Non-B-form DNA tends to form in centromeric regions and has undergone changes in polyploid oat subgenomes.

Centromeres are the specialized regions of the chromosomes that direct faithful chromosome segregation during cell division. Despite their functional conservation, centromeres display features of rapidly evolving DNA and wide evolutionary diversity in size and organization. Previous work found that the noncanonical B-form DNA structures are abundant in the centromeres of several eukaryotic species with a possible implication for centromere specification. Thus far, systematic studies into the organization and function of non-B-form DNA in plants remain scarce. Here, we applied the oat system to investigate the role of non-B-form DNA in centromeres. We conducted chromatin immunoprecipitation sequencing using an antibody to the centromere-specific histone H3 variant (CENH3); this accurately positioned oat centromeres with different ploidy levels and identified a series of centromere-specific sequences including minisatellites and retrotransposons. To define genetic characteristics of oat centromeres, we surveyed the repeat sequences and found that dyad symmetries were abundant in oat centromeres and were predicted to form non-B-DNA structures in vivo. These structures including bent DNA, slipped DNA, Z-DNA, G-quadruplexes, and R-loops were prone to form within CENH3-binding regions. Dynamic conformational changes of predicted non-B-DNA occurred during the evolution from diploid to tetraploid to hexaploid oat. Furthermore, we applied the single-molecule technique of AFM and DNA:RNA immunoprecipitation with deep sequencing to validate R-loop enrichment in oat centromeres. Centromeric retrotransposons exhibited strong associations with R-loop formation. Taken together, our study elucidates the fundamental character of non-B-form DNA in the oat genome and reveals its potential role in centromeres.

Pan-centromere reveals widespread centromere repositioning of soybean genomes.

Centromere repositioning refers to a de novo centromere formation at another chromosomal position without sequence rearrangement. This phenomenon was frequently encountered in both mammalian and plant species and has been implicated in genome evolution and speciation. To understand the dynamic of centromeres on soybean genome, we performed the pan-centromere analysis using CENH3-ChIP-seq data from 27 soybean accessions, including 3 wild soybeans, 9 landraces, and 15 cultivars. Building upon the previous discovery of three centromere satellites in soybean, we have identified two additional centromere satellites that specifically associate with chromosome 1. These satellites reveal significant rearrangements in the centromere structures of chromosome 1 across different accessions, consequently impacting the localization of CENH3. By comparative analysis, we reported a high frequency of centromere repositioning on 14 out of 20 chromosomes. Most newly emerging centromeres formed in close proximity to the native centromeres and some newly emerging centromeres were apparently shared in distantly related accessions, suggesting their emergence is independent. Furthermore, we crossed two accessions with mismatched centromeres to investigate how centromere positions would be influenced in hybrid genetic backgrounds. We found that a significant proportion of centromeres in the S9 generation undergo changes in size and position compared to their parental counterparts. Centromeres preferred to locate at satellites to maintain a stable state, highlighting a significant role of centromere satellites in centromere organization. Taken together, these results revealed extensive centromere repositioning in soybean genome and highlighted how important centromere satellites are in constraining centromere positions and supporting centromere function.

Autoploid origin and rapid diploidization of the tetraploid Thinopyrum elongatum revealed by genome differentiation and chromosome pairing in meiosis.

Polyploidy is a common mode of evolution in flowering plants. Both the natural tetraploid Thinopyrum elongatum and the diploid one from the same population show a diploid-like pairing in meiosis. However, debate on the chromosome composition and origin of the tetraploid Th. elongatum is ongoing. In the present study, we obtained the induced tetraploid Th. elongatum and found that the induced and natural tetraploids are morphologically close, except for slower development and lower seed setting. Using probes developed from single chromosome microdissection and a Fosmid library, obvious differentiations were discovered between two chromosome sets (E1 and E2 ) of the natural tetraploid Th. elongatum but not the induced one. Interestingly, hybrid F1 derived from the two different wheat-tetraploid Th. elongatum amphiploids 8802 and 8803 produced seeds well. More importantly, analysis of meiosis in F2 individuals revealed that chromosomes from E1 and E2 could pair well on the durum wheat background with the presence of Ph1. No chromosome set differentiation on the FISH level was discovered from the S1 to S4 generations in the induced one. In metaphase of the meiosis first division in the natural tetraploid, more pairings were bivalents and fewer quadrivalents with ratio of 13.94 II + 0.03 IV (n = 31). Chromosome pairing configuration in the induced tetraploid is 13.05 II + 0.47 IV (n = 19), with the quadrivalent ratio being only slightly higher than the ratio in the natural tetraploid. Therefore, the natural tetraploid Th. elongatum is of autoploid origin and the induced tetraploid Th. elongatum evolutionarily underwent rapid diploidization in the low generation.

