An antisense intragenic lncRNA SEAIRa mediates transcriptional and epigenetic repression of SERRATE in Arabidopsis.

SERRATE (SE) is a core protein for microRNA (miRNA) biogenesis as well as for mRNA alternative splicing. Investigating the regulatory mechanism of SE expression is hence critical to understanding its detailed function in diverse biological processes. However, little about the control of SE expression has been clarified, especially through long noncoding RNA (lncRNA). Here, we identified an antisense intragenic lncRNA transcribed from the 3' end of SE, named SEAIRa. SEAIRa repressed SE expression, which in turn led to serrated leaves. SEAIRa recruited plant U-box proteins PUB25/26 with unreported RNA binding ability and a ubiquitin-like protein related to ubiquitin 1 (RUB1) for H2A monoubiquitination (H2Aub) at exon 11 of SE. In addition, PUB25/26 helped cleave SEAIRa and release the 5' domain fragment, which recruited the PRC2 complex for H3 lysine 27 trimethylation (H3K27me3) deposition at the first exon of SE. The distinct modifications of H2Aub and H3K27me3 at different sites of the SE locus cooperatively suppressed SE expression. Collectively, our results uncover an epigenetic mechanism mediated by the lncRNA SEAIRa that modulates SE expression, which is indispensable for plant growth and development.

Characterization of sucrose nonfermenting-1-related protein kinase 2 (SnRK2) gene family in Haynaldia villosa demonstrated SnRK2.9-V enhances drought and salt stress tolerance of common wheat.

BACKGROUND: The sucrose nonfermenting-1-related protein kinase 2 (SnRK2) plays a crucial role in responses to diverse biotic/abiotic stresses. Currently, there are reports on these genes in Haynaldia villosa, a diploid wild relative of wheat. RESULTS: To understand the evolution of SnRK2-V family genes and their roles in various stress conditions, we performed genome-wide identification of the SnRK2-V gene family in H. villosa. Ten SnRK2-V genes were identified and characterized for their structures, functions and spatial expressions. Analysis of gene exon/intron structure further revealed the presence of evolutionary paths and replication events of SnRK2-V gene family in the H. villosa. In addition, the features of gene structure, the chromosomal location, subcellular localization of the gene family were investigated and the phylogenetic relationship were determined using computational approaches. Analysis of cis-regulatory elements of SnRK2-V gene members revealed their close correlation with different phytohormone signals. The expression profiling revealed that ten SnRK2-V genes expressed at least one tissue (leave, stem, root, or grain), or in response to at least one of the biotic (stripe rust or powdery mildew) or abiotic (drought or salt) stresses. Moreover, SnRK2.9-V was up-regulated in H. villosa under the drought and salt stress and overexpressing of SnRK2.9-V in wheat enhanced drought and salt tolerances via enhancing the genes expression of antioxidant enzymes, revealing a potential value of SnRK2.9-V in wheat improvement for salt tolerance. CONCLUSION: Our present study provides a basic genome-wide overview of SnRK2-V genes in H. villosa and demonstrates the potential use of SnRK2.9-V in enhancing the drought and salt tolerances in common wheat.

Genome-wide characterization of i-motifs and their potential roles in the stability and evolution of transposable elements in rice.

I-motifs (iMs) are non-canonical DNA secondary structures that fold from cytosine (C)-rich genomic DNA regions termed putative i-motif forming sequences (PiMFSs). The structure of iMs is stabilized by hemiprotonated C-C base pairs, and their functions are now suspected in key cellular processes in human cells such as genome stability and regulation of gene transcription. In plants, their biological relevance is still largely unknown. Here, we characterized PiMFSs with high potential for i-motif formation in the rice genome by developing and applying a protocol hinging on an iMab antibody-based immunoprecipitation (IP) coupled with high-throughput sequencing (seq), consequently termed iM-IP-seq. We found that PiMFSs had intrinsic subgenomic distributions, cis-regulatory functions and an intricate relationship with DNA methylation. We indeed found that the coordination of PiMFSs with DNA methylation may affect dynamics of transposable elements (TEs) among different cultivated Oryza subpopulations or during evolution of wild rice species. Collectively, our study provides first and unique insights into the biology of iMs in plants, with potential applications in plant biotechnology for improving important agronomic rice traits.

