BAC library for the amphipod crustacean,Parhyale hawaiensis

Bacterial artificial chromosomes (BACs) are capable of propagating large fragments of DNA and have become an invaluable tool for studying genome biology. To fill a phylogenetic gap in available genomic sequence and to complement ongoing molecular and genetic studies, we have generated a BAC library for the marine amphipod crustacean, Parhyale hawaiensis. The library was generated from genomic DNA isolated from whole adult animals and comprises 129,024 individual clones. The median insert size is ～ 140 kb and the genomic coverage is approximately five genome equivalents. We have screened the Parhyale BAC library for developmentally relevant genes and characterized the genomic organization of four genes: a hedgehog ortholog, and three Pax3/7 paralogs. Preliminary analysis suggests that introns are larger and more prevalent in Parhyale than in other arthropods whose genomes have been sequenced, which may partly account for the large genome size in Parhyale.     

An Integrated Genetic and Cytogenetic Map for Zhikong Scallop,Chlamys farreri,Based on Microsatellite Markers

The reliability of genome analysis and proficiency of genetic manipulation requires knowledge of the correspondence between the genetic and cytogenetic maps. In the present study, we integrated cytogenetic and microsatellite-based linkage maps for Zhikong scallop, Chlamys farreri. Thirty-eight marker-anchored BAC clones standing for the 19 linkage groups were used to be FISH probes. Of 38 BAC clones, 30 were successfully located on single chromosome by FISH and used to integrate the genetic and cytogenetic map. Among the 19 linkage groups, 12 linkage groups were physically anchored by 2 markers, 6 linkage groups were anchored by 1 marker, and one linkage group was not anchored any makers by FISH. In addition, using two-color FISH, six linkage groups were distinguished by different chromosomal location; linkage groups LG6 and LG16 were placed on chromosome 10, LG8 and LG18 on chromosome 14. As a result, 18 of 19 linkage groups were localized to 17 pairs of chromosomes of C. farreri. We first integrated genetic and cytogenetic map for C. farreri. These 30 chromosome specific BAC clones in the cytogenetic map could be used to identify chromosomes of C. farreri. The integrated map will greatly facilitate molecular genetic studies that will be helpful for breeding applications in C. farreri and the upcoming genome projects of this species.     

Construction and characterization of a BAC library from the Coffea arabica genotype Timor Hybrid CIFC 832/2

Most of the world’s coffee production originates from Coffea arabica, an allotetraploid species with low genetic diversity and for which few genomic resources are available. Genomic libraries with large DNA fragment inserts are useful tools for the study of plant genomes, including the production of physical maps, integration studies of physical and genetic maps, genome structure analysis and gene isolation by positional cloning. Here, we report the construction and characterization of a Bacterial Artificial Chromosome (BAC) library from C. arabica Timor Hybrid CIFC 832/2, a parental genotype for several modern coffee cultivars. The BAC library consists of 56,832 clones with an average insert size of 118 kb, which represents a dihaploid genome coverage of five to sixfold. The content of organellar DNA was estimated at 1.04 and 0.5 % for chloroplast and mitochondrial DNA, respectively. The BAC library was screened for the NADPH-dependent mannose-6-phosphate reductase gene (CaM6PR) with markers positioned on four linkage groups of a partial C. arabica genetic map. A mixed approach using PCR and membrane hybridization of BAC pools allowed for the discovery of nine BAC clones with the CaM6PR gene and 53 BAC clones that were anchored to the genetic map with simple sequence repeat markers. This library will be a useful tool for future studies on comparative genomics and the identification of genes and regulatory elements controlling major traits in this economically important crop species.     

Construction and characterization of a BAC library from a gynogenetic channel catfish Ictalurus punctatus

A bacterial artificial chromosome (BAC) library was constructed by cloning HindIII-digested high molecular weight DNA from a gynogenetic channel catfish, Ictalurus punctatus, into the vector pBeloBAC11. Approximately 53 500 clones were arrayed in 384-well plates and stored at -80°C (CCBL1), while clones from a smaller insert size fraction were stored at -80°C without arraying (CCBL2). Pulsed-field gel electrophoresis of 100 clones after NotI digestion revealed an average insert size of 165 kb for CCBL1 and 113 kb for CCBL2. Further characterization of CCBL1 demonstrated that 10% of the clones did not contain an insert. CCBL1 provides a 7.2-fold coverage of the channel catfish haploid genome. PCR-based screening demonstrated that 68 out of 74 unique loci were present in the library. This represents a 92% chance to find a unique sequence. These libraries will be useful for physical mapping of the channel catfish genome, and identification of genes controlling major traits in this economically important species.     

