Zhenglong Ren

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.

Genetic mapping of a putative Thinopyrum intermedium-derived stripe rust resistance gene on wheat chromosome 1B.

Key messageStripe rust resistance transferred fromThinopyrum intermediuminto common wheat was controlled by a single dominant gene, which mapped to chromosome 1B nearYr26and was designatedYrL693.AbstractStripe rust caused by Puccinia striiformis f. sp. tritici (Pst) is a highly destructive disease of wheat (Triticum aestivum). Stripe rust resistance was transferred from Thinopyrum intermedium to common wheat, and the resulting introgression line (L693) exhibited all-stage resistance to the widely virulent and predominant Chinese pathotypes CYR32 and CYR33 and to the new virulent pathotype V26. There was no cytological evidence that L693 had alien chromosomal segments from Th. intermedium. Genetic analysis of stripe rust resistance was performed by crossing L693 with the susceptible line L661. F1, F2, and F2:3 populations from reciprocal crosses showed that resistance was controlled by a single dominant gene. A total 479 F2:3 lines and 781 pairs of genomic simple sequence repeat (SSR) primers were employed to determine the chromosomal location of the resistance gene. The gene was linked to six publicly available and three recently developed wheat genomic SSR markers. The linked markers were localized to wheat chromosome 1B using Chinese Spring nulli-tetrasomic lines, and the resistance gene was localized to chromosome 1B based on SSR and wheat genomic information. A high-density genetic map was also produced. The pedigree, molecular marker data, and resistance response indicated that the stripe rust resistance gene in L693 is a novel gene, which was temporarily designated YrL693. The SSR markers that co-segregate with this gene (Xbarc187-1B, Xbarc187-1B-1, Xgwm18-1B, and Xgwm11-1B) have potential application in marker-assisted breeding of wheat, and YrL693 will be useful for broadening the genetic basis of stripe rust resistance in wheat.

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.

The Selection of Transgenic Recipients from New Elite Wheat Cultivars and Study on Its Plant Regeneration System

Abstract In the protocol of wheat transformation, to use elite wheat cultivars as exogenous gene recipients can speed up the process of commercial field applications of transgenic wheat. However, it is necessary to screen wheat cultivars with good tissue culture response (TCR) continuously from plenty of elite wheat cultivars released for wheat transformation, and it is also important to find a plant regeneration system that is suitable for these cultivars. So, the TCR of mature and immature embryos of six wheat cultivars Chuannong 11 (CN11), Chuannong 12 (CN12), Chuannong 17 (CN17), Chuannong 18 (CN18), Chuannong 19 (CN19), and Chuannong 21 (CN21), which possess superior agronomic traits, were investigated by using a good TCR wheat cultivar Bobwhite as control. The results indicated that only the immature and mature embryos of CN12, CN17, and CN18 exhibited good TCR compared with Bobwhite. No significant differences were observed between embryos of Bobwhite and of the three cultivars in TCR. Mature embryo-derived calli of CN12 were used as explants for transformation by particle bombardment of SAMDC gene. Seven transformants were obtained and the efficiency was 2.3%. This research supplies three new elite recipient cultivars for wheat transformation. The wheat plant regeneration system used in this research is different from those successful ones reported previously and it could be a reference for other wheat genotypes. Furthermore, Bobwhite and the three wheat cultivars were proved to be 1RS/1BL translocation, by methods of A-PAGE, C-banding, and genomic in situ hybridization (GISH). These results imply that probably there is some relationship between 1RS/1BL translocation and TCR of wheat embryos. So this research gives us a hint that we should pay more attention to the 1RS/1BL translocations when we screen the wheat cultivars with good TCR and also that the mechanism of the effect of 1RS/1BL translocation on TCR is worthy of being investigated.

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.

Characterization of a new wheat-Aegilops biuncialis addition line conferring quality-associated HMW glutenin subunits.

In this study, a new disomic addition line, 12-5-2, with 44 chromosomes that was derived from BC3F2 descendants of the hybridization between Triticum aestivum cv. CN19 and Aegilops biuncialis was created and reported. 12-5-2 was immune to both powdery mildew and stripe rust and has stable fertility. Fluorescence in situ hybridization and C-banding revealed that 12-5-2 was a 1U(b) disomic addition line (ADL1U(b)). The seed storage protein electrophoresis showed that 12-5-2 presented all high molecular weight glutenin subunits (7 + 8 and 2 + 12) of CN19 and 2 new subunits that were designated Ux and Uy. Additionally, the flour quality parameters showed that the protein content, Zeleny sedimentation value, wet gluten content, and grain hardness of 12-5-2 were significantly higher than those of its parent CN19. Moreover, 5 pairs of the chromosome 1U(b)-specific polymerase chain reaction-based landmark unique gene markers, TNAC1021, TNAC1041, TNAC1071, TNAC1-01, and TNAC1-04, were also obtained. The new ADL1U(b) 12-5-2 could be a valuable source for wheat improvement, especially for wheat end-product quality and resistance to disease.

Identification of Novel miRNAs and miRNA Expression Profiling in Wheat Hybrid Necrosis

MicroRNAs (miRNAs) play essential roles in a vast array of biological processes, including growth and development, defense against viral infection, and responses to environmental changes in plant. Wheat hybrid necrosis is an interesting genetic phenomenon observed frequency and it is lethal or semi lethal, resulting in gradual death or loss of productivity. However, the molecular basis and mechanisms associated with hybrid necrosis in wheat are still not well understood. Here, we report the population and expression profiles of miRNAs in wheat hybrid necrosis. We identified a total of 57 conserved miRNA families as well as 182 putative novel miRNAs. Expression profiling revealed that expression of 49 known miRNAs and 165 novel miRNAs was changed in hybrid necrosis. And the expression levels of some miRNAs and their predicated targets have been confirmed by qRT-PCR. These results indicate that these miRNAs, especially miR159, miR166, miR167 and miR5072 could be involved in the extensive regulation of gene expression in response to hybrid necrosis.

Diversity resistance to Puccinia striiformis f. sp Tritici in rye chromosome arm 1RS expressed in wheat.

The 1BL.1RS wheat-rye translocation contained in the Russian cultivar Aurora has been the most widespread alien translocation in wheat-breeding programs all over the world. However, following the prevalence of new biotypes of the pathogens, disease-resistance genes in this translocation chromosome have been overcome and consequently they have been eliminated in modern wheat-breeding programs. In this paper, we report on 12 new primary 1BL.1RS translocation lines derived from the crosses of a Chinese high yield wheat cv. Mianyang 11 with three rye cultivars collected from China. GISH, C-banding and PCR techniques using the specific primers for 1BS, 1RS and centromeres of wheat and rye were applied to identify the constitution of chromosomes. The results confirmed that all 1BL.1RS chromosomes in the 12 primary translocation lines contained integrated 1RS chromosome arms. In the resistance analysis using five kinds of Pst pathotypes, the 12 primary translocation lines showed diversity resistance to stripe rust, which contained at least five different new genes (alleles), significantly different from the Yr9 gene coming from Russian wheat cultivar Aurora. The results indicated that the chromosome arm 1RS in the rye population carries abundant yet untapped genes (alleles) for resistance to wheat stripe rust, which would originate from the neutral diversity in the natural population of rye. It is suggested that creating more primary translocation lines in genome modification will be extremely important to use the diversity of alien R-genes, which was generated by long-term neutral mutation and maintained in the population of alien species.