scholarly journals PCR identification of durum wheat BAC clones containing genes coding for carotenoid biosynthesis enzymes and their chromosome localization

Genome ◽  
2004 ◽  
Vol 47 (5) ◽  
pp. 911-917 ◽  
Author(s):  
A Cenci ◽  
S Somma ◽  
N Chantret ◽  
J Dubcovsky ◽  
A Blanco
Keyword(s):  
Tetraploid Wheat ◽  
Bac Library ◽  
Pcr Amplification ◽  
Carotenoid Biosynthesis ◽  
Quality Trait ◽  
Substitution Lines ◽  
D Genome ◽  
Pcr Screening ◽  
Bac Clones ◽  
Essential Components

Carotenoids are essential components in all plants. Their accumulation in wheat seed determines the endosperm colour, which is an important quality trait in wheat. In this study, we report the isolation of BAC clones containing genes coding for three different enzymes of the carotenoid biosynthesis pathway: phytoene synthase (PSY), phytoene desaturase (PDS), and ζ-carotene desaturase (ZDS). Primers were designed on the basis of wheat ESTs similar to the sequences of these three genes in other species, and used to screen a BAC library from Triticum turgidum var. durum (2n = 28, genomes AABB). Eight, six, and nine 384-well plates containing at least one positive clone were found for PSY, PDS, and ZDS, respectively. BACs selected for each of these genes were then divided in two groups corresponding to the A and B genomes of tetraploid wheat, based on differences in the length of the PCR amplification products, conformation-sensitive gel electrophoresis (CSGE), or cleavage amplification polymorphisms. Positive clones were then assigned to chromosomes using a set of D genome substitution lines in T. turgidum var. durum 'Langdon'. PSY clones were localized on chromosomes 5A and 5B, PDS on chromosomes 4A and 4B, and ZDS on chromosomes 2A and 2B. The strategies used for the PCR screening of large BAC libraries and for the differentiation of BAC clones from different genomes in a polyploid species are discussed.Key words: wheat, carotenoid biosynthesis, BAC.

scholarly journals Utilization of Super BAC Pools and Fluidigm Access Array Platform for High-Throughput BAC Clone Identification: Proof of Concept

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Peter J. Maughan ◽  
Scott M. Smith ◽  
Joshua A. Raney
Keyword(s):  
Bac Library ◽  
Pcr Amplification ◽  
Artificial Chromosome ◽  
Nucleotide Polymorphisms ◽  
Bac Clone ◽  
Pcr Screening ◽  
Clone Identification ◽  
Bac Clones ◽  
Array Platform ◽  
Fluidigm Access Array

Bacterial artificial chromosome (BAC) libraries are critical for identifying full-length genomic sequences, correlating genetic and physical maps, and comparative genomics. Here we describe the utilization of the Fluidigm access array genotyping system in conjunction with KASPar genotyping technology to identify individual BAC clones corresponding to specific single-nucleotide polymorphisms (SNPs) from an Amplicon Express seven-plate super pooledAmaranthus hypochondriacusBAC library. Ninety-six SNP loci, spanning the length ofA. hypochondriacuslinkage groups 1, 2, and 15, were simultaneously tested for clone identification from four BAC super pools, corresponding to 28 384-well plates, using a single Fluidigm integrated fluidic chip (IFC). Forty-six percent of the SNPs were associated with a single unambiguous identified BAC clone. PCR amplification and next-generation sequencing of individual BAC clones confirmed the IFC clone identification. Utilization of the Fluidigm Dynamic array platform allowed for the simultaneous PCR screening of 10,752 BAC pools for 96 SNP tag sites in less than three hours at a cost of~$0.05per reaction.