New insights on the evolution of nucleolar dominance in newly resynthesized hexaploid wheat Triticum zhukovskyi.

Nucleolar dominance (ND) is a widespread epigenetic phenomenon in hybridizations where nucleolus transcription fails at the nucleolus organizer region (NOR). However, the dynamics of NORs during the formation of Triticum zhukovskyi (GGAu Au Am Am ), another evolutionary branch of allohexaploid wheat, remains poorly understood. Here, we elucidated genetic and epigenetic changes occurring at the NOR loci within the Am , G, and D subgenomes during allopolyploidization by synthesizing hexaploid wheat GGAu Au Am Am and GGAu Au DD. In T. zhukovskyi, Au genome NORs from T. timopheevii (GGAu Au ) were lost, while the second incoming NORs from T. monococcum (Am Am ) were retained. Analysis of the synthesized T. zhukovskyi revealed that rRNA genes from the Am genome were silenced in F1 hybrids (GAu Am ) and remained inactive after genome doubling and subsequent self-pollinations. We observed increased DNA methylation accompanying the inactivation of NORs in the Am genome and found that silencing of NORs in the S1 generation could be reversed by a cytidine methylase inhibitor. Our findings provide insights into the ND process during the evolutionary period of T. zhukovskyi and highlight that inactive rDNA units may serve as a 'first reserve' in the form of R-loops, contributing to the successful evolution of T. zhukovskyi.

Systemic development of wheat-Thinopyrum elongatum translocation lines and their deployment in wheat breeding for Fusarium head blight resistance.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is one of the most destructive diseases of wheat (Triticum aestivum) around the world. FHB causes significant yield losses and reduces grain quality. The lack of resistance resources is a major bottleneck for wheat FHB resistance breeding. As a wheat relative, Thinopyrum elongatum contains many genes that can be used for wheat improvement. Although the novel gene Fhb-7EL was mapped on chromosome 7EL of Th. elongatum, successful transfer of the FHB resistance gene into commercial wheat varieties has not been reported. In this study, we developed 836 wheat-Th. elongatum translocation lines of various types by irradiating the pollen of the wheat-Th. elongatum addition line CS-7EL at the flowering stage, among which 81 were identified as resistant to FHB. By backcrossing the FHB-resistant lines with the main cultivar Jimai 22, three wheat-Th. elongatum translocation lines, Zhongke 1878, Zhongke 166, and Zhongke 545, were successfully applied in wheat breeding without yield penalty. Combining karyotype and phenotype analyses, we mapped the Fhb-7EL gene to the distal end of chromosome 7EL. Five molecular markers linked with the FHB resistance interval were developed, which facilitates molecular marker-assisted breeding. Altogether, we successfully applied alien chromatin with FHB resistance from Th. elongatum in wheat breeding without yield penalty. These newly developed FHB-resistant wheat-Th. elongatum translocation lines, Zhongke 1878, Zhongke 166, and Zhongke 545, can be used as novel resistance resources for wheat breeding.

Genome-wide mapping reveals R-loops associated with centromeric repeats in maize.

