Physical organization of repetitive sequences and chromosome diversity of barley revealed by fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) using oligonucleotides is a simple and convenient method for chromosome research. In this study, 34 of 46 previously developed oligonucleotides produced signals in barley. Together with two plasmid clones and one PCR-amplified cereal centromere repeat (CCS1) probe, 37 repetitive sequences were chromosomally located produced three types of signals covering different positions on the chromosomes. The centromeric and pericentric regions had a more complex genomic organization and sequence composition probably indicative of higher contents of heterochromatin. An efficient multi-plex probe containing eight oligonucleotides and a plasmid clone of 45S rDNA was developed. Thirty-three barley karyotypes were developed and compared. Among them, 11 irradiation-induced mutants of cultivar 08-49 showed no chromosomal variation, whereas 22 cultivar and landrace accessions contained 28 chromosomal polymorphisms. Chromosome 4H was the most variable and 6H was the least variable based on chromosome polymorphic information content (CPIC). Five polymorphic chromosomes (1H-2, 2H-1, 3H-3, 5H-2, and 6H-2) were dominant types, each occurring in more than 50% of accessions. The multi-plex probe should facilitate identification of further chromosomal polymorphisms in barley.

High-resolution chromosome painting with repetitive and single-copy oligonucleotides in Arachis species identifies structural rearrangements and genome differentiation.

BACKGROUND: Arachis contains 80 species that carry many beneficial genes that can be utilized in the genetic improvement of peanut (Arachis hypogaea L. 2n = 4x = 40, genome AABB). Chromosome engineering is a powerful technique by which these genes can be transferred and utilized in cultivated peanut. However, their small chromosomes and insufficient cytological markers have made chromosome identification and studies relating to genome evolution quite difficult. The development of efficient cytological markers or probes is very necessary for both chromosome engineering and genome discrimination in cultivated peanut. RESULTS: A simple and efficient oligonucleotide multiplex probe to distinguish genomes, chromosomes, and chromosomal aberrations of peanut was developed based on eight single-stranded oligonucleotides (SSONs) derived from repetitive sequences. High-resolution karyotypes of 16 Arachis species, two interspecific F1 hybrids, and one radiation-induced M1 plant were then developed by fluorescence in situ hybridization (FISH) using oligonucleotide multiplex, 45S and 5S rDNAs, and genomic in situ hybridization (GISH) using total genomic DNA of A. duranensis (2n = 2x = 20, AA) and A. ipaënsis (2n = 2x = 20, BB) as probes. Genomes, chromosomes, and aberrations were clearly identifiable in the established karyotypes. All eight cultivars had similar karyotypes, whereas the eight wild species exhibited various chromosomal variations. In addition, a chromosome-specific SSON library was developed based on the single-copy sequence of chromosome 6A of A. duranensis. In combination with repetitive SSONs and rDNA FISH, the single-copy SSON library was applied to identify the corresponding A3 chromosome in the A. duranensis karyotype. CONCLUSIONS: The development of repetitive and single-copy SSON probes for FISH and GISH provides useful tools for the differentiation of chromosomes and identification of structural chromosomal rearrangement. It facilitates the development of high-resolution karyotypes and detection of chromosomal variations in Arachis species. To our knowledge, the methodology presented in this study demonstrates for the first time the correlation between a sequenced chromosome region and a cytologically identified chromosome in peanut.

Structural chromosome rearrangements and polymorphisms identified in Chinese wheat cultivars by high-resolution multiplex oligonucleotide FISH.

KEY MESSAGE: High-resolution multiplex oligonucleotide FISH revealed the frequent occurrence of structural chromosomal rearrangements and polymorphisms in widely grown wheat cultivars and their founders. Over 2000 wheat cultivars including 19 founders were released and grown in China from 1949 to 2000. To understand the impact of breeding selection on chromosome structural variations, high-resolution karyotypes of Chinese Spring (CS) and 373 Chinese cultivars were developed and compared by FISH (fluorescence in situ hybridization) using an oligonucleotide multiplex probe based on repeat sequences. Among them, 148 (39.7%) accessions carried 14 structural rearrangements including three single translocations (designated as T), eight reciprocal translocations (RT), one pericentric inversion (perInv), and two combined variations having both the deletion and single translocations. Five rearrangements were traced to eight founders, including perInv 6B detected in 57 cultivars originating from Funo, Abbondanza, and Fan 6, T 1RS∙1BL in 47 cultivars derived from the Lovrin series, RT 4AS∙4AL-1DS/1DL∙1DS-4AL in 31 varieties from Mazhamai and Bima 4, RT 1RS∙7DL/7DS∙1BL in three cultivars was from Aimengniu, and RT 5BS∙5BL-5DL/5DS∙5DL-5BL was only detected in Youzimai. In addition to structural rearrangements, 167 polymorphic chromosome blocks (defined as unique signal patterns of oligonucleotide repeat probes distributed within chromosomes) were identified, and 59 were present in one or more founders. Some specific types were present at high frequencies indicating selective blocks in Chinese wheat varieties. All cultivars and CS were clustered into four groups and 15 subgroups at chromosome level. Common block patterns occurred in the same subgroup. Origin, geographic distribution, probable adaptation to specific environments, and potential use of these chromosomal rearrangements and blocks are discussed.