Chromosome painting reveals inter-chromosomal rearrangements and evolution of subgenome D of wheat.

Aegilops species represent the most important gene pool for breeding bread wheat (Triticum aestivum). Thus, understanding the genome evolution, including chromosomal structural rearrangements and syntenic relationships among Aegilops species or between Aegilops and wheat, is important for both basic genome research and practical breeding applications. In the present study, we attempted to develop subgenome D-specific fluorescence in situ hybridization (FISH) probes by selecting D-specific oligonucleotides based on the reference genome of Chinese Spring. The oligo-based chromosome painting probes consisted of approximately 26 000 oligos per chromosome and their specificity was confirmed in both diploid and polyploid species containing the D subgenome. Two previously reported translocations involving two D chromosomes have been confirmed in wheat varieties and their derived lines. We demonstrate that the oligo painting probes can be used not only to identify the translocations involving D subgenome chromosomes, but also to determine the precise positions of chromosomal breakpoints. Chromosome painting of 56 accessions of Ae. tauschii from different origins led us to identify two novel translocations: a reciprocal 3D-7D translocation in two accessions and a complex 4D-5D-7D translocation in one accession. Painting probes were also used to analyze chromosomes from more diverse Aegilops species. These probes produced FISH signals in four different genomes. Chromosome rearrangements were identified in Aegilops umbellulata, Aegilops markgrafii, and Aegilops uniaristata, thus providing syntenic information that will be valuable for the application of these wild species in wheat breeding.

Recognition of glycoside hydrolase 12 proteins by the immune receptor RXEG1 confers Fusarium head blight resistance in wheat.

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease in wheat (Triticum aestivum) that results in substantial yield losses and mycotoxin contamination. Reliable genetic resources for FHB resistance in wheat are lacking. In this study, we characterized glycoside hydrolase 12 (GH12) family proteins secreted by F. graminearum. We established that two GH12 proteins, Fg05851 and Fg11037, have functionally redundant roles in F. graminearum colonization of wheat. Furthermore, we determined that the GH12 proteins Fg05851 and Fg11037 are recognized by the leucine-rich-repeat receptor-like protein RXEG1 in the dicot Nicotiana benthamiana. Heterologous expression of RXEG1 conferred wheat responsiveness to Fg05851 and Fg11037, enhanced wheat resistance to F. graminearum and reduced levels of the mycotoxin deoxynivalenol in wheat grains in an Fg05851/Fg11037-dependent manner. In the RXEG1 transgenic lines, genes related to pattern-triggered plant immunity, salicylic acid, jasmonic acid, and anti-oxidative homeostasis signalling pathways were upregulated during F. graminearum infection. However, the expression of these genes was not significantly changed during infection by the deletion mutant ΔFg05851/Fg11037, suggesting that the recognition of Fg05851/Fg11037 by RXEG1 triggered plant resistance against FHB. Moreover, introducing RXEG1 into three other different wheat cultivars via crossing also conferred resistance to F. graminearum. Expression of RXEG1 did not have obvious deleterious effects on plant growth and development in wheat. Our study reveals that N. benthamiana RXEG1 remains effective when transferred into wheat, a monocot, which in turn suggests that engineering wheat with interfamily plant immune receptor transgenes is a viable strategy for increasing resistance to FHB.

Epigenomic features of DNA G-quadruplexes and their roles in regulating rice gene transcription.