R-loops are stable chromatin structures comprising a DNA:RNA hybrid and a displaced single-stranded DNA. R-loops have been implicated in gene expression and chromatin structure, as well as in replication blocks and genome instability. Here, we conducted a genome-wide identification of R-loops and identified more than 700,000 R-loop peaks in the maize (Zea mays) genome. We found that sense R-loops were mainly enriched in promoters and transcription termination sites and relatively less enriched in gene bodies, which is different from the main gene-body localization of sense R-loops in Arabidopsis and Oryza sativa At the chromosome scale, maize R-loops were enriched in pericentromeric heterochromatin regions, and a significant portion of R-loops were derived from transposable elements. In centromeres, R-loops preferentially formed within the binding regions of the centromere-specific histone CENH3, and centromeric retrotransposons were strongly associated with R-loop formation. Furthermore, centromeric retrotransposon R-loops were observed by applying the single-molecule imaging technique of atomic force microscopy. These findings elucidate the fundamental character of R-loops in the maize genome and reveal the potential role of R-loops in centromeres.

Young retrotransposons and non-B DNA structures promote the establishment of dominant rye centromere in the 1RS.1BL fused centromere.

The fine centromere structure in Robertsonian wheat-rye translocation chromosomes exhibits variation among different translocation genotypes. Within extensively employed wheat-rye 1RS.1BL translocation lines in wheat breeding, their translocated chromosomes frequently display fused centromere. Nevertheless, the mechanism governing the functionality of the fused centromere in 1RS.1BL translocated chromosomes remains to be clarified. In this study, we investigated the fine centromere structure of the 1RS.1BL translocated chromosome through a combination of cytological and genomics methods. We found that only the rye-derived centromere exhibits functional activity, whether in breeding applications or artificially synthesized translocation chromosomes. The active rye-derived centromere had higher proportion of young full-length long terminal repeat retrotransposons (flLTR-RTs) and more stable non-B DNA structures, which may be beneficial toward transcription of centromeric repeats and CENH3 loading to maintain the activity of rye centromeres. High levels of DNA methylation and H3K9me2 were found in the inactive wheat-derived centromeres, suggesting that it may play a crucial role in maintaining the inactive status of the wheat centromere. Our works elucidate the fine structure of 1RS.1BL translocations and the potential mechanism of centromere inactivation in the fused centromere, contributing knowledge to the application of fused centromere in wheat breeding formation of new wheat-rye translocation lines.

Reshaped DNA methylation cooperating with homoeolog-divergent expression promotes improved root traits in synthesized tetraploid wheat.

Polyploidization is a major event driving plant evolution and domestication. However, how reshaped epigenetic modifications coordinate gene transcription to generate phenotypic variations during wheat polyploidization is currently elusive. Here, we profiled transcriptomes and DNA methylomes of two diploid wheat accessions (SlSl and AA) and their synthetic allotetraploid wheat line (SlSlAA), which displayed elongated root hair and improved root capability for nitrate uptake and assimilation after tetraploidization. Globally decreased DNA methylation levels with a reduced difference between subgenomes were observed in the roots of SlSlAA. DNA methylation changes in first exon showed strong connections with altered transcription during tetraploidization. Homoeolog-specific transcription was associated with biased DNA methylation as shaped by homoeologous sequence variation. The hypomethylated promoters showed significantly enriched binding sites for MYB, which may affect gene transcription in response to root hair growth. Two master regulators in root hair elongation pathway, AlCPC and TuRSL4, exhibited upregulated transcription levels accompanied by hypomethylation in promoter, which may contribute to the elongated root hair. The upregulated nitrate transporter genes, including NPFs and NRTs, also are significantly associated with hypomethylation, indicating an epigenetic-incorporated regulation manner in improving nitrogen use efficiency. Collectively, these results provided new insights into epigenetic changes in response to crop polyploidization and underscored the importance of epigenetic regulation in improving crop traits.

Cytological analysis of the diploid-like inheritance of newly synthesized allotetraploid wheat.