Molecular characterization and chromosome-specific TRAP-marker development for Langdon durum D-genome disomic substitution lines

Genome ◽  
2006 ◽  
Vol 49 (12) ◽  
pp. 1545-1554 ◽  
Author(s):  
J. Li ◽  
D.L. Klindworth ◽  
F. Shireen ◽  
X. Cai ◽  
J. Hu ◽  
...  
Keyword(s):  
Triticum Turgidum ◽  
Tetraploid Wheat ◽  
Substitution Lines ◽  
Genome Chromosome ◽  
D Genome ◽  
Single Chromosome ◽  
B Genome ◽  
The Individual ◽  
Trap Marker

The aneuploid stocks of durum wheat ( Triticum turgidum L. subsp. durum (Desf.) Husnot) and common wheat ( T. aestivum L.) have been developed mainly in ‘Langdon’ (LDN) and ‘Chinese Spring’ (CS) cultivars, respectively. The LDN-CS D-genome chromosome disomic substitution (LDN-DS) lines, where a pair of CS D-genome chromosomes substitute for a corresponding homoeologous A- or B-genome chromosome pair of LDN, have been widely used to determine the chromosomal locations of genes in tetraploid wheat. The LDN-DS lines were originally developed by crossing CS nulli-tetrasomics with LDN, followed by 6 backcrosses with LDN. They have subsequently been improved with 5 additional backcrosses with LDN. The objectives of this study were to characterize a set of the 14 most recent LDN-DS lines and to develop chromosome-specific markers, using the newly developed TRAP (target region amplification polymorphism)-marker technique. A total of 307 polymorphic DNA fragments were amplified from LDN and CS, and 302 of them were assigned to individual chromosomes. Most of the markers (95.5%) were present on a single chromosome as chromosome-specific markers, but 4.5% of the markers mapped to 2 or more chromosomes. The number of markers per chromosome varied, from a low of 10 (chromosomes 1A and 6D) to a high of 24 (chromosome 3A). There was an average of 16.6, 16.6, and 15.9 markers per chromosome assigned to the A-, B-, and D-genome chromosomes, respectively, suggesting that TRAP markers were detected at a nearly equal frequency on the 3 genomes. A comparison of the source of the expressed sequence tags (ESTs), used to derive the fixed primers, with the chromosomal location of markers revealed that 15.5% of the TRAP markers were located on the same chromosomes as the ESTs used to generate the fixed primers. A fixed primer designed from an EST mapped on a chromosome or a homoeologous group amplified at least 1 fragment specific to that chromosome or group, suggesting that the fixed primers might generate markers from target regions. TRAP-marker analysis verified the retention of at least 13 pairs of A- or B-genome chromosomes from LDN and 1 pair of D-genome chromosomes from CS in each of the LDN-DS lines. The chromosome-specific markers developed in this study provide an identity for each of the chromosomes, and they will facilitate molecular and genetic characterization of the individual chromosomes, including genetic mapping and gene identification.

scholarly journals A Census of rRNA Genes and Linked Genomic Sequences within a Soil Metagenomic Library

2003 ◽  
Vol 69 (5) ◽  
pp. 2684-2691 ◽  
Author(s):  
Mark R. Liles ◽  
Brian F. Manske ◽  
Scott B. Bintrim ◽  
Jo Handelsman ◽  
Robert M. Goodman
Keyword(s):  
16S Rrna ◽  
Bac Library ◽  
Metagenomic Library ◽  
Pcr Amplification ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Acid Uptake ◽  
Direct Pcr ◽  
Bac Clone ◽  
Bac Clones

ABSTRACT We have analyzed the diversity of microbial genomes represented in a library of metagenomic DNA from soil. A total of 24,400 bacterial artificial chromosome (BAC) clones were screened for 16S rRNA genes. The sequences obtained from BAC clones were compared with a collection generated by direct PCR amplification and cloning of 16S rRNA genes from the same soil. The results indicated that the BAC library had substantially lower representation of bacteria among the Bacillus, α-Proteobacteria, and CFB groups; greater representation among the β- and γ-Proteobacteria, and OP10 divisions; and no rRNA genes from the domains Eukaryota and Archaea. In addition to rRNA genes recovered from the bacterial divisions Proteobacteria, Verrucomicrobia, Firmicutes, Cytophagales, and OP11, we identified many rRNA genes from the BAC library affiliated with the bacterial division Acidobacterium; all of these sequences were affiliated with subdivisions that lack cultured representatives. The complete sequence of one BAC clone derived from a member of the Acidobacterium division revealed a complete rRNA operon and 20 other open reading frames, including predicted gene products involved in cell division, cell cycling, folic acid biosynthesis, substrate metabolism, amino acid uptake, DNA repair, and transcriptional regulation. This study is the first step in using genomics to reveal the physiology of as-yet-uncultured members of the Acidobacterium division.