A DNA G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure involved in many biological processes in mammals. The current knowledge on plant DNA G4s, however, is limited; whether and how DNA G4s impact gene expression in plants is still largely unknown. Here, we applied a protocol referred to as BG4-DNA-IP-seq followed by a comprehensive characterization of DNA G4s in rice (Oryza sativa L.); we next integrated dG4s (experimentally detectable G4s) with existing omics data and found that dG4s exhibited differential DNA methylation between transposable element (TE) and non-TE genes. dG4 regions displayed genic-dependent enrichment of epigenomic signatures; finally, we showed that these sites displayed a positive association with expression of DNA G4-containing genes when located at promoters, and a negative association when located in the gene body, suggesting localization-dependent promotional/repressive roles of DNA G4s in regulating gene transcription. This study reveals interrelations between DNA G4s and epigenomic signatures, as well as implicates DNA G4s in modulating gene transcription in rice. Our study provides valuable resources for the functional characterization or bioengineering of some of key DNA G4s in rice.

Isoprenylation modification is required for HIPP1-mediated powdery mildew resistance in wheat.

Powdery mildew (Pm), caused by Blumeria graminis f.sp. tritici (Bgt), is one of the most important wheat diseases. Heavy-metal-associated isoprenylated plant protein (HIPP1) has been proved playing important roles in response to biotic and a biotic stress. In present study, we proved HIPP1-V from Haynalidia villosa is a positive regulator in Pm resistance. HIPP1-V was rapidly induced by Bgt. Transiently or stably heterologous overexpressing HIPP1-V in wheat suppressed the haustorium formation and enhanced Pm resistance. HIPP1-V isoprenylation was critical for plasma membrane (PM) localization, interaction with E3-ligase CMPG1-V and function in Pm resistance. Bgt infection recruited the isoprenylated HIPP1-V and CMPG1s on PM; blocking the HIPP1 isoprenylation reduced such recruitment and compromised the resistance of OE-CMPG1-V and OE-HIPP1-V. Overexpressing HIPP1-VC148G could not enhance Pm resistance. These indicated the Pm resistance was dependent on HIPP1-V's isoprenylation. DGEs related to the ROS and SA pathways were remarkably enriched in OE-HIPP1-V, revealing their involvement in Pm resistance. Our results provide evidence on the important role of protein isoprenylation in plant defense.

Rapid development of wheat-Dasypyrum villosum compensating translocations resistant to powdery mildew using a triple marker strategy conducted on a large ph1b-induced population.

KEY MESSAGE: Twenty-two compensating wheat-Dasypyrum villosum translocations carrying the powdery mildew resistance gene PmV were developed using a triple marker selection strategy in a large homozygous ph1bph1b population. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive wheat disease in China. Currently, nearly all resistant varieties grown in the middle and lower reaches of the Yangtze River carry Pm21 which is present in a wheat-Dasypyrum villosum T6V#2S·6AL translocation. Its widespread use poses a strong risk of loss of effectiveness if the pathogen were to change. PmV, a Pm21 homolog carried by a wheat-D. villosum T6V#4S·6DL translocation, is also resistant to powdery mildew but is less transmittable and exploited in cultivars. To utilize PmV more effectively, a new recombinant translocation T6V#4S-6V#2S·6AL carrying PmV with a higher transmission rate was used as a basic material for inducing smaller alien translocations. A locally adapted ph1b-carrying line, Yangmai 23-ph1b, was crossed with T6V#4S-6V#2S·6AL to generate a homozygous ph1bph1b population of 6300 F3 individuals. A modified triple marker strategy based on three co-dominant markers including the functional marker MBH1 for PmV in combination with distal and proximal markers 6VS-GX4 and 6VS-GX17, respectively, was used to screen for new recombinants efficiently. Forty-eight compensating translocations were identified, 22 of which carried PmV. Two translocation lines, Dv6T25 with the shortest distal segment carrying PmV and Dv6T31 with the shortest proximal segment carrying PmV were identified, both expressed normal transmission and therefore could promote PmV in wheat breeding. This work exemplifies a model for rapid development of wheat-alien compensating translocations.

Fine mapping of two recessive powdery mildew resistance genes from Aegilops tauschii accession CIae8.

