Shulan Fu

Alterations and abnormal mitosis of wheat chromosomes induced by wheat-rye monosomic addition lines.

Background Wheat-rye addition lines are an old topic. However, the alterations and abnormal mitotic behaviours of wheat chromosomes caused by wheat-rye monosomic addition lines are seldom reported. Methodology/Principal Findings Octoploid triticale was derived from common wheat T. aestivum L. ‘Mianyang11’×rye S. cereale L. ‘Kustro’ and some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ followed by self-fertilization. Genomic in situ hybridization (GISH) using rye genomic DNA and fluorescence in situ hybridization (FISH) using repetitive sequences pAs1 and pSc119.2 as probes were used to analyze the mitotic chromosomes of these progeny. Strong pSc119.2 FISH signals could be observed at the telomeric regions of 3DS arms in ‘Mianyang11’. However, the pSc119.2 FISH signals were disappeared from the selfed progeny of 4R monosomic addition line and the changed 3D chromosomes could be transmitted to next generation stably. In one of the selfed progeny of 7R monosomic addition line, one 2D chromosome was broken and three 4A chromosomes were observed. In the selfed progeny of 6R monosomic addition line, structural variation and abnormal mitotic behaviour of 3D chromosome were detected. Additionally, 1A and 4B chromosomes were eliminated from some of the progeny of 6R monosomic addition line. Conclusions/Significance These results indicated that single rye chromosome added to wheat might cause alterations and abnormal mitotic behaviours of wheat chromosomes and it is possible that the stress caused by single alien chromosome might be one of the factors that induced karyotype alteration of wheat.

Oligonucleotide Probes for ND-FISH Analysis to Identify Rye and Wheat Chromosomes

Genomic in situ hybridization (GISH) has been widely used to detect rye (Secale cereale L.) chromosomes in wheat (Triticum aestivum L.) introgression lines. The routine procedure of GISH using genomic DNA of rye as a probe is time-consuming and labor-intensive because of the preparation and labeling of genomic DNA of rye and denaturing of chromosomes and probes. In this study, new oligonucleotide probes Oligo-1162, Oligo-pSc200 and Oligo-pSc250 were developed. The three new probes can be used for non-denaturing fluorescence in situ hybridization (ND-FISH) assays and replace genomic DNA of rye as a probe to discriminate rye chromosomes in wheat backgrounds. In addition, previously developed oligonucleotide probes Oligo-pSc119.2-1, Oligo-pSc119.2-2, Oligo-pTa535-1, Oligo-pTa535-2, Oligo-pTa71-2, Oligo-pAWRC.1 and Oligo-CCS1 can also be used for ND-FISH of wheat and rye. These probes have provided an easier, faster and more cost-effective method for the FISH analysis of wheat and hybrids derived from wheat × rye.

New Types of Wheat Chromosomal Structural Variations in Derivatives of Wheat-Rye Hybrids

Background Chromosomal rearrangements induced by wheat-rye hybridization is a very well investigated research topic. However, the structural alterations of wheat chromosomes in wheat-rye hybrids are seldom reported. Methodology/Principal Findings Octoploid triticale lines were derived from common wheat Triticum. aestivum L. ‘Mianyang11’×rye Secale cereale L. ‘Kustro’. Some progeny were obtained by the controlled backcrossing of triticale with ‘Mianyang11’ and common wheat T. aestivum L. ‘Chuannong27’ followed by self-fertilization. Fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) using Oligo-pSc119.2-1, Oligo-pTa535-1 and rye genomic DNA as probes were used to analyze the mitotic chromosomes of these progeny. Alterations of wheat chromosomes including 5A, 6A, 1B, 2B, 6B, 7B, 1D, 3D and 7D were observed. 5AL arm carrying intercalary Oligo-pSc119.2-1, Oligo-pTa535-1 or both Oligo-pSc119.2-1 and Oligo-pTa535-1 signals, 6AS, 1BS and 1DL arms containing terminal Oligo-pSc119.2-1 signal, 6BS and 3DS arms without terminal Oligo-pSc119.2-1 signal, 7BS without subtelomeric Oligo-pSc119.2-1 signal and 7DL with intercalary Oligo-pSc119.2-1 signal have been observed. However, these changed wheat chromosomes have not been detected in ‘Mianyang11’ and Chuannong 27. The altered 5A, 6A, 7B and 7D chromosomes in this study have not been reported and represent several new karyotype structures of common wheat chromosomes. Conclusions/Significance These rearranged wheat chromosomes in the present study afford some new genetic variations for wheat breeding program and are valuable materials for studying the biological function of tandem repetitive DNA sequences.