Langdon durum disomic substitution lines and aneuploid analysis in tetraploid wheat

Genome ◽  
1988 ◽  
Vol 30 (2) ◽  
pp. 222-228 ◽  
Author(s):  
L. R. Joppa ◽  
N. D. Williams
Keyword(s):  
Chromosomal Location ◽  
Triticum Turgidum ◽  
Wheat Cultivar ◽  
Tetraploid Wheat ◽  
Phenotypic Characteristics ◽  
Substitution Lines ◽  
D Genome ◽  
Chromosome Transmission ◽  
Complete Set ◽  
Aneuploid Analysis

A complete set of disomic substitution lines have been developed in the tetraploid wheat cultivar Langdon (Triticum turgidum L. var. durum). These aneuploid lines each have a pair of durum wheat homoeologues replaced by a pair of D-genome chromosomes transferred from 'Chinese Spring' hexaploid wheat. They can be used to determine the chromosomal location of genes, to transfer chromosomes from one cultivar or line of tetraploid wheat to another, to study the cytogenetics of tetraploid wheat, to determine gene linkages, and to identify chromosomes involved in translocations. Their phenotypic characteristics, their cytogenetic behavior, and suggested methods for their use are described.Key words: cytogenetics, monosomic, chromosome transmission, telosomic, chromosome substitution.

Chromosomal location of genes for supernumerary spikelet in tetraploid wheat

Genome ◽  
1990 ◽  
Vol 33 (4) ◽  
pp. 515-520 ◽  
Author(s):  
D. L. Klindworth ◽  
N. D. Williams ◽  
L. R. Joppa
Keyword(s):  
Chromosomal Location ◽  
Triticum Turgidum ◽  
Tetraploid Wheat ◽  
Substitution Lines ◽  
D Genome ◽  
Strong Inhibitor ◽  
Branched Spike ◽  
Chinese Spring Wheat ◽  
Monosomic Addition ◽  
A Minor

The supernumerary spikelet (SS) trait of durum wheat (Triticum turgidum L.), including the ramified and four-rowed spike traits, is characterized by an increased number of spikelets per spike. Chromosomal location of the SS gene(s) was determined by crossing the ramified spike line PI349056 to the set of 'Langdon' D-genome disomic substitution lines. Double monosomic F1 plants were backcrossed to PI349056 and the testcross F1 plants were classified for chromosome pairing and spike type. Segregation for spike type was observed in the testcross F2. Data indicated that the major SS gene was located on chromosome 2A. Subsequent crosses with the 'Langdon' 2A telosomics indicated that the major SS gene was located on the short arm of chromosome 2A. Segregation of the testcross F2 indicated that a minor SS gene was located on chromosome 2B. Results also indicated that inhibitors of SS may be located on the D-genome chromosomes and an additional experiment was designed to test this hypothesis. Eight D-genome monosomic addition lines were developed by backcrossing PI349056 from one to three times to plants containing D-genome univalents. The test populations contained two cytological types of plants, disomics having 14 pairs of durum chromosomes and D-genome monosomic additions having 14 pairs of durum chromosomes plus a D-genome monosome. Comparison of these two types of plants indicated that chromosome 2D (from 'Chinese Spring' wheat) had a strong inhibitor of SS expression.Key words: Triticum, branched spike, ramified spike, four-rowed spike, cytogenetics.