KEY MESSAGE: Two recessive powdery mildew resistance loci pmAeCIae8_2DS and pmAeCIae8_7DS from Aegilops tauschii were mapped and two synthesized hexaploid wheat lines were developed by distant hybridization. Wheat powdery mildew (Pm), one of the worldwide destructive fungal diseases, causes significant yield loss up to 30%. The identification of new Pm resistance genes will enrich the genetic diversity of wheat breeding for Pm resistance. Aegilops tauschii is the ancestor donor of sub-genome D of hexaploid wheat. It provides beneficial genes that can be easily transferred into wheat by producing synthetic hexaploid wheat followed by genetic recombination. We assessed the Pm resistance level of 35 Ae. tauschii accessions from different origins. Accession CIae8 exhibited high Pm resistance. Inheritance analysis and gene mapping were performed using F2 and F2:3 populations derived from the cross between CIae8 and a Pm susceptible accession PI574467. The Pm resistance of CIae8 was controlled by two independent recessive genes. Bulked segregate analysis using a 55 K SNP array revealed the SNPs were mainly enriched into genome regions, i.e. 2DS (13.5-20 Mb) and 7DS (4.0-15.5 Mb). The Pm resistance loci were named as pmAeCIae8_2DS and pmAeCIae8_7DS, respectively. By recombinant screening, we narrowed the pmAeCIae8_2DS into a 370-kb interval flanked by markers CINAU-AE7800 (14.89 Mb) and CINAU-AE20 (15.26 Mb), and narrowed the pmAeCIae8_7DS into a 260-kb interval flanked by markers CINAU-AE58 (4.72 Mb) and CINAU-AE25 (4.98 Mb). The molecular markers closely linked with the resistance loci were developed, and two synthesized hexaploid wheat (SHW) lines were produced. These laid the foundation for cloning of the two resistance loci and for transferring the resistance into common wheat.

Transferring a new Fusarium head blight resistance locus FhbRc1 from Roegneria ciliaris into wheat by developing alien translocation lines.

KEY MESSAGE: A new FHB resistance locus FhbRc1 was identified from the R. ciliaris chromosome 7Sc and transferred into common wheat by developing alien translocation lines. Fusarium head blight (FHB) caused by multiple Fusarium species is a globally destructive disease of common wheat. Exploring and utilization of resources with FHB resistance are the most effective and environmentally beneficial approach for the disease control. Roegneria ciliaris (Trin.) Nevski (2n = 4x = 28, ScScYcYc), a tetraploid wheat wild relative, possesses high resistance to FHB. In the previous study, a complete set of wheat-R. ciliaris disomic addition (DA) lines were evaluated for FHB resistance. DA7Sc had stable FHB resistance, which was confirmed to be derived from alien chromosome 7Sc. We tentatively designated the resistant locus as FhbRc1. For better utilization of the resistance in wheat breeding, we developed translocations by inducing chromosome structural aberrations using iron irradiation and the homologous pairing gene mutant ph1b. Totally, 26 plants having various 7Sc structural aberrations were identified. By marker analysis, a cytological map of 7Sc was constructed and 7Sc was dissected into 16 cytological bins. Seven alien chromosome aberration lines, which all had the bin 7Sc-1 on the long arm of 7Sc, showed enhanced FHB resistance. Thus, FhbRc1 was mapped to the distal region of 7ScL. A homozygous translocation line T4BS·4BL-7ScL (NAURC001) was developed. It showed improved FHB resistance, while had no obvious genetic linkage drag for the tested agronomic traits compared with the recurrent parent Alondra's. When transferring the FhbRc1 into three different wheat cultivars, the derived progenies having the translocated chromosome 4BS·4BL-7ScL all showed improved FHB resistance. This revealed the potential value of the translocation line in wheat breeding for FHB resistance.

Resistance of QYm.nau-2D to wheat yellow mosaic virus was derived from an alien introgression into common wheat.