LOSS OF PARENTAL CODING SEQUENCES IN AN EARLY GENERATION OF WHEAT-RYE ALLOPOLYPLOID

During wheat-rye allopolyploidization, the characteristics of the sequences in the bands that appear in both parents and progeny are still unclear. In this study, two different combinations of wheat (Triticum aestivum L.) and rye (Secale cereale L.), including F1 hybrids and the first and second allopolyploid generations, were analyzed by PCR and sequencing using 60 wheat expressed sequence tag (EST)–derived single-sequence repeat markers and EST-derived sequence-tagged site markers. Thirty markers produced the same bands from parental plants, F1 plants, and amphiploids. Seven of the 30 markers amplified identical sequences from wheat and rye parents. Most of these sequences have high similarity between the two parental plants. The variation patterns of sequences in the bands produced by the seven markers were observed. In the F1 hybrids and amphiploids, loss of parental sequences was observed and the frequency of losing rye sequences was higher than that of losing wheat sequences. In addition, a few sequences in these bands exhibited significant differences, indicating that parental sequences changed drastically during allopolyploidization. Therefore, the fact that the parents and progeny contained the same bands should not be regarded as conservation. The results in this study add to the investigations dealing with variation patterns of coding sequences during wheat-rye allopolyploidization.

Genetic and epigenetic variations induced by wheat-rye 2R and 5R monosomic addition lines.

Background Monosomic alien addition lines (MAALs) can easily induce structural variation of chromosomes and have been used in crop breeding; however, it is unclear whether MAALs will induce drastic genetic and epigenetic alterations. Methodology/Principal Findings In the present study, wheat-rye 2R and 5R MAALs together with their selfed progeny and parental common wheat were investigated through amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) analyses. The MAALs in different generations displayed different genetic variations. Some progeny that only contained 42 wheat chromosomes showed great genetic/epigenetic alterations. Cryptic rye chromatin has introgressed into the wheat genome. However, one of the progeny that contained cryptic rye chromatin did not display outstanding genetic/epigenetic variation. 78 and 49 sequences were cloned from changed AFLP and MSAP bands, respectively. Blastn search indicated that almost half of them showed no significant similarity to known sequences. Retrotransposons were mainly involved in genetic and epigenetic variations. Genetic variations basically affected Gypsy-like retrotransposons, whereas epigenetic alterations affected Copia-like and Gypsy-like retrotransposons equally. Genetic and epigenetic variations seldom affected low-copy coding DNA sequences. Conclusions/Significance The results in the present study provided direct evidence to illustrate that monosomic wheat-rye addition lines could induce different and drastic genetic/epigenetic variations and these variations might not be caused by introgression of rye chromatins into wheat. Therefore, MAALs may be directly used as an effective means to broaden the genetic diversity of common wheat.

Isolation of rye-specific DNA fragment and genetic diversity analysis of rye genus Secale L. using wheat SSR markers

Rye (Secale cereale L.) is one of the most widely utilized sources in wheat breeding. Biochemical (Dhaliwal et al. 1988) and cytological (Belyayev et al. 2001) strategies have been used to identify rye chromatins in wheat backgrounds. The major limitation of these techniques is that they are highly technical and time-consuming. Species-specific PCR-based markers are useful and convenient tools for detecting alien chromosome segments incorporated into wheat genomes. Some rye-specific PCR-based markers have been successfully developed by random amplified polymorphic DNA (RAPD) analysis (Iqbal and Rayburn 1995; Katto et al. 2004; Liu et al. 2008; Jia et al. 2009). Simple sequence repeat (SSR) markers are another useful tool to develop species-specific DNA fragments. As previously reported, many wheat SSR markers were successfully applied to the amplification of DNA from several related species, such as triticale (Kuleung et al. 2004), Aegilops (Adonina et al. 2005) and Haynaldia (Zhang et al. 2006). In addition, some reports have already indicated that wheat SSR markers can be amplified in rye (Röder et al. 1995; Kuleung et al. 2004). Thereby, wheat SSR can be used to enrich ryespecific PCR-based markers. However, until now, data were rarely available about using wheat SSR markers to develop rye-specific markers. The objective of this study is to develop rye-specific PCR-based markers using wheat SSR markers. The phenomenon that rye-specific bands amplified by wheat SSR markers could display genetic diversity among genus Secale has also been observed and discussed.

Diversity and evolution of four dispersed repetitive DNA sequences in the genus Secale

We present the first characterization of 360 sequences in six species of the genus Secale of both cultivated and wild accessions. These include four distinct kinds of dispersed repetitive DNA sequences named pSc20H, pSc119.1, pSaO5(411), and pSaD15(940) belonging to the Revolver family. During the evolution of the genus Secale from wild to cultivated accessions, the pSaO5(411)-like sequences became shorter mainly because of the deletion of a trinucleotide tandem repeating unit, the pSc20H-like sequences displayed apparent homogenization in cultivated rye, and the second intron of Revolver became longer. In addition, the pSc20H-, pSc119.1-, and pSaO5(411)-like sequences cloned from wild rye and cultivated rye could be divided into two large clades. No single case of the four kinds of repetitive elements has been inherited by each Secale accession from a lone ancestor. It is reasonable to consider the vertical transmission of the four repetitive elements during the evolution of the genus Secale. The pSc20H- and pSaO5(411)-like sequences showed evolutionary elimination at specific chromosomal locations from wild species to cultivated species. These cases imply that different repetitive DNA sequences have played different roles in the chromosome development and genomic evolution of rye. The present study adds important information to the investigations dealing with characterization of dispersed repetitive elements in wild and cultivated rye.