Genome Assembly of Alfalfa Cultivar Zhongmu-4 and Identification of SNPs Associated with Agronomic Traits

Alfalfa(Medicago sativa L.) is the most important legume forage crop worldwide with high nutritional value and yield.For a long time,the breeding of alfalfa was hampered by lacking reliable information on the autotetraploid genome and molecular markers linked to important agronomic traits.We herein reported the de novo assembly of the allele-aware chromosome-level genome of Zhongmu-4,a cultivar widely cultivated in China,and a comprehensive database of genomic variations based on resequencing of...     

A BAC library of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L. for the targeted isolation of centromeric DNA and molecular cytogenetics of <Emphasis Type="Italic">Beta</Emphasis> species

We constructed a sugar beet (Beta vulgaris) bacterial artificial chromosome (BAC) library of the monosomic addition line PAT2. This chromosomal mutant carries a single additional chromosome fragment (minichromosome) derived from the wild beet Beta patellaris. Restriction analysis of the mutant line by pulsed-field gel electrophoresis was used to determine HindIII as a suitable enzyme for partial digestion of genomic DNA to generate large-insert fragments which were cloned into the vector pCC1. The library consists of 36,096 clones with an average insert size of 120 kb, and 2.2% of the clones contain mitochondrial or chloroplast DNA. Based on a haploid genome size of 758 Mbp, the library represents 5.7 genome equivalents providing the probability of 99.67% that any sequence of the PAT2 genome can be found in the library. Hybridization to high-density filters was used to isolate 89 BACs containing arrays of the centromere-associated satellite repeats pTS5 and pTS4.1. Using the identified BAC clones in fluorescent in situ hybridization experiments with PAT2 and Beta patellaris chromosome spreads their wild beet origin and centromeric localization was demonstrated. Multi-colour FISH with differently labelled satellite repeats pTS5 and pTS4.1 was used to investigate the large-scale organization of the centromere of the PAT2 minichromosome in detail. FISH studies showed that the centromeric satellite pTS5 is flanked on both sides by pTS4.1 arrays and the arms of the minichromosome are terminated by the Arabidopsis-type telomeric sequences. FISH with a BAC, selected from high-density filters after hybridization with an RFLP marker of the genetic linkage group I, demonstrated that it is feasible to correlate genetic linkage groups with chromosomes. Therefore, the PAT2 BAC library provides a useful tool for the characterization of Beta centromeres and a valuable resource for sugar beet genome analysis.     

Construction and characterization of a soybean yeast artificial chromosome library and identification of clones for the<Emphasis Type="Italic"> Rps6</Emphasis> region

We report the construction and characterization of the first soybean yeast artificial chromosome (YAC) library using high-molecular weight DNA isolated from leaf nuclei of the cultivar Conrad 94 that carries Phytophthora resistance genes Rps1-k and Rps6. The quality of this library has been evaluated through analysis of 393 randomly selected YAC clones. The library consists of 36,864 clones, of which 19,956 carry single soybean YACs with an average size of about 285 kb. The library represents approximately five soybean genome equivalents. The probability of finding any soybean sequences from this library is about 0.99. The library was screened for 43 SSR markers representing the whole soybean genome. We were able to identify positive YAC pools for 95% of the SSR markers. Two YAC clones carrying molecular markers linked to the Rps6 gene were identified. The YAC library reported here would be a useful resource for map-based cloning of agronomically important soybean genes and also to complement the effort towards construction of the physical map for the soybean genome.     

Construction and characterization of a bacterial artificial chromosome library for <Emphasis Type="Italic">Camellia sinensis</Emphasis>

Tea is a popular and natural non-alcoholic beverage, and is produced from fresh leaves of Camellia sinensis. Tea leaves contain many bioactive compounds that have significant health benefits. We constructed a high quality bacterial artificial chromosome (BAC) library by using the fresh petals of C. sinensis “Shuchazao” for genome sequencing and improvement of genomic assembly. BAC library is still a significant tool for studies of functional genomes and preservation of precious genetic resources. The BAC library contains 161,280 clones with an average insert size of 113 kb, which represents approximately 6.2-fold coverage of haploid genome equivalents of C. sinensis. We characterized 20 complete BAC clones and 738 BAC end sequences (BESs) ranging from 105 to 917 bp. In addition, we predicted cis-regulatory elements of LAR (leucoanthocyanidin reductase), TCS (caffeine synthase), and TS (theanine synthetase) involved in tea characteristic metabolite synthesis and identified a larger number of light-responsive cis-acting elements in these three genes. Meanwhile, we analyzed alternative splicing of these three genes. Furthermore, 12 pairs of SSR primers were successfully amplified in tea plant DNA. The tea BAC library was a critical resource to accomplish de novo whole-genome sequencing, accelerate gene discovery and enhance molecular breeding of C. sinensis.     