KEY MESSAGE: The QYm.nau-2D locus conferring wheat yellow mosaic virus resistance is an exotic introgression and we developed 11 diagnostic markers tightly linked to QYm.nau-2D. Wheat yellow mosaic virus (WYMV) is a serious disease of winter wheat in China. Breeding resistant varieties is the most effective strategy for WYMV control. A WYMV resistant locus QYm.nau-2D on the chromosome arm 2DL has been repeatedly reported but the mapped region is large. In the present study, we screened recombinants using a biparental population and mapped QYm.nau-2D into an 18.8 Mb physical interval. By genome-wide association studies of 372 wheat varieties for WYMV resistance in four environments, we narrowed down QYm.nau-2D into a 16.4 Mb interval. Haplotype analysis indicated QYm.nau-2D were present as six different states due to recombination during hybridization breeding. QYm.nau-2D was finally mapped into a linkage block of 11.2 Mb. Chromosome painting using 2D specific probes and collinearity analysis among the published sequences corresponding to QYm.nau-2D region indicated the block was an exotic introgression. The Illumina-sequenced reads of four diploid Aegilops species were mapped to the sequence of Fielder, a variety having the introgression. The mapping reads were significantly increased at the putative introgression regions of Fielder. Ae. uniaristata (NN) had the highest mapping reads, suggesting that QYm.nau-2D was possibly an introgression from genome N. We investigated the agronomic performances of different haplotypes and observed no linkage drag of the alien introgression for the 15 tested traits. For marker-assisted selection of QYm.nau-2D, we developed 11 diagnostic markers tightly linked to the locus. This research provided a case study of an exotic introgression, which has been utilized in wheat improvement for WYMV resistance.

Characterization of functional relationships of R-loops with gene transcription and epigenetic modifications in rice.

We conducted genome-wide identification of R-loops followed by integrative analyses of R-loops with relation to gene expression and epigenetic signatures in the rice genome. We found that the correlation between gene expression levels and profiled R-loop peak levels was dependent on the positions of R-loops within gene structures (hereafter named "genic position"). Both antisense only (ASO)-R-loops and sense/antisense (S/AS)-R-loops sharply peaked around transcription start sites (TSSs), and these peak levels corresponded positively with transcript levels of overlapping genes. In contrast, sense only (SO)-R-loops were generally spread over the coding regions, and their peak levels corresponded inversely to transcript levels of overlapping genes. In addition, integrative analyses of R-loop data with existing RNA-seq, chromatin immunoprecipitation sequencing (ChIP-seq), DNase I hypersensitive sites sequencing (DNase-seq), and whole-genome bisulfite sequencing (WGBS or BS-seq) data revealed interrelationships and intricate connections among R-loops, gene expression, and epigenetic signatures. Experimental validation provided evidence that the demethylation of both DNA and histone marks can influence R-loop peak levels on a genome-wide scale. This is the first study in plants that reveals novel functional aspects of R-loops, their interrelations with epigenetic methylation, and roles in transcriptional regulation.

Cell autonomous and non-autonomous functions of plant intracellular immune receptors in stomatal defense and apoplastic defense.

Stomatal closure defense and apoplastic defense are two major immunity mechanisms restricting the entry and propagation of microbe pathogens in plants. Surprisingly, activation of plant intracellular immune receptor NLR genes, while enhancing whole plant disease resistance, was sometimes linked to a defective stomatal defense in autoimmune mutants. Here we report the use of high temperature and genetic chimera to investigate the inter-dependence of stomatal and apoplastic defenses in autoimmunity. High temperature inhibits both stomatal and apoplastic defenses in the wild type, suppresses constitutive apoplastic defense responses and rescues the deficiency of stomatal closure response in autoimmune mutants. Chimeric plants have been generated to activate NLR only in guard cells or the non-guard cells. NLR activation in guard cells inhibits stomatal closure defense response in a cell autonomous manner likely through repressing ABA responses. At the same time, it leads to increased whole plant resistance accompanied by a slight increase in apoplastic defense. In addition, NLR activation in both guard and non-guard cells affects stomatal aperture and water potential. This study thus reveals that NLR activation has a differential effect on immunity in a cell type specific matter, which adds another layer of immune regulation with spatial information.