Polyploidization is a process which is related to species hybridization and whole genome duplication. It is widespread among angiosperm evolution and is essential for speciation and diversification. Allopolyploidization is mainly derived from interspecific hybridization and is believed to pose chromosome imbalances and genome instability caused by meiotic irregularity. However, the self-compatible allopolyploid in wild nature is cytogenetically and genetically stable. Whether this stabilization form was achieved in initial generation or a consequence of long term of evolution was largely unknown. Here, we synthesized a series of nascent allotetraploid wheat derived from three diploid genomes of A, S*, and D. The chromosome numbers of the majority of the progeny derived from these newly formed allotetraploid wheat plants were found to be relatively consistent, with each genome containing 14 chromosomes. In meiosis, bivalent was the majority of the chromosome configuration in metaphase I which supports the stable chromosome number inheritance in the nascent allotetraploid. These findings suggest that diploidization occurred in the newly formed synthetic allotetraploid wheat. However, we still detected aneuploids in a proportion of newly formed allotetraploid wheat, and meiosis of these materials present more irregular chromosome behavior than the euploid. We found that centromere pairing and centromere clustering in meiosis was affected in the aneuploids, which suggest that aneuploidy may trigger the irregular interactions of centromere in early meiosis which may take participate in promoting meiosis stabilization in newly formed allotetraploid wheat.

Knl1 participates in spindle assembly checkpoint signaling in maize.

The Knl1-Mis12-Ndc80 (KMN) network is an essential component of the kinetochore-microtubule attachment interface, which is required for genomic stability in eukaryotes. However, little is known about plant Knl1 proteins because of their complex evolutionary history. Here, we cloned the Knl1 homolog from maize (Zea mays) and confirmed it as a constitutive central kinetochore component. Functional assays demonstrated their conserved role in chromosomal congression and segregation during nuclear division, thus causing defective cell division during kernel development when Knl1 transcript was depleted. A 145 aa region in the middle of maize Knl1, that did not involve the MELT repeats, was associated with the interaction of spindle assembly checkpoint (SAC) components Bub1/Mad3 family proteins 1 and 2 (Bmf1/2) but not with the Bmf3 protein. They may form a helical conformation with a hydrophobic interface with the TPR domain of Bmf1/2, which is similar to that of vertebrates. However, this region detected in monocots shows extensive divergence in eudicots, suggesting that distinct modes of the SAC to kinetochore connection are present within plant lineages. These findings elucidate the conserved role of the KMN network in cell division and a striking dynamic of evolutionary patterns in the SAC signaling and kinetochore network.

Sequence of the supernumerary B chromosome of maize provides insight into its drive mechanism and evolution.

B chromosomes are enigmatic elements in thousands of plant and animal genomes that persist in populations despite being nonessential. They circumvent the laws of Mendelian inheritance but the molecular mechanisms underlying this behavior remain unknown. Here we present the sequence, annotation, and analysis of the maize B chromosome providing insight into its drive mechanism. The sequence assembly reveals detailed locations of the elements involved with the cis and trans functions of its drive mechanism, consisting of nondisjunction at the second pollen mitosis and preferential fertilization of the egg by the B-containing sperm. We identified 758 protein-coding genes in 125.9 Mb of B chromosome sequence, of which at least 88 are expressed. Our results demonstrate that transposable elements in the B chromosome are shared with the standard A chromosome set but multiple lines of evidence fail to detect a syntenic genic region in the A chromosomes, suggesting a distant origin. The current gene content is a result of continuous transfer from the A chromosomal complement over an extended evolutionary time with subsequent degradation but with selection for maintenance of this nonvital chromosome.

The Cohesin Complex Subunit ZmSMC3 Participates in Meiotic Centromere Pairing in Maize.

Meiosis consists of two highly conserved nuclear divisions, which allow eukaryotes to maintain their chromosome number through sexual reproduction. The successful completion of meiosis depends on homologous chromosome pairing. Centromere interactions during early meiotic prophase I facilitate homologous chromosome pairing, but the underlying mechanism is unclear. Here, we performed chromatin immunoprecipitation-mass spectrometry analysis of maize (Zea mays) anthers during early meiotic prophase I using anti-centromeric histone H3 (CENH3) antibodies and determined that the cohesin subunit Structural Maintenance of Chromosome3 (SMC3) interacts with CENH3 during this period. SMC3 is enriched at centromeres and along chromosome arms in threads from leptotene to pachytene and might promote interactions between homologous centromeres. We observed dysfunctional SMC3 assembly in meiotic-specific maize mutants with defective centromere pairing. In SMC3 RNAi meiocytes, centromere pairing defects were observed during early meiotic prophase I, SMC3 was weakly associated with centromeres, and SMC3 did not localize to the chromosome arms. In wild-type mitosis, SMC3 is associated with chromatin and is enriched at centromeres from prophase to anaphase. CRISPR-Cas9-induced Zmsmc3 mutants showed premature loss of sister chromatid cohesion and mis-segregation of chromosomes in mitotic spreads. Our findings suggest that in addition to sister chromatid cohesion, ZmSMC3 participates in meiotic centromere pairing.