Aneuploid analyses of hybrid necrosis and hybrid chlorosis in tetraploid wheats using the D genome chromosome substitution lines of durum wheat

Genome ◽  
1992 ◽  
Vol 35 (4) ◽  
pp. 594-601 ◽  
Author(s):  
Koichiro Tsunewaki
Keyword(s):  
Durum Wheat ◽  
Tetraploid Wheat ◽  
Hybrid Necrosis ◽  
Chromosome Substitution ◽  
Substitution Lines ◽  
Genome Chromosome ◽  
D Genome ◽  
Chromosome Substitution Lines ◽  
Complementary Genes ◽  
Tetraploid Wheats

Chromosomal locations of the Ne1 gene, one of the two complementary genes for type 1 hybrid necrosis, and two complementary genes, Cs1 and Cs2, for type 2 hybrid chlorosis in tetraploid wheats were determined by aneuploid analyses employing the D genome chromosome substitution lines of 'Langdon' durum wheat. The Ne1 gene of 'Langdon' is located on chromosome 5B, whereas the Cs1 gene of Triticum dicoccum 'Hokudai' and the Cs2 gene of T. timopheevi are located on chromosomes 5A and 4G, respectively. Chromosomes 4B and 4G show almost complete functional compensation, though they rarely pair with each other, but chromosome 4D of T. aestivum 'Chinese Spring' has only half the ability of chromosome 4G in compensating for chromosome 4B on the fertilization ability of the male gamete. The results have demonstrated the usefulness of the D genome chromosome substitution lines of durum wheat for determining the chromosomes carrying major genes in tetraploid wheat. The results of these studies support the reallocation of chromosome 4A to the B genome.Key words: durum wheat, hybrid necrosis, hybrid chlorosis, aneuploid analyses, chromosome substitution lines.

Highly Recombinogenic Regions at Seed Storage Protein Loci on Chromosome 1DS of Aegilops tauschii, the D-Genome Donor of Wheat

Genetics ◽  
2000 ◽  
Vol 155 (1) ◽  
pp. 361-367 ◽  
Author(s):  
Wolfgang Spielmeyer ◽  
Odile Moullet ◽  
André Laroche ◽  
Evans S Lagudah
Keyword(s):  
Genetic Distance ◽  
Storage Protein ◽  
Seed Storage Protein ◽  
Aegilops Tauschii ◽  
Bac Library ◽  
Seed Storage ◽  
D Genome ◽  
Rflp Map ◽  
Genome Donor ◽  
The Relationship

Abstract A detailed RFLP map was constructed of the distal end of the short arm of chromosome 1D of Aegilops tauschii, the diploid D-genome donor species of hexaploid wheat. Ae. tauschii was used to overcome some of the limitations commonly associated with molecular studies of wheat such as low levels of DNA polymorphism. Detection of multiple loci by most RFLP probes suggests that gene duplication events have occurred throughout this chromosomal region. Large DNA fragments isolated from a BAC library of Ae. tauschii were used to determine the relationship between physical and genetic distance at seed storage protein loci located at the distal end of chromosome 1DS. Highly recombinogenic regions were identified where the ratio of physical to genetic distance was estimated to be <20 kb/cM. These results are discussed in relation to the genome-wide estimate of the relationship between physical and genetic distance.

A cytological and molecular analysis of D-genome chromosome retention following F2–F6 generations of hexaploid×tetraploid wheat crosses

2018 ◽  
Vol 69 (2) ◽  
pp. 121 ◽  
Author(s):  
Sriram Padmanaban ◽  
Peng Zhang ◽  
Mark W. Sutherland ◽  
Noel L. Knight ◽  
Anke Martin
Keyword(s):  
Durum Wheat ◽  
Tetraploid Wheat ◽  
Triticum Aestivum L ◽  
Food Crops ◽  
Cytological Analysis ◽  
Genome Chromosome ◽  
D Genome ◽  
Ploidy Levels ◽  
F2 Generation ◽  
Global Food