MagnaportheDB: a federated solution for integrating physical and genetic map data with BAC end derived sequences for the rice blast fungus Magnaporthe grisea

We have created a federated database for genome studies of Magnaporthe grisea, the causal agent of rice blast disease, by integrating end sequence data from BAC clones, genetic marker data and BAC contig assembly data. A library of 9216 BAC clones providing >25-fold coverage of the entire genome was end sequenced and fingerprinted by HindIII digestion. The Image/FPC software package was then used to generate an assembly of 188 contigs covering >95% of the genome. The database contains the results of this assembly integrated with hybridization data of genetic markers to the BAC library. AceDB was used for the core database engine and a MySQL relational database, populated with numerical representations of BAC clones within FPC contigs, was used to create appropriately scaled images. The database is being used to facilitate sequencing efforts. The database also allows researchers mapping known genes or other sequences of interest, rapid and easy access to the fundamental organization of the M.grisea genome. This database, MagnaportheDB, can be accessed on the web at http://www.cals.ncsu.edu/fungal_genomics/mgdatabase/int.htm.     

Phylogenetic analysis in some Hordeum species (Triticeae; Poaceae) based on two single-copy nuclear genes encoding acetyl-CoA carboxylase

We investigate the phylogenetic relationship, and evolutionary history of 18 diploid and polyploid Hordeum species including 22 taxa based on two single-copy nuclear ACC1 and ACC2 genes using maximum parsimony, maximum likelihood, and Bayesian inference. The results of molecular phylogenetic analysis demonstrated genetic relationships among taxa and origin of polyploids. Our phylogenetic analyses revealed a clear alloploid origin of Hordeum capense, with Eurasian Hordeum marinum subsp. gussoneanum as the Xa genome donor and diploid Asian Hordeum roshevitzii as the H genome donor. The formation of hexaploid Hordeum lechleri likely involves hybridization between tetraploid Hordeum brachyantherum subsp. brachyantherum and a diploid possessing the I genome. The Acc1 and Acc2 gene data analyses suggested that Siberian Hordeum bogdanii might have be the common ancestor of the diploid New World Hordeum species. Perennial diploid South American species, Hordeum comosum was the first-diverging group within the clade of diploid American species in the analyses.     

Construction and Characterization of aMagnaporthe griseaBacterial Artificial Chromosome Library

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of aMagnaporthe griseabacterial artificial chromosome library.Fungal Genet. Biol.20,280–288. A bacterial artificial chromosome (BAC) library ofMagnaporthe griseacontaining 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents ofM. griseaand has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using alys1-3auxotroph, we have shown that BAC clones at least 113 kb can be transformed intoM. griseato screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes.     

Construction and characterization of two rice bacterial artificial chromosome libraries from the parents of a permanent recombinant inbred mapping population

Rice is a leading grain crop and the staple food for over half of the world population. Rice is also an ideal species for genetic and biological studies of cereal crops and other monocotyledonous plants because of its small genome and well developed genetic system. To facilitate rice genome analysis leading to physical mapping, the identification of molecular markers closely linked to economic traits, and map-based cloning, we have constructed two rice bacterial artificial chromosome (BAC) libraries from the parents of a permanent mapping population (Lemont and Teqing) consisting of 400 F9 recombinant inbred lines (RILs). Lemont (japonica) and Teqing (indica) represent the two major genomes of cultivated rice, both are leading commercial varieties and widely used germplasm in rice breeding programs. The Lemont library contains 7296 clones with an average insert size of 150 kb, which represents 2.6 rice haploid genome equivalents. The Teqing library contains 14208 clones with an average insert size of 130 kb, which represents 4.4. rice haploid genome equivalents. Three single-copy DNA probes were used to screen the libraries and at least two overlapping BAC clones were isolated with each probe from each library, ranging from 45 to 260 kb in insert size. Hybridization of BAC clones with chloroplast DNA probes and fluorescent in situ hybridization using BAC DNA as probes demonstrated that both libraries contain very few clones of chloroplast DNA origin and are likely free of chimeric clones. These data indicate that both BAC libraries should be suitable for map-based cloning of rice genes and physical mapping of the rice genome.     

Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup

Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.”     