A simple and efficient non-denaturing FISH method for maize chromosome differentiation using single-strand oligonucleotide probes.

Single-strand oligonucleotides (SSONs hereafter) as probes are becoming a powerful method of chromosome painting in many species. In this study, nine SSONs ((ACT)10, (ACT)19, Knob-1, Knob-2, Knob-3, CentC69-1, MR68-3, K10-72-1, and TR1-357-2) were developed and used for chromosome identification in 16 maize (Zea mays L., 2n = 20) inbred lines and hybrids by non-denaturing fluorescence in situ hybridization (ND-FISH). Each SSON produced clear signals on 2-10 chromosomes of inbred lines B73 and Mo17. A multiplex probe set containing four SSONs ((ACT)10, Knob-2, CentC69-1, and MR68-3) clearly characterized all maize chromosomes in the 16 lines by a single round of ND-FISH and revealed genetic variation at a chromosome level. For example, unique signals on chromosome 6 clearly distinguished all 16 genotypes. The SSONs and multiplex probe developed in this research will facilitate genotype identification and chromosome research in maize.

Development of oligonucleotides and multiplex probes for quick and accurate identification of wheat and Thinopyrum bessarabicum chromosomes.

In comparison with general FISH for preparing probes in terms of time and cost, synthesized oligonucleotide (oligo hereafter) probes for FISH have many advantages such as ease of design, synthesis, and labeling. Low cost and high sensitivity and resolution of oligo probes greatly simplify the FISH procedure as a simple, fast, and efficient method of chromosome identification. In this study, we developed new oligo and oligo multiplex probes to accurately and efficiently distinguish wheat (Triticum aestivum, 2n = 6x, AABBDD) and Thinopyrum bessarabicum (2n = 2x = 14, JJ) chromosomes. The oligo probes contained more nucleotides or more repeat units that produced stronger signals for more efficient chromosome painting. Four Th. bessarabicum-specific oligo probes were developed based on genomic DNA sequences of Th. bessarabicum chromosome arm 4JL, and one of them (oligo DP4J27982) was pooled with the oligo multiplex #1 to simultaneously detect wheat and Th. bessarabicum chromosomes for quick and accurate identification of Chinese Spring (CS) - Th. bessarabicum alien chromosome introgression lines. Oligo multiplex #4 revealed chromosome variations among CS and eight wheat cultivars by a single round of FISH analysis. This research demonstrated the high efficiency of using oligos and oligo multiplexes in chromosome identification and manipulation.

Chromosome aberrations induced by zebularine in triticale.

Chromosome engineering is an important approach for generating wheat germplasm. Efficient development of chromosome aberrations will facilitate the introgression and application of alien genes in wheat. In this study, zebularine, a DNA methylation transferase inhibitor, was successfully used to induce chromosome aberrations in the octoploid triticale cultivar Jinghui#1. Dry seeds were soaked in zebularine solutions (250, 500, and 750 µmol/L) for 24 h, and the 500 µmol/L treatment was tested in three additional treatment times, i.e., 12, 36, and 48 h. All treatments induced aberrations involving wheat and rye chromosomes. Of the 920 cells observed in 67 M1 plants, 340 (37.0%) carried 817 aberrations with an average of 0.89 aberrations per cell (range: 0-12). The aberrations included probable deletions, telosomes and acentric fragments (49.0%), large segmental translocations (28.9%), small segmental translocations (17.1%), intercalary translocations (2.6%), long chromosomes that could carry more than one centromere (2.0%), and ring chromosomes (0.5%). Of 510 M2 plants analyzed, 110 (21.6%) were found to carry stable aberrations. Such aberrations included 79 with varied rye chromosome numbers, 7 with wheat and rye chromosome translocations, 15 with possible rye telosomes/deletions, and 9 with complex aberrations involving variation in rye chromosome number and wheat-rye translocations. These indicated that aberrations induced by zebularine can be steadily transmitted, suggesting that zebularine is a new efficient agent for chromosome manipulation.

