genome size 475 Mb) through the successful construction of four bacterial artificial chromosome (BAC) libraries from three major cultivars, Cabernet Sauvignon (Cabernet S), Syrah and two different clones of Pinot Noir (Pinot N). Depending on the library, the genome coverage represented 4.5–14.8 genome equivalents with clones having a mean insert size of 93–158 kb. BAC pools suitable for PCR screening were constructed for two of these BAC libraries [Cabernet S and Pinot N clone (cl) 115] and subsequently used to confirm the genome coverage of both libraries by PCR anchoring of 74 genetic markers sampled from the 19 linkage groups. For ten of these markers, two bands on separate BAC pools were differentiated that could correspond either to different alleles or to a duplication of the locus being studied. Finally, a preliminary assessment of the correspondence between genetic and physical distances was made through the anchoring of all the markers mapped along linkage group 1 of the V. vinifera genetic map. A pair of markers, 2.1 cM apart, anchored the same BAC clones, which allowed us to estimate that 1 cM corresponded in this particular region to a maximum length of 130 kb.     

Genetic mapping and BAC assignment of EST-derived SSR markers shows non-uniform distribution of genes in the barley genome

A set of 111,090 barley expressed sequence tags (ESTs) was searched for the presence of microsatellite motifs [simple sequence repeat (SSRs)] and yielded 2,823 non-redundant SSR-containing ESTs (SSR–ESTs). From this, a set of 754 primer pairs was designed of which 525 primer pairs yielded an amplicon and as a result, 185 EST-derived microsatellite loci (EST–SSRs) were placed onto a genetic map of barley. The markers show a uniform distribution along all seven linkage groups ranging from 21 (7H) to 35 (3H) markers. Polymorphism information content values ranged from of 0.24 to 0.78 (average 0.48). To further investigate the physical distribution of the EST–SSRs in the barley genome, a bacterial artificial chromosomes (BAC) library was screened. Out of 129 markers tested, BAC addresses were obtained for 127 EST–SSR markers. Twenty-seven BACs, forming eight contigs, were hit by two or three EST–SSRs each. This unexpectedly high incidence of EST–SSRs physically linked at the sub-megabase level provides additional evidence of an uneven distribution of genes and the segmentation of the barley genome in gene-rich and gene-poor regions.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.Primer sequences for developed SSR markers are available upon request from the corresponding author (A. Graner).     

Development of microsatellite markers in autopolyploid sugarcane and comparative analysis of conserved microsatellites in sorghum and sugarcane

Sugarcane has become an increasingly important first-generation biofuel crop in tropical and subtropical regions. It has a large, complex, polyploid genome that has hindered the progress of genomic research and marker-assisted selection. Genetic mapping and ultimately genome sequence assembly require a large number of DNA markers. Simple sequence repeats (SSRs) are widely used in genetic mapping because of their abundance, high rates of polymorphism, and ease of use. The objectives of this study were to develop SSR markers for construction of a saturated genetic map and to characterize the frequency and distribution of SSRs in a polyploid genome. SSR markers were mined from expressed sequence tag (EST), reduced representation library genomic sequences, and bacterial artificial chromosome (BAC) sequences. A total of 5,675 SSR markers were surveyed in a segregating population. The overall successful amplification and polymorphic rates were 87.9 and 16.4%, respectively. The trinucleotide repeat motifs were most abundant, with tri- and hexanucleotide motifs being the most abundant for the ESTs. BAC and genomic SSRs were mostly AT-rich while the ESTs were relatively GC-rich due to codon bias. These markers were also aligned to the sorghum genome, resulting in 1,203 markers mapped in the sorghum genome. This set of SSRs conserved in sugarcane and sorghum would be the most informative for mapping quantitative trait loci in sugarcane and for comparative genomic analyses. This large collection of SSR markers is a valuable resource for sugarcane genomic research and crop improvement.     