Development and application of oligonucleotide-based chromosome painting for chromosome 4D of Triticum aestivum L.

Chromosome painting is a useful technique for distinguishing specific chromosomes (fragments), elucidating the genetic relationships of different genomes or chromosomes, and identifying chromosomal rearrangements. The development of chromosome- or genome-specific probes is fundamental for chromosome painting. The possibility for developing such probes specifically painting homoeologous chromosomes in allopolyploid species has been questioned since that chromosomes belonging to the same homoeologous group share highly conserved sequences. In the present study, we attempted to construct a wheat chromosome 4D-specific oligo probe library by selecting 4D-specific sequences in reference genome of common wheat cv. Chinese Spring (CS, 2n = 6x = 42, AABBDD). The synthesized library contains 27,392 oligos. Oligo painting using the probe library confirmed its specificity, shown by that only chromosome 4D could be painted in three wheat genotypes and CS nulli-tetrasomic line N4AT4D. Oligo painting was successfully used to define the 4D breakpoints in CS deletion lines involving 4D and two wheat-Haynaldia villosa 4D-4V translocation lines. Thirteen wheat relatives and a Triticum durum-H. villosa amphiploid were used for oligo painting. Except the 4D in two Aegilops tauschii accessions, the 4M in Ae. comosa and 4U in Ae. umbellulata could be painted. In tetraploid Ae. ventricosa, both 4D and 4M could be painted; however, the signal intensity of 4M was less compared with 4D. No painted chromosome was observed for the other alien species. This indicated that the relationship among D/M/U was closer than that among D/A/B as well as D with genomes H/R/Ss/Sc/Y/P/N/J. Our successful development of 4D-specific oligo probe library may serve as a model for developing oligo probes specific for other homoeologous chromosomes.

Dynamic and reversible DNA methylation changes induced by genome separation and merger of polyploid wheat.

BACKGROUND: Wheat is a powerful genetic model for studying polyploid evolution and crop domestication. Hexaploid bread wheat was formed by two rounds of interspecific hybridization and polyploidization, processes which are often accompanied by genetic and epigenetic changes, including DNA methylation. However, the extent and effect of such changes during wheat evolution, particularly from tetraploid-to-hexaploid wheat, are currently elusive. RESULTS: Here we report genome-wide DNA methylation landscapes in extracted tetraploid wheat (ETW, AABB), natural hexaploid wheat (NHW, AABBDD), resynthesized hexaploid wheat (RHW, AABBDD), natural tetraploid wheat (NTW, AABB), and diploid (DD). In the endosperm, levels of DNA methylation, especially in CHG (H=A, T, or C) context, were dramatically decreased in the ETW relative to natural hexaploid wheat; hypo-differentially methylated regions (DMRs) (850,832) were 24-fold more than hyper-DMRs (35,111). Interestingly, those demethylated regions in ETW were remethylated in the resynthesized hexaploid wheat after the addition of the D genome. In ETW, hypo-DMRs correlated with gene expression, and TEs were demethylated and activated, which could be silenced in the hexaploid wheat. In NHW, groups of TEs were dispersed in genic regions of three subgenomes, which may regulate the expression of TE-associated genes. Further, hypo-DMRs in ETW were associated with reduced H3K9me2 levels and increased expression of histone variant genes, suggesting concerted epigenetic changes after separation from the hexaploid. CONCLUSION: Genome merger and separation provoke dynamic and reversible changes in chromatin and DNA methylation. These changes correlate with altered gene expression and TE activity, which may provide insights into polyploid genome and wheat evolution.

Genome-Wide Identification of GDSL-Type Esterase/Lipase Gene Family in Dasypyrum villosum L. Reveals That DvGELP53 Is Related to BSMV Infection.