Physical mapping of chromosome 4J of Thinopyrum bessarabicum using gamma radiation-induced aberrations.

KEY MESSAGE: Gamma radiation induced a series of structural aberrations involving Thinopyrum bessarabicum chromosome 4J. The aberrations allowed for deletion mapping of 101 4J-specific markers and fine mapping of blue-grained gene BaThb. Irradiation can induce translocations and deletions to assist physically locating genes and markers on chromosomes. In this study, a 12-Gy dosage of (60)Co-γ was applied to pollen and eggs of a wheat (Triticum aestivum) landrace Chinese Spring (CS)-Thinopyrum bessarabicum chromosome 4J disomic addition line (DA4J), and the gametes from irradiated plants were fertilized with normal CS eggs or pollen to produce M1 seeds. Based on genomic in situ hybridization analysis of 261 M1 plants, we identified 74 lines carrying structural aberrations involving chromosome 4J with the higher aberration rate in treated pollen (31.2 %) than in the treated eggs (21.3 %). We further identified 43 (53.8 %) lines with structural aberrations on chromosome 4J by analyzing another 80 M1 plants with 74 4J-specific markers, indicating that combining molecular and cytological methods was more efficient for detecting chromosome aberrations. Marker analysis thus was performed prior to cytogenetic identification on M2-M4 seeds to detect chromosome structural aberrations. Sixty-eight M3 lines with structural aberrations on chromosome 4J and six previously obtained chromosome 4J alien lines were then analyzed using 101 chromosome 4J-specific markers. After combining marker results with chromosome aberrations in each line, chromosome 4J was physically divided into 24 segmental blocks with 7 in the short arm and 17 in the long arm. The blue-grained gene BaThb was further mapped into the region corresponding to block 4JL-11. The chromosome aberrations and the physical map developed in this research provide useful stocks and tools for introgression of genes on chromosome 4J into wheat.

Characterization of a wheat-Thinopyrum bessarabicum (T2JS-2BS.2BL) translocation line.

Thinopyrum bessarabicum (2n = 2x = 14, JJ or E(b)E(b)) is an important genetic resource for wheat improvement due to its salinity tolerance and disease resistance. Development of wheat-Th. bessarabicum translocation lines will facilitate its practical utilization in wheat improvement. In this study, a novel wheat-Th. bessarabicum translocation line T2JS-2BS.2BL, which carries a segment of Th. bessarabicum chromosome arm 2JS was identified and further characterized using sequential chromosome C-banding, genomic in situ hybridization (GISH), dual-color fluorescent in situ hybridization (FISH) and DNA markers. The translocation breakpoint was mapped within bin C-2BS1-0.53 of chromosome 2B through marker analysis. Compared to the Chinese Spring (CS) parent and to CS-type lines, the translocation line has more fertile spikes per plant, longer spikes, more grains per spike and higher yield per plant, which suggests that the alien segment carries yield-related genes. However, plants with the translocation are also taller, head later and have lower 1,000-kernel weight than CS or CS-type lines. By using markers specific to the barley photoperiod response gene Ppd-H1, it was determined that the late heading date was conferred by a recessive allele located on the 2JS segment. In addition, four markers specific for the translocated segment were identified, which can be used for marker-aided screening.

[Development and identification of a set of Triticum aestivum-Thinopyrum bessarabicum disomic alien addition lines].

In order to transfer the genes for salt tolerance and disease resistance from Thinopyrum bessarabicum into wheat, the hybrid progenies between T. aestivum cv. Chinese Spring and T. aestivum cv. Chinese Spring-amphiploid Th. bessarabicum were screened. A set of T. aestivum-Th. bessarabicum disomic addition lines was developed with the assistance of mitotic chromosome C-banding and genomic in situ hybridization (GISH), as well as GISH on meiotic M I chromosome preparations. The results indicated that all the wheat chromosomes in the lines remained unchanged karyotypically, while the added Th. bessarabicum chromosomes paired regularly in meiosis. The developed disomic addition lines were designated temporarily as DAJ1, DAJ2, DAJ3, DAJ4, DAJ5, DAJ6 and DAJ7 respectively. Determination of the homoelogous groups of the added Th. bessarabicum chromosomes and localization of the genes for salt tolerance and disease resistance are undergoing.