A first generation physical map of the medaka genome in BACs essential for positional cloning and clone-by-clone based genomic sequencing

In order to realize the full potential of the medaka as a model system for developmental biology and genetics, characterized genomic resources need to be established, culminating in the sequence of the medaka genome. To facilitate the map-based cloning of genes underlying induced mutations and to provide templates for clone-based genomic sequencing, we have created a first-generation physical map of the medaka genome in bacterial artificial chromosome (BAC) clones. In particular, we exploited the synteny to the closely related genome of the pufferfish, Takifugu rubripes, by marker content mapping. As a first step, we clustered 103,144 public medaka EST sequences to obtain a set of 21,121 non-redundant sequence entities. Avoiding oversampling of gene-dense regions, 11,254 of EST clusters were successfully matched against the draft sequence of the fugu genome, and 2363 genes were selected for the BAC map project. We designed 35mer oligonucleotide probes from the selected genes and hybridized them against 64,500 BAC clones of strains Cab and Hd-rR, representing 14-fold coverage of the medaka genome. Our data set is further supplemented with 437 results generated from PCR-amplified inserts of medaka cDNA clones and BAC end-fragment markers. Our current, edited, first generation medaka BAC map consists of 902 map segments that cover about 74% of the medaka genome. The map contains 2721 markers. Of these, 2534 are from expressed sequences, equivalent to a non-redundant set of 2328 loci. The 934 markers (724 different) are anchored to the medaka genetic map. Thus, genetic map assignments provide immediate access to underlying clones and contigs, simplifying molecular access to candidate gene regions and their characterization.     

甘蓝型油菜A基因组候选BAC克隆的筛选及其插入片段大小分析

选择甘蓝型油菜A基因组10个连锁群上特有的85个SSR分子标记,合成其引物序列,采用四维PCR法筛选甘蓝型油菜-新疆野生油菜二体异附加系BAC文库,成功筛选到甘蓝型油菜A基因组39个BAC克隆,其插入片段介于50～300 kb之间,平均为120 kb。甘蓝型油菜A基因组10个连锁群BAC克隆的获得,对后续开展甘蓝型油菜A基因组染色体识别、基因染色体定位、遗传距离与物理距离间关系分析等均具有重要价值。     

Genetic mapping of new cotton fiber loci using EST-derived microsatellites in an interspecific recombinant inbred line cotton population

There is an immediate need for a high-density genetic map of cotton anchored with fiber genes to facilitate marker-assisted selection (MAS) for improved fiber traits. With this goal in mind, genetic mapping with a new set of microsatellite markers [comprising both simple (SSR) and complex (CSR) sequence repeat markers] was performed on 183 recombinant inbred lines (RILs) developed from the progeny of the interspecific cross Gossypium hirsutum L. cv. TM1 × Gossypium barbadense L. Pima 3-79. Microsatellite markers were developed using 1557 ESTs-containing SSRs (≥10 bp) and 5794 EST-containing CSRs (≥12 bp) obtained from ~14,000 consensus sequences derived from fiber ESTs generated from the cultivated diploid species Gossypium arboreum L. cv AKA8401. From a total of 1232 EST-derived SSR (MUSS) and CSR (MUCS) primer-pairs, 1019 (83%) successfully amplified PCR products from a survey panel of six Gossypium species; 202 (19.8%) were polymorphic between the G. hirsutum L. and G. barbadense L. parents of the interspecific mapping population. Among these polymorphic markers, only 86 (42.6%) showed significant sequence homology to annotated genes with known function. The chromosomal locations of 36 microsatellites were associated with 14 chromosomes and/or 13 chromosome arms of the cotton genome by hypoaneuploid deficiency analysis, enabling us to assign genetic linkage groups (LG) to specific chromosomes. The resulting genetic map consists of 193 loci, including 121 new fiber loci not previously mapped. These fiber loci were mapped to 19 chromosomes and 11 LG spanning 1277 cM, providing approximately 27% genome coverage. Preliminary quantitative trait loci analysis suggested that chromosomes 2, 3, 15, and 18 may harbor genes for traits related to fiber quality. These new PCR-based microsatellite markers derived from cotton fiber ESTs will facilitate the development of a high-resolution integrated genetic map of cotton for structural and functional study of fiber genes and MAS of genes that enhance fiber quality. Electronic Supplementary Material Supplementary material is available for this article at Names are necessary to report factually on available data, however, the USDA neither guarantees nor warrants the standard of products or service, and the use of the name by the USDA implies no approval of the products or service to the exclusion of others that may also be suitable.     

Development and mapping of 2240 new SSR markers for rice (Oryza sativa L.).