Construction and Characterization of a Bacterial Artificial Chromosome (BAC) Library of Pacific White Shrimp, Litopenaeus vannamei

The pacific white shrimp (Litopenaeus vannamei) is one of the most economically important marine aquaculture species in the world. To facilitate gene cloning and characterization, genome analysis, physical mapping, and molecular selection breeding of marine shrimp, we have developed the techniques to isolate high-quality megabase-sized DNA from hemocyte nuclear DNA of female shrimp and constructed a bacterial artificial chromosome (BAC) genomic library for the species. The library was constructed in the Hind III site of the vector pECBAC1, consisting of 101,760 clones arrayed in 265 384-well microtiter plates, with an average insert size of 101 kb, and covering the genome approximately fivefold. To characterize the library, 92,160 clones were spotted onto high-density nylon filters for hybridization screening. A set of 18 pairs of overgo probes designed from eight cDNA sequences of L. vannamei genes were used in hybridization screening, and 35 positive clones were identified. These results suggest that the shrimp BAC libraries will provide a useful resource for screening of genomic regions of interest candidate genes, gene families, or large-sized synthetic DNA region and promote future works on comparative genomics, physical mapping, and large-scale genome sequencing in the species.     

Molecular cloning of a cysteine synthase cDNA from Citrullus vulgaris (watermelon) by genetic complementation in an Escherichia coli Cys− auxotroph

We have isolated cDNA clones encoding cysteine synthase (CSase, EC 4.2.99.8), which catalyzes the terminal step in cysteine biosynthesis, by direct genetic complementation of a Cys^? mutation in Escherichia coli with an expression library of Citrullus vulgaris (watermelon) cDNA. The library was constructed from 8-day-old etiolated seedlings of C. vulgaris in the λZAPII vector, converted to a plasmid library by in vivo excision, and then used for transformation of cysteine auxotroph E. coli NK3, which lacks the cysK and cysM loci. The complementing cDNA containing a 560 by 5′-untranslated region encodes a polypeptide of 325 amino acids of M_r 34342. The translational product reacted with an antibody raised against CSase A of Spinacia oleracea. CSase and β-pyrazolealanine synthase activities were demonstrated in vitro in extracts from E. coli cells expressing the cDNA. Genomic DNA blot analysis indicated the presence of a single copy of the gene, designated cysA, in the C. vulgaris genome. RNA blot hybridization indicated constitutive expression of cysA in cotyledons, hypocotyls and radicles of green and etiolated seedlings. These data suggested that this cDNA clone encodes CSase A the homolog of which in spinach is localized in the cytoplasm. The molecular phylogenetic tree of the amino acid sequences of CSaes from plants and bacteria suggested that there are three families in the CSase superfamily; the plant CSase A family, the plant CSase B family and the bacterial CSase family. The proteins in the plant CSase A family are the most conserved relative to the ancestral CSase protein.     

Integrated physical,genetic and genome map of chickpea (Cicer arietinum L.)

Physical map of chickpea was developed for the reference chickpea genotype (ICC 4958) using bacterial artificial chromosome (BAC) libraries targeting 71,094 clones (~12× coverage). High information content fingerprinting (HICF) of these clones gave high-quality fingerprinting data for 67,483 clones, and 1,174 contigs comprising 46,112 clones and 3,256 singletons were defined. In brief, 574 Mb genome size was assembled in 1,174 contigs with an average of 0.49 Mb per contig and 3,256 singletons represent 407 Mb genome. The physical map was linked with two genetic maps with the help of 245 BAC-end sequence (BES)-derived simple sequence repeat (SSR) markers. This allowed locating some of the BACs in the vicinity of some important quantitative trait loci (QTLs) for drought tolerance and reistance to Fusarium wilt and Ascochyta blight. In addition, fingerprinted contig (FPC) assembly was also integrated with the draft genome sequence of chickpea. As a result, ~965 BACs including 163 minimum tilling path (MTP) clones could be mapped on eight pseudo-molecules of chickpea forming 491 hypothetical contigs representing 54,013,992 bp (~54 Mb) of the draft genome. Comprehensive analysis of markers in abiotic and biotic stress tolerance QTL regions led to identification of 654, 306 and 23 genes in drought tolerance “QTL-hotspot” region, Ascochyta blight resistance QTL region and Fusarium wilt resistance QTL region, respectively. Integrated physical, genetic and genome map should provide a foundation for cloning and isolation of QTLs/genes for molecular dissection of traits as well as markers for molecular breeding for chickpea improvement.     