Rapid Birth or Death of Centromeres on Fragmented Chromosomes in Maize.

Comparative genomics has revealed common occurrences in karyotype evolution such as chromosomal end-to-end fusions and insertions of one chromosome into another near the centromere, as well as many cases of de novo centromeres that generate positional polymorphisms. However, how rearrangements such as dicentrics and acentrics persist without being destroyed or lost remains unclear. Here, we sought experimental evidence for the frequency and timeframe for inactivation and de novo formation of centromeres in maize (Zea mays). The pollen from plants with supernumerary B chromosomes was gamma-irradiated and then applied to normal maize silks of a line without B chromosomes. In ∼8,000 first-generation seedlings, we found many B-A translocations, centromere expansions, and ring chromosomes. We also found many dicentric chromosomes, but a fraction of these show only a single primary constriction, which suggests inactivation of one centromere. Chromosomal fragments were found without canonical centromere sequences, revealing de novo centromere formation over unique sequences; these were validated by immunolocalization with Thr133-phosphorylated histone H2A, a marker of active centromeres, and chromatin immunoprecipitation-sequencing with the CENH3 antibody. These results illustrate the regular occurrence of centromere birth and death after chromosomal rearrangement during a narrow window of one to potentially only a few cell cycles for the rearranged chromosomes to be recognized in this experimental regime.

Effects of Aegilops longissima chromosome 1Sl on wheat bread-making quality in two types of translocation lines.

KEY MESSAGE: Two wheat-Ae. longissima translocation chromosomes (1BS·1SlL and 1SlS·1BL) were transferred into three commercial wheat varieties, and the new advanced lines showed improved bread-making quality compared to their recurrent parents. Aegilops longissima chromosome 1Sl encodes specific types of gluten subunits that may positively affect wheat bread-making quality. The most effective method of introducing 1Sl chromosomal fragments containing the target genes into wheat is chromosome translocation. Here, a wheat-Ae. longissima 1BS·1SlL translocation line was developed using molecular marker-assisted chromosome engineering. Two types of translocation chromosomes developed in a previous study, 1BS·1SlL and 1SlS·1BL, were introduced into three commercial wheat varieties (Ningchun4, Ningchun50, and Westonia) via backcrossing with marker-assisted selection. Advanced translocation lines were confirmed through chromosome in situ hybridization and genotyping by target sequencing using the wheat 40 K system. Bread-making quality was found to be improved in the two types of advanced translocation lines compared to the corresponding recurrent parents. Furthermore, 1SlS·1BL translocation lines displayed better bread-making quality than 1BS·1SlL translocation lines in each genetic background. Further analysis revealed that high molecular weight glutenin subunit (HMW-GS) contents and expression levels of genes encoding low molecular weight glutenin subunits (LMW-GSs) were increased in 1SlS·1BL translocation lines. Gliadin and gluten-related transcription factors were also upregulated in the grains of the two types of advanced translocation lines compared to the recurrent parents. This study clarifies the impacts of specific glutenin subunits on bread-making quality and provides novel germplasm resources for further improvement of wheat quality through molecular breeding.

Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize.

