Zengjun Qi

Erratum to: Identification and development of diagnostic markers for a powdery mildew resistance gene on chromosome 2R of Chinese rye cultivar Jingzhouheimai

Under the heading Discussion the sentence ‘‘Hysing et al. (2007) developed Hamlet, a resistant 2BS-2RL translocation line, by using a susceptible wheat cultivar and resistant rye cv. Chaupon’’ should be replaced by ‘‘A new wheat-rye translocation (T2BS2RL, SLU) developed by Merker and Forsström (2000) showed resistance to multiple wheat diseases after evaluation by Hysing et al. (2007).’’ The following reference should be included in the reference list of the article: ‘‘Merker A, and Forsström PO (2000) Isolation of mildew resistant wheat-rye translocations from a double substitution line. Euphytica 115:167–172.’’

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.

Physical localization of a novel blue-grained gene derived from Thinopyrum bessarabicum

Blue wheat grain contains different groups of pigments that can be used for making specialty foods or as food colorants. Thinopyrum bessarabicum, a wild relative of wheat, carries a blue-grained gene on chromosome 4J. In this study, we analyzed the mitotic chromosomes of 159 F7 lines derived from the cross between Triticum aestivum cv. Chinese Spring (CS) and a CS–Th. bessarabicum amphiploid by using multi-color fluorescence in situ hybridization, genomic in situ hybridization, and newly developed chromosome 4J-specific DNA markers. Intact chromosome 4J and various 4J chromosomal segments were identified in the 159 lines. The blue-grained gene of Th. bessarabicum was physically localized to the region between the centromere and FL0.52 on chromosome arm 4JL. The chromosomal location of this gene differed from the location of previously reported blue-grained genes. In addition, a strong dosage effect was observed with this gene. These results suggest that the blue-grained gene in Th. bessarabicum represents a novel gene locus for blue aleurone, designated BaThb. The wheat lines and 4J chromosome-specific molecular markers developed in this study will facilitate the introgression and utilization of BaThb for wheat nutritional quality improvement.

A new secondary reciprocal translocation discovered in Chinese wheat

A new wheat-rye secondary reciprocal translocation involving T1RS·7DL and T7DS·1BL was detected by chromosome C-banding and genomic in situ hybridization (GISH). The meiotic configuration analysis combined with C-banding and GISH on F1 hybrids of this newly discovered translocation with T1RS·1BL and Chinese Spring Dt7DS indicated that the new translocation probably resulted from a secondary reciprocal translocation between the primary translocation T1RS·1BL and 7D in the progenies of Aifeng3//Mengxian201/Neuzucht. On the basis of the cytological analysis of progenies and recombinant inbred lines (RILs) (derived from a cross between T1RS·7DL, T7DS·1BL and T1RS·1BL), the translocation chromosomes T1RS·7DL and T7DS·1BL transmitted readily, and appeared in most of the progenies.

Multiple structural aberrations and physical mapping of rye chromosome 2R introgressed into wheat

Multiple structural aberrations produced by chromosome breakage and reunion not only provide new germplasm for enriching genetic diversity but are also helpful for physical mapping of alien chromosomes introgressed into wheat. In this study, mass structural aberrations of rye chromosome 2R were induced by means of spontaneous breakage and reunion, gametocidal chromosome action and irradiation. A total of 88 chromosome 2R aberrations were identified in 65 plants. From the self-pollinated progenies of plants carrying these aberrations, 19 stable lines with different segments of chromosome 2R which included seven whole arm, six small segmental and three large segmental translocations, one deletion and two ditelosomic additions, were subsequently identified and characterized using cytogenetic and molecular markers. Based on these lines, 88 markers specific for chromosome 2R were physically mapped to 13 different blocks of 2R with three in arm 2RS and 10 in arm 2RL. The powdery mildew resistance gene PmJZHM2RL was located to a region corresponding to the block 2RL-7. A total of eighteen 2R-specific EST markers were located in the same block where ten were derived from genes that were up-regulated during powdery mildew infection. Potential use of these stable aberrations and the colinearity of chromosome 2R with corresponding chromosomes in the other model monocot species were discussed.

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.

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.

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

Key messageHigh-resolution multiplex oligonucleotide FISH revealed the frequent occurrence of structural chromosomal rearrangements and polymorphisms in widely grown wheat cultivars and their founders.AbstractOver 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.

RNA-seq facilitates development of chromosome-specific markers and transfer of rye chromatin to wheat

Transcriptome shotgun sequencing (RNA-seq) provides an abundant resource for developing molecular markers specifically related to functional genes. In this study, transcriptomes of Chinese rye cultivar Jingzhouheimai (JZHM) challenged with powdery mildew pathogen Bgt (Blumeria graminis f. sp. tritici) were obtained and used to develop expressed sequence tag (EST)-based simple sequence repeat (SSR) and sequence-tagged site (STS) markers. A total of 866 primer sets for EST-SSRs and STSs were designed, from which we developed 401 rye-specific markers. The highest level of polymorphism was observed in EST-SSRs (56.73%) followed by STS2 (49.07%) designed via rye-specific contigs and STS1 (35.90%) primers designed from upregulated contigs. Genotyping with newly developed markers along with cytogenetic techniques allowed us to identify nine wheat alien chromosome lines from the cross of Zhoumai 18/Jinghui 1 (Jinghui 1 is an amphiploid of wheat landrace Huixianhong and JZHM) carrying rye chromosome 6R or 6R segments of different length, which permitted preliminary location of the powdery mildew resistance gene PmJZHM6RL and 12 specific markers to 6RL FL 0.51–1.0. 5R-specific markers and genomic in situ hybridization/fluorescence in situ hybridization detected MtA5RL and T5AS·5AL-5RL chromosomes among 41 F5 plants from the cross CS ph1bph1b/MA5R, and aberrations permitted the location of the hairy peduncle gene (Hp) and marker XLFZ3473 to the region 5RL FL 0.78–1.0 with another nine markers locating to 5RL 0.0–0.78. The chromosome-specific markers and chromosome aberrations developed in this study will facilitate the introgression of rye chromatin into wheat.

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

BackgroundArachis 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.ResultsA 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.ConclusionsThe 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.