A total of 2414 new di-, tri- and tetra-nucleotide non-redundant SSR primer pairs, representing 2240 unique marker loci, have been developed and experimentally validated for rice (Oryza sativa L.). Duplicate primer pairs are reported for 7% (174) of the loci. The majority (92%) of primer pairs were developed in regions flanking perfect repeats > or = 24 bp in length. Using electronic PCR (e-PCR) to align primer pairs against 3284 publicly sequenced rice BAC and PAC clones (representing about 83% of the total rice genome), 65% of the SSR markers hit a BAC or PAC clone containing at least one genetically mapped marker and could be mapped by proxy. Additional information based on genetic mapping and "nearest marker" information provided the basis for locating a total of 1825 (81%) of the newly designed markers along rice chromosomes. Fifty-six SSR markers (2.8%) hit BAC clones on two or more different chromosomes and appeared to be multiple copy. The largest proportion of SSRs in this data set correspond to poly(GA) motifs (36%), followed by poly(AT) (15%) and poly(CCG) (8%) motifs. AT-rich microsatellites had the longest average repeat tracts, while GC-rich motifs were the shortest. In combination with the pool of 500 previously mapped SSR markers, this release makes available a total of 2740 experimentally confirmed SSR markers for rice, or approximately one SSR every 157 kb.     

水稻第六染色体长臂亚端粒区遗传图与物理图的整合

生物染色体亚闰区域在物种翰经过程中是高度活跃的。为了认识水稻染色体亚端粒区域的组织结构,用水稻第六染色体长臂亚端料区的ＲＦＬＰ标记Ｇ３４２和Ｒ１１６７作探针筛选ＢＡＣ文库,以得到的阳笥ＢＡＣ克隆为起点进行染色体眇地,构建了覆盖这２个分子标记区约５００ｋｂ的ＢＡＣ跨叠克隆群,将这一区域的跗图和物理图进行了整合。对１４个ＢＡＣ克隆插入末端进行了亚克隆,鉴定的７个亚克隆 端为单考贝或抵考贝序列,其中５个     

A sunflower BAC library suitable for PCR screening and physical mapping of targeted genomic regions

A sunflower BAC library consisting of 147,456 clones with an average size of 118 kb has been constructed and characterized. It represents approximately 5× sunflower haploid genome equivalents. The BAC library has been arranged in pools and superpools of DNA allowing screening with various PCR-based markers. Each of the 32 superpools contains 4,608 clones and corresponds to a 36 matrix pools. Thus, the screening of the entire library could be accomplished in less than 80 PCR reactions including positive and negative controls. As a demonstration of the feasibility of the concept, a set of 24 SSR markers covering about 36 cM in the sunflower SSR map (Tang et al. in Theor Appl Genet 105:1124–1136, 2002) have been used to screen the BAC library. About 125 BAC clones have been identified and then organized in 23 contigs by HindIII digestion. The contigs are anchored on the SSR map and thus constitutes a first-generation physical map of this region. The utility of this BAC library as a genomic resource for physical mapping and map-based cloning in sunflower is discussed.     

A human-horse comparative map based on equine BAC end sequences

In an effort to increase the density of sequence-based markers for the horse genome we generated 9473 BAC end sequences (BESs) from the CHORI-241 BAC library with an average read length of 677 bp. BLASTN searches with the BESs revealed 4036 meaningful hits (E 相似文献   

Microsatellite markers spanning the apple (Malus x domestica Borkh.) genome

A new set of 148 apple microsatellite markers has been developed and mapped on the apple reference linkage map Fiesta x Discovery. One-hundred and seventeen markers were developed from genomic libraries enriched with the repeats GA, GT, AAG, AAC and ATC; 31 were developed from EST sequences. Markers derived from sequences containing dinucleotide repeats were generally more polymorphic than sequences containing trinucleotide repeats. Additional eight SSRs from published apple, pear, and Sorbus torminalis SSRs, whose position on the apple genome was unknown, have also been mapped. The transferability of SSRs across Maloideae species resulted in being efficient with 41% of the markers successfully transferred. For all 156 SSRs, the primer sequences, repeat type, map position, and quality of the amplification products are reported. Also presented are allele sizes, ranges, and number of SSRs found in a set of nine cultivars. All this information and those of the previous CH-SSR series can be searched at the apple SSR database () to which updates and comments can be added. A large number of apple ESTs containing SSR repeats are available and should be used for the development of new apple SSRs. The apple SSR database is also meant to become an international platform for coordinating this effort. The increased coverage of the apple genome with SSRs allowed the selection of a set of 86 reliable, highly polymorphic, and overall the apple genome well-scattered SSRs. These SSRs cover about 85% of the genome with an average distance of one marker per 15 cM.E. Silfverberg-Dilworth and C. L. Matasci contributed equally to this work.     