Development of SSR markers to study diversity in the genus Cymbidium

Cymbidium spp. are important potted flowers with extremely high ornamental and economic value. The present study reports the development of 14 new simple sequence repeat (SSR) markers through the construction of an enriched Cymbidium goeringii library and cross-amplification in Cymbidium sinensis and Cymbidium hybridium. Of 525, 322 (61.33%) clones had SSR motifs and among motifs di-nucleotides were predominant and followed by tri-nucleotide and tetra-nucleotide type. In polymorphic analysis using 14 newly developed SSRs, a total of 201 alleles across 96 Cymbidium accessions were detected with an average of 14.4 per locus. The average heterozygosity was 0.394. The average gene diversity and polymorphism information content values were 0.394 and 0.639, respectively. The mean genetic similarity coefficient was 0.4297, indicating a wide genetic variation among the Cymbidium accessions. These newly developed SSRs will be useful tools for genotype identification, germplasm conservation, molecular breeding, and assessments of genetic diversity and population structure in Cymbidium.     

Creation of BAC genomic resources for cocoa (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.) for physical mapping of RGA containing BAC clones

We have constructed and validated the first cocoa (Theobroma cacao L.) BAC library, with the aim of developing molecular resources to study the structure and evolution of the genome of this perennial crop. This library contains 36,864 clones with an average insert size of 120 kb, representing approximately ten haploid genome equivalents. It was constructed from the genotype Scavina-6 (Sca-6), a Forastero clone highly resistant to cocoa pathogens and a parent of existing mapping populations. Validation of the BAC library was carried out with a set of 13 genetically-anchored single copy and one duplicated markers. An average of nine BAC clones per probe was identified, giving an initial experimental estimation of the genome coverage represented in the library. Screening of the library with a set of resistance gene analogues (RGAs), previously mapped in cocoa and co-localizing with QTL for resistance to Phytophthora traits, confirmed at the physical level the tight clustering of RGAs in the cocoa genome and provided the first insights into the relationships between genetic and physical distances in the cocoa genome. This library represents an available BAC resource for structural genomic studies or map-based cloning of genes corresponding to important QTLs for agronomic traits such as resistance genes to major cocoa pathogens like Phytophthora spp (palmivora and megakarya), Crinipellis perniciosa and Moniliophthora roreri.Communicated by J.W. Snape     

Human BAC library: construction and rapid screening

We have constructed a human genomic bacterial artificial chromosome (BAC) library using high molecular weight DNA from a pre-pro-B cell line, FLEB14-14, with a normal male diploid karyotype. This BAC library consists of 96 000 clones with an average DNA insert size of 110 kb, covering the human genome approximately 3 times. The library can be screened by three different methods. (1) Probe hybridization to 31 high-density replica (HDR) filters: each filter contains 3072 BAC clones which were gridded in a 6×6 pattern. (2) Probe hybridization to two Southern blot filters to which 31 HindIII digests of the pooled 3072 BAC clones were loaded. This identifies a particular HDR filter for which further probe hybridization is performed to identify a particular clone(s). (3) Two-step polymerase chain reaction (PCR). First, PCR is applied to DNA samples prepared from ten superpools of 9600 BAC clones each to identify a particular superpool and the second PCR is applied to 40 unique DNA samples prepared from the four-dimensionally assigned BAC clones of the particular superpool. We present typical examples of the library screening using these three methods. The two-step PCR screening is particularly powerful since it allows us to isolate a desired BAC clone(s) within a day or so. The theoretical consideration of the advantage of this method is presented. Furthermore, we have adapted Vectorette method to our BAC library for the isolation of terminal sequences of the BAC DNA insert to facilitate contig formation by BAC walking.     

Members of the ribosomal protein S6 (RPS6) family act as pro-viral factor for tomato spotted wilt orthotospovirus infectivity in Nicotiana benthamiana

To identify host factors for tomato spotted wilt orthotospovirus (TSWV), a virus-induced gene silencing (VIGS) screen using tobacco rattle virus (TRV) was performed on Nicotiana benthamiana for TSWV susceptibility. To rule out any negative effect on the plants’ performance due to a double viral infection, the method was optimized to allow screening of hundreds of clones in a standardized fashion. To normalize the results obtained in and between experiments, a set of controls was developed to evaluate in a consist manner both VIGS efficacy and the level of TSWV resistance. Using this method, 4532 random clones of an N. benthamiana cDNA library were tested, resulting in five TRV clones that provided nearly complete resistance against TSWV. Here we report on one of these clones, of which the insert targets a small gene family coding for the ribosomal protein S6 (RPS6) that is part of the 40S ribosomal subunit. This RPS6 family is represented by three gene clades in the genome of Solanaceae family members, which were jointly important for TSWV susceptibility. Interestingly, RPS6 is a known host factor implicated in the replication of different plant RNA viruses, including the negative-stranded TSWV and the positive-stranded potato virus X.