In most plants, centromeric DNA contains highly repetitive sequences, including tandem repeats and retrotransposons; however, the roles of these sequences in the structure and function of the centromere are unclear. Here, we found that multiple RNA sequences from centromeric retrotransposons (CRMs) were enriched in maize (Zea mays) centromeres, and back-spliced RNAs were generated from CRM1. We identified 3 types of CRM1-derived circular RNAs with the same back-splicing site based on the back-spliced sequences. These circular RNAs bound to the centromere through R-loops. Two R-loop sites inside a single circular RNA promoted the formation of chromatin loops in CRM1 regions. When RNA interference (RNAi) was used to target the back-splicing site of the circular CRM1 RNAs, the levels of R-loops and chromatin loops formed by these circular RNAs decreased, while the levels of R-loops produced by linear RNAs with similar binding sites increased. Linear RNAs with only one R-loop site could not promote chromatin loop formation. Higher levels of R-loops and lower levels of chromatin loops in the CRM1 regions of RNAi plants led to a reduced localization of the centromeric H3 variant (CENH3). Our work reveals centromeric chromatin organization by circular CRM1 RNAs via R-loops and chromatin loops, which suggested that CRM1 elements might help build a suitable chromatin environment during centromere evolution. These results highlight that R-loops are integral components of centromeric chromatin and proper centromere structure is essential for CENH3 localization.

Genomic sequencing of Thinopyrum elongatum chromosome arm 7EL, carrying fusarium head blight resistance, and characterization of its impact on the transcriptome of the introgressed line CS-7EL.

BACKGROUND: The tall wheatgrass species Thinopyrum elongatum carries a strong fusarium head blight (FHB) resistance locus located on the long arm of chromosome 7 (7EL) as well as resistance to leaf and stem rusts, all diseases with a significant impact on wheat production. Towards understanding the contribution of Th. elongatum 7EL to improvement of disease resistance in wheat, the genomic sequence of the 7EL fragment present in the wheat Chinese Spring (CS) telosomic addition line CS-7EL was determined and the contribution and impact of 7EL on the rachis transcriptome during FHB infection was compared between CS and CS-7EL. RESULTS: We assembled the Th. elongatum 7EL chromosome arm using a reference-guided approach. Combining this assembly with the available reference sequence for CS hexaploid wheat provided a reliable reference for interrogating the transcriptomic differences in response to infection conferred by the 7EL fragment. Comparison of the transcriptomes of rachis tissues from CS and CS-7EL showed expression of Th. elongatum transcripts as well as modulation of wheat transcript expression profiles in the CS-7EL line. Expression profiles at 4 days after infection with Fusarium graminearum, the causal agent of FHB, showed an increased in expression of genes associated with an effective defense response, in particular glucan endo-1,3-beta-glucosidases and chitinases, in the FHB-resistant line CS-7EL while there was a larger increase in differential expression for genes associated with the level of fungal infection in the FHB-susceptible line CS. One hundred and seven 7EL transcripts were expressed in the smallest 7EL region defined to carry FHB resistance. CONCLUSION: 7EL contributed to CS-7EL transcriptome by direct expression and through alteration of wheat transcript profiles. FHB resistance in CS-7EL was associated with transcriptome changes suggesting a more effective defense response. A list of candidate genes for the FHB resistance locus on 7EL has been established.

Centromeres: From chromosome biology to biotechnology applications and synthetic genomes in plants.

Centromeres are the genomic regions that organize and regulate chromosome behaviours during cell cycle, and their variations are associated with genome instability, karyotype evolution and speciation in eukaryotes. The highly repetitive and epigenetic nature of centromeres were documented during the past half century. With the aid of rapid expansion in genomic biotechnology tools, the complete sequence and structural organization of several plant and human centromeres were revealed recently. Here, we systematically summarize the current knowledge of centromere biology with regard to the DNA compositions and the histone H3 variant (CENH3)-dependent centromere establishment and identity. We discuss the roles of centromere to ensure cell division and to maintain the three-dimensional (3D) genomic architecture in different species. We further highlight the potential applications of manipulating centromeres to generate haploids or to induce polyploids offspring in plant for breeding programs, and of targeting centromeres with CRISPR/Cas for chromosome engineering and speciation. Finally, we also assess the challenges and strategies for de novo design and synthesis of centromeres in plant artificial chromosomes. The biotechnology applications of plant centromeres will be of great potential for the genetic improvement of crops and precise synthetic breeding in the future.

Centromere Satellite Repeats Have Undergone Rapid Changes in Polyploid Wheat Subgenomes.