Anchoring of rice BAC clones to the rice genetic map in silico

A wealth of molecular resources have been developed for rice genomics, including dense genetic maps, expressed sequence tags (ESTs), yeast artificial chromosome maps, bacterial artificial chromosome (BAC) libraries and BAC end sequence databases. Integration of genetic and physical maps involves labor-intensive empirical experiments. To accelerate the integration of the bacterial clone resources with the genetic map for the International Rice Genome Sequencing Project, we cleaned and filtered the available EST and BAC end sequences for repetitive sequences and then searched all available rice genetic markers with our filtered databases. We identified 418 genetic markers that aligned with at least one BAC end sequence with >95% sequence identity, providing a set of large insert clones with an average separation of 1 Mb that can serve as nucleation points for the sequencing phase of the International Rice Genome Sequencing Project.     

Development of Chromosome-specific Cytogenetic Markers and Merging of Linkage Fragments in Papaya

Carica papaya L. is a tropical and sub-tropical fruit-tree crop with a small genome and nine pairs of chromosomes. The transgenic cultivar ‘SunUp’ has been sequenced and three high-density genetic maps are available for mapping agronomically and economically-important traits. However, the small size and similar morphology of papaya chromosomes hinder their identification and few cytological resources are available for integration of genetic and cytogenetic information. Fluorescence in situ hybridization (FISH) was performed on mitotic metaphase chromosomes using BAC clones harboring mapped simple sequence repeat (SSR) markers as probes. A total of 104 BAC clones covering all 12 linkage groups (LGs) were tested and 12 of them, that gave a single specific signal, were chosen as representative of the 12 LGs of the SSR genetic map. This set of chromosome-specific DNA markers acted as a foundation for papaya chromosome karyotyping and re-assigning orientation of LGs. Chromosome-specific markers allowed us to assign the minor LGs 10, 11, and 12 to major LGs 8, 9, and 7, respectively. We thus reduced the number of LGs in the genetic map to nine, corresponding to the haploid number of papaya chromosomes. We also tested the relative order of DNA markers on minor LGs 10 and 11 to place them on top of LGs 8 and 9 in the correct orientation. Ribosomal DNAs (rDNAs), a set of major cytogenetic markers, were positioned on specific papaya chromosomes. The 25S rDNA showed strong signals at the constriction site of a single pair of chromosomes identified as LG 2 by LG 2-specific BAC clone. The 5S rDNA showed strong signals on two pairs of chromosomes that are syntenic with LG 4- and LG 5-specific BAC clones. This integrated map will facilitate genome assembly, quantitative trait locus (QTL) mapping, and the study of cytological, physical and genetic distance relationships between papaya chromosomes.     

Physical mapping of an adult plant stripe rust resistance gene from Triticum monococcum

Stripe rust (Puccinia striiformis f. sp. tritici) is one of the major devastating disease which causes large reduction in wheat yield. T. monococcum is an attractive diploid species for gene discovery in wheat with smaller genome size of 5700 Mb compared to 17,300 Mb of bread wheat. An adult plant stripe rust resistance QTL QYrtm.pau-2A was mapped on chromosome 2A flanked by two SSR markers Xwmc170 and Xwmc407. In the present study, two gene based markers Pau_Ta2AL_Gene45 and Pau_Ta2AL_Gene54 developed from 2A specific ESTs were found to map close to QYrtmpau-2A to narrow down the region for candidate gene identification. Utilizing sequence information of these two markers, four BAC clones were identified from the Minimum Tiling Path of 2AL assembly and were sequenced. SSR markers were designed from these BAC sequences and mapped to chromosome 2A. A 50 Mb region of wheat chromomse 2A was identified to harbor stripe rust resistance gene of T. monococcum. Gene based markers identified in the present investigation can be used for marker assisted introgression of QYrtm.pau-2A from T. monococcum to cultivated wheat.     