Both hexaploid bread wheat (AABBDD) (Triticum aestivum L.) and tetraploid durum wheat (AABB) (T. turgidum spp. durum) are highly significant global food crops. Crossing these two wheats with different ploidy levels results in pentaploid (AABBD) F1 lines. This study investigated the differences in the retention of D chromosomes between different hexaploid × tetraploid crosses in subsequent generations by using molecular and cytological techniques. Significant differences (P < 0.05) were observed in the retention of D chromosomes in the F2 generation depending on the parents of the original cross. One of the crosses, 2WE25 × 950329, retained at least one copy of each D chromosome in 48% of its F2 lines. For this cross, the retention or elimination of D chromosomes was determined through several subsequent self-fertilised generations. Cytological analysis indicated that D chromosomes were still being eliminated at the F5 generation, suggesting that in some hexaploid × tetraploid crosses, D chromosomes are unstable for many generations. This study provides information on the variation in D chromosome retention in different hexaploid × tetraploid wheat crosses and suggests efficient strategies for utilising D genome retention or elimination to improve bread and durum wheat, respectively.

scholarly journals Identification and Molecular Mapping of a Gene Conferring Resistance to Pyrenophora tritici-repentis Race 3 in Tetraploid Wheat

2006 ◽  
Vol 96 (8) ◽  
pp. 885-889 ◽  
Author(s):  
P. K. Singh ◽  
J. L. Gonzalez-Hernandez ◽  
M. Mergoum ◽  
S. Ali ◽  
T. B. Adhikari ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Molecular Mapping ◽  
Recombinant Inbred Lines ◽  
Tetraploid Wheat ◽  
Recessive Gene ◽  
Tan Spot ◽  
Substitution Lines ◽  
Disease Reaction ◽  
Pyrenophora Tritici Repentis ◽  
The Cross

Race 3 of the fungus Pyrenophora tritici-repentis, causal agent of tan spot, induces differential symptoms in tetraploid and hexaploid wheat, causing necrosis and chlorosis, respectively. This study was conducted to examine the genetic control of resistance to necrosis induced by P. tritici-repentis race 3 and to map resistance genes identified in tetraploid wheat (Triticum turgidum). A mapping population of recombinant inbred lines (RILs) was developed from a cross between the resistant genotype T. tur-gidum no. 283 (PI 352519) and the susceptible durum cv. Coulter. Based on the reactions of the Langdon-T. dicoccoides (LDN[DIC]) disomic substitution lines, chromosomal location of the resistance genes was determined and further molecular mapping of the resistance genes for race 3 was conducted in 80 RILs of the cross T. turgidum no. 283/Coulter. Plants were inoculated at the two-leaf stage and disease reaction was assessed 8 days after inoculation based on lesion type. Disease reaction of the LDN(DIC) lines and molecular mapping on the T. turgidum no. 283/Coulter population indicated that the gene, designated tsn2, conditioning resistance to race 3 is located on the long arm of chromosome 3B. Genetic analysis of the F2 generation and of the F4:5 and F6:7 families indicated that a single recessive gene controlled resistance to necrosis induced by race 3 in the cross studied.

Construction and characterization of bacterial artificial chromosome library of black-handed spider monkey (Ateles geoffroyi)

Genome ◽  
2004 ◽  
Vol 47 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Yaping Qian ◽  
Li Jin ◽  
Bing Su
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Large Scale ◽  
Bac Library ◽  
Artificial Chromosome ◽  
Spider Monkey ◽  
Ateles Geoffroyi ◽  
Comparative Genomic ◽  
Average Insert Size ◽  
Bac Clones ◽  
Genomic Study

The large-insert genomic DNA library is a critical resource for genome-wide genetic dissection of target species. We constructed a high-redundancy bacterial artificial chromosome (BAC) library of a New World monkey species, the black-handed spider monkey (Ateles geoffroyi). A total of 193 152 BAC clones were generated in this library. The average insert size of the BAC clones was estimated to be 184.6 kb with the small inserts (50-100 kb) accounting for less than 3% and the non-recombinant clones only 1.2%. Assuming a similar genome size with humans, the spider monkey BAC library has about 11× genome coverage. In addition, by end sequencing of randomly selected BAC clones, we generated 367 sequence tags for the library. When blasted against human genome, they showed a good correlation between the number of hit clones and the size of the chromosomes, an indication of unbiased chromosomal distribution of the library. This black-handed spider monkey BAC library would serve as a valuable resource in comparative genomic study and large-scale genome sequencing of nonhuman primates.Key words: black-handed spider monkeys, Ateles geoffroyi, BAC library.

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