New Oligonucleotide Probes for ND-FISH Analysis to Identify Barley Chromosomes and to Investigate Polymorphisms of Wheat Chromosomes

Oligonucleotide probes that can be used for non-denaturing fluorescence in situ hybridization (ND-FISH) analysis are convenient tools for identifying chromosomes of wheat (Triticum aestivum L.) and its relatives. New oligonucleotide probes, Oligo-HvT01, Oligo-pTa71-1, Oligo-s120.1, Oligo-s120.2, Oligo-s120.3, Oligo-275.1, Oligo-275.2, Oligo-k566 and Oligo-713, were designed based on the repetitive sequences HVT01, pTa71, pTa-s120, pTa-275, pTa-k566 and pTa-713. All these probes can be used for ND-FISH analysis and some of them can be used to detect polymorphisms of wheat chromosomes. Probes Oligo-HvT01, Oligo-pTa71-1, Oligo-s120.3, Oligo-275.1, Oligo-k566 and Oligo-713 can, respectively, replace the roles of their original sequences to identify chromosomes of some barley (Hordeum vulgare ssp. vulgare) and the common wheat variety Chinese Spring. Oligo-s120.1, Oligo-s120.2 and Oligo-275.2 produced different hybridization patterns from the ones generated by their original sequences. In addition, Oligo-s120.1, Oligo-s120.2 and Oligo-s120.3, which were derived from pTa-s120, revealed different signal patterns. Likewise, Oligo-275.1 and Oligo-275.2, which were derived from pTa-275, also displayed different hybridization patterns. These results imply that differently arranged or altered structural statuses of tandem repeats might exist on different chromosome regions. These new oligonucleotide probes provide extra convenience for identifying some wheat and barley chromosomes, and they can display polymorphisms of wheat chromosomes.

Identification and Physical Mapping of New PCR-Based Markers Specific for the Long Arm of Rye (Secale cereale L.) Chromosome 6

To effectively use elite genes on the long arm of rye chromosome 6 (the 6RL arm) in wheat breeding programs, precise and fast identification of 6RL chromatin in wheat backgrounds is necessary. PCR-based 6RL-specific markers can facilitate the detection of elite genes on 6RL in wheat breeding. However, only a limited number of 6RL-specific markers have been developed. In the present study, 300 new PCR-based 6RL-specific markers were identified using specific length amplified fragment sequencing (SLAF-seq) technology, and were further physically mapped to four regions on the 6RL arm using 6R and 6RL deletion lines. Interestingly, 127 of the 300 markers were physically localized to a region from the site between 2.3 and 2.5 to the telomere, the same region where the powdery mildew resistance gene was mapped. In addition, 95 of the 300 markers exhibit polymorphisms, which can be used to investigate the diversity of rye 6RL arms. The markers developed in this study can be used to identify given segments of 6RL in wheat backgrounds and accelerate the utilization of elite genes on 6RL in wheat breeding.

Oligonucleotides and ND-FISH Displaying Different Arrangements of Tandem Repeats and Identification of Dasypyrum villosum Chromosomes in Wheat Backgrounds

Oligonucleotide probes and the non-denaturing fluorescence in situ hybridization (ND-FISH) technique are widely used to analyze plant chromosomes because they are convenient tools. New oligonucleotide probes, Oligo-Ku, Oligo-3B117.1, Oligo-3B117.2, Oligo-3B117.2.1, Oligo-3B117.3, Oligo-3B117.4, Oligo-3B117.5, Oligo-3B117.6, Oligo-pTa71A-1, Oligo-pTa71A-2, Oligo-pTa71B-1, Oligo-pTa71B-2, Oligo-pTa71C-1, Oligo-pTa71C-2, Oligo-pTa71C-3 and Oligo-pTa71D were designed based on the repetitive sequences KU.D15.15, pSc119.2-like sequence 3B117 and pTa71. Oligonucleotide probe (GT)7 was also used. Oligo-Ku and (GT)7 can be together used to identify Dasypyrum villosum from wheat chromosomes and to distinguish individual D. villosum chromosomes. The oligonucleotide probes that were derived from the same repeat sequence displayed different signal intensity and hybridization sites on the same chromosomes. Both the length and the nucleotide composition of oligonucleotide probes determined their signal intensity. For example, Oligo-3B117.2 (25 bp) and Oligo-pTa71A-2 (46 bp) produced the strongest signals on chromosomes of wheat (Triticum aestivum L.), rye (Secale cereale L.), barley (Hordeum vulgare ssp. vulgare) or D. villosum, the signal of Oligo-3B117.4 (18 bp) on the short arm of 7B chromosome was weaker than that of Oligo-3B117.2.1 (15 bp) and Oligo-3B117.3 (16 bp), and Oligo-pTa71A-1 (38 bp) produced the same strong signals as Oligo-pTa71A-2 did on 1B and 6B chromosomes, but its signals on 1R and 1V chromosomes were weaker than the ones of Oligo-pTa71A-2. Oligonucleotide probes and ND-FISH analysis can reflect the distribution and structural statues of different segments of tandem repeats on chromosomes. The possible reasons why different segments derived from the same repeat sequence produced different signal patterns are discussed.