Centromeres mediate the pairing of homologous chromosomes during meiosis; this pairing is particularly challenging for polyploid plants such as hexaploid bread wheat (Triticum aestivum), as their meiotic machinery must differentiate homologs from similar homoeologs. However, the sequence compositions (especially functional centromeric satellites) and evolutionary history of wheat centromeres are largely unknown. Here, we mapped T. aestivum centromeres by chromatin immunoprecipitation sequencing using antibodies to the centromeric-specific histone H3 variant (CENH3); this identified two types of functional centromeric satellites that are abundant in two of the three subgenomes. These centromeric satellites had unit sizes greater than 500 bp and contained specific sites with highly phased binding to CENH3 nucleosomes. Phylogenetic analysis revealed that the satellites have diverged in the three T. aestivum subgenomes, and the more homogeneous satellite arrays are associated with CENH3. Satellite signals decreased and the degree of satellites variation increased from diploid to hexaploid wheat. Moreover, several T. aestivum centromeres lack satellite repeats. Rearrangements, including local expansion and satellite variations, inversions, and changes in gene expression, occurred during the evolution from diploid to tetraploid and hexaploid wheat. These results reveal the asymmetry in centromere organization among the wheat subgenomes, which may play a role in proper homolog pairing during meiosis.

Establishment of a set of wheat-rye addition lines with resistance to stem rust.

KEY MESSAGE: Complete new wheat-rye disomic, telosomic addition lines and various chromosomal aberrations were developed and characterized by molecular cytogenetic method as novel chromosome engineering materials. A new stem rust resistance (Ug99) gene was located on 3RL. Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a devastating fungal disease worldwide. A recently emerged great threat to global wheat production is Pgt strain Ug99 and its derivatives, which have overcome most of the commonly used resistance genes. Rye (Secale cereale L.), closely related to wheat (Triticum aestivum L.), is a significant and valuable resource of resistance genes for wheat germplasm improvement. It is of great importance and urgency to identify new resistance gene sources of rye and transfer them into wheat. In this study, two complete sets of wheat-rye addition lines were established through wide hybridization, chromosome doubling and backcrossing. A wheat-rye 3RL telosomic addition line was identified with high resistance to stem rust strain Ug99. PCR-based markers specific for the rye chromosome were developed. Furthermore, abundant chromosomal aberrations such as minichromosomes, ring chromosomes as well as centromere reduction and expansion were identified in the progeny of wheat-rye addition lines by multicolor GISH and FISH. The line carrying a novel resistance gene to stem rust can be utilized as a bridge material for wheat disease resistance breeding. The chromosomal and centromeric variation within the wheat-rye hybrids can further contribute to genetic diversity of their offspring.

De novo centromere formation on chromosome fragments with an inactive centromere in maize (Zea mays).

The B chromosome of maize undergoes nondisjunction at the second pollen mitosis as part of its accumulation mechanism. Previous work identified 9-Bic-1 (9-B inactivated centromere-1), which comprises an epigenetically silenced B chromosome centromere that was translocated to the short arm of chromosome 9(9S). This chromosome is stable in isolation, but when normal B chromosomes are added to the genotype, it will attempt to undergo nondisjunction during the second pollen mitosis and usually fractures the chromosome in 9S. These broken chromosomes allow a test of whether the inactive centromere is reactivated or whether a de novo centromere is formed elsewhere on the chromosome to allow recovery of fragments. Breakpoint determination on the B chromosome and chromosome 9 showed that mini chromosome B1104 has the same breakpoint as 9-Bic-1 in the B centromere region and includes a portion of 9S. CENH3 binding was found on the B centromere region and on 9S, suggesting both centromere reactivation and de novo centromere formation. Another mini chromosome, B496, showed evidence of rearrangement, but it also only showed evidence for a de novo centromere. Other mini chromosome fragments recovered were directly derived from the B chromosome with breakpoints concentrated near the centromeric knob region, which suggests that the B chromosome is broken at a low frequency due to the failure of the sister chromatids to separate at the second pollen mitosis. Our results indicate that both reactivation and de novo centromere formation could occur on fragments derived from the progenitor possessing an inactive centromere.