Development and genetic mapping of microsatellite markers from genome survey sequences in Brassica napus

Microsatellite or simple sequence repeat (SSR) markers are routinely used for tagging genes and assessing genetic diversity. In spite of their importance, there are limited numbers of SSR markers available for Brassica crops. A total of 627 new SSR markers (designated BnGMS) were developed based on publicly available genome survey sequences and used to survey polymorphisms among six B. napus cultivars that serve as parents for established populations. Among these SSR markers, 591 (94.3%) successfully amplified at least one fragment and 434 (73.4%) detected polymorphism among the six B. napus cultivars. No correlation was observed between SSR motifs, repeat number or repeat length with polymorphism levels. A linkage map was constructed using 163 newly developed BnGMS marker loci and anchored with 164 public SSRs in a doubled haploid population. These new markers are evenly distributed over all linkage groups (LGs). Given that the majority of these SSRs are derived from bacterial artificial chromosome (BAC) end sequences, they will be useful in the assignment of their cognate BACs to LGs and facilitate the integration of physical maps with genetic maps for genome sequencing in B. napus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of three maize bacterial artificial chromosome libraries toward anchoring of the physical map to the genetic map using high-density bacterial artificial chromosome filter hybridization

Three maize (Zea mays) bacterial artificial chromosome (BAC) libraries were constructed from inbred line B73. High-density filter sets from all three libraries, made using different restriction enzymes (HindIII, EcoRI, and MboI, respectively), were evaluated with a set of complex probes including the 185-bp knob repeat, ribosomal DNA, two telomere-associated repeat sequences, four centromere repeats, the mitochondrial genome, a multifragment chloroplast DNA probe, and bacteriophage lambda. The results indicate that the libraries are of high quality with low contamination by organellar and lambda-sequences. The use of libraries from multiple enzymes increased the chance of recovering each region of the genome. Ninety maize restriction fragment-length polymorphism core markers were hybridized to filters of the HindIII library, representing 6x coverage of the genome, to initiate development of a framework for anchoring BAC contigs to the intermated B73 x Mo17 genetic map and to mark the bin boundaries on the physical map. All of the clones used as hybridization probes detected at least three BACs. Twenty-two single-copy number core markers identified an average of 7.4 +/- 3.3 positive clones, consistent with the expectation of six clones. This information is integrated into fingerprinting data generated by the Arizona Genomics Institute to assemble the BAC contigs using fingerprint contig and contributed to the process of physical map construction.     

Palaeohexaploid ancestry for Caryophyllales inferred from extensive gene-based physical and genetic mapping of the sugar beet genome (Beta vulgaris)

Sugar beet (Beta vulgaris) is an important crop plant that accounts for 30% of the world's sugar production annually. The genus Beta is a distant relative of currently sequenced taxa within the core eudicotyledons; the genomic characterization of sugar beet is essential to make its genome accessible to molecular dissection. Here, we present comprehensive genomic information in genetic and physical maps that cover all nine chromosomes. Based on this information we identified the proposed ancestral linkage groups of rosids and asterids within the sugar beet genome. We generated an extended genetic map that comprises 1127 single nucleotide polymorphism markers prepared from expressed sequence tags and bacterial artificial chromosome (BAC) end sequences. To construct a genome-wide physical map, we hybridized gene-derived oligomer probes against two BAC libraries with 9.5-fold cumulative coverage of the 758 Mbp genome. More than 2500 probes and clones were integrated both in genetic maps and the physical data. The final physical map encompasses 535 chromosomally anchored contigs that contains 8361 probes and 22 815 BAC clones. By using the gene order established with the physical map, we detected regions of synteny between sugar beet (order Caryophyllales) and rosid species that involves 1400-2700 genes in the sequenced genomes of Arabidopsis, poplar, grapevine, and cacao. The data suggest that Caryophyllales share the palaeohexaploid ancestor proposed for rosids and asterids. Taken together, we here provide extensive molecular resources for sugar beet and enable future high-resolution trait mapping, gene identification, and cross-referencing to regions sequenced in other plant species.     

棉花BAC文库快速筛选法

目的:构建棉花细菌人工染色体(Bacterial Artificial Chromosome,BAC)文库的快速筛选法,以期从BAC文库中大量、快速、高效筛选出特定BAC克隆,为从事基因组测序、分离和分析特定基因、构建物理图谱及基因图位克隆等生物学技术研究奠定基础。方法:构建了整板、行、列的三维混合池,以菌液PCR为基础,从BAC文库中筛选出含有特定DNA片段的BAC单克隆。结果:从BAC文库的3 456个克隆中,共筛选出16个阳性单克隆,涉及13条染色体、11个SSR标记。结论:该文构建的棉花BAC文库筛选体系,筛选快速、准确,适合从BAC文库中大量筛选BAC单克隆。结合当前的多种BAC文库筛选方法进行探讨,根据不同的实验目的选择更合适的筛选方法和操作步骤。