GDSL-type esterase/lipase proteins (GELPs) characterized by a conserved GDSL motif at their N-terminus belong to the lipid hydrolysis enzyme superfamily. In plants, GELPs play an important role in plant growth, development and stress response. The studies of the identification and characterization of the GELP gene family in Triticeae have not been reported. In this study, 193 DvGELPs were identified in Dasypyrum villosum and classified into 11 groups (clade A-K) by means of phylogenetic analysis. Most DvGELPs contain only one GDSL domain, only four DvGELPs contain other domains besides the GDSL domain. Gene structure analysis indicated 35.2% DvGELP genes have four introns and five exons. In the promoter regions of the identified DvGELPs, we detected 4502 putative cis-elements, which were associated with plant hormones, plant growth, environmental stress and light responsiveness. Expression profiling revealed 36, 44 and 17 DvGELPs were highly expressed in the spike, the root and the grain, respectively. Further investigation of a root-specific expressing GELP, DvGELP53, indicated it was induced by a variety of biotic and abiotic stresses. The knockdown of DvGELP53 inhibited long-distance movement of BSMV in the tissue of D. villosum. This research provides a genome-wide glimpse of the D. villosum GELP genes and hints at the participation of DvGELP53 in the interaction between virus and plants.

Research progress of plant cytogenetics in Jiangsu province.

Cytogenetics was established based on the "Chromosome theory of inheritance", proposed by Boveri and Sutton and evidenced by Morgan's lab in early stage of the 20 th centrary. With rapid development of related research areas, especially molecular genetics, cytogenetics developed from traditional into a new era, molecular cytogenetics in late 1960s. Featured by an established technique named DNA in situ hybridization (ISH), molecular cytogenetics has been applied in various research areas. ISH provids vivid and straightforward figures showing the virtual presence of DNA, RNA or proteins. In combination with genomics and cell biology tools, ISH and derived techniques have been widely used in studies of the origin, evolution, domestication of human, animal and plant, as well as wide hybridization and chromosome engineering. The physical location and order of DNA sequences revealed by ISH enables the detection of chromosomal re-arrangments among related species and gaps of assembled genome sequences. In addition, ISH using RNA or protein probes can reveal the location and quantification of transcripted RNA or translated protein. Since the 1970s, scientists from universities or institutes belonging to the Jiangsu Society of Genetics have initiated cytogenetics researches using various plant species. In recent years, research platforms for molecular cytogenetics have also been well established in Nanjing Agricultural University, Yangzhou University, Nanjing Forestry University, Jiangsu Xuhuai Academy of Agricultural Sciences, and Jiangsu Normal University. The application of molecular cytogenetics in plant evolution, wide hybridization, chromosome engineering, chromosome biology, genomics has been successful. Significant progresses have been achieved, both in basic and applied researches. In this paper, we will review main research progresses of plant cytogenetics in Jiangsu province, and discuss the potential development of this research area.

Fine mapping of wheat powdery mildew resistance gene Pm6 using 2B/2G homoeologous recombinants induced by the ph1b mutant.

KEY MESSAGE: Using the ph1b mutant, the recombination frequency between the homoeologous region of 2B and 2G was significantly increased. By this, we narrowed Pm6 to a 0.9 Mb physical region. The powdery mildew (Pm) resistance gene Pm6 from Triticum timopheevii (2n = 48, AAGG) was mapped to the long arm of chromosome 2G and introduced into common wheat in the form of 2B-2G introgressions. The introgression line IGV1-465 has the shortest 2G segment, which is estimated 37 Mb in size when referring to 2BL genome reference of Chinese Spring (CS). The further fine mapping of Pm6 was impeded by the inhibition of allogeneic chromosome recombination between 2B and 2G in the Pm6 region. In the present study, to overcome 2B/2G recombination suppression, a ph1b-based strategy was employed to produce introgressions with reduced 2G fragments for the fine mapping of Pm6. IGV1-465 was crossed and backcrossed to the CSph1b mutant to produce plants with increased 2B/2G chromosome pairing frequency at the Pm6 region. A total of 182 allogeneic recombinants were obtained through two-round screening, i.e., first round of screening of 820 BC1F2:3 progenies using the flanking markers CIT02g-14/CIT02g-19 and second round of screening of 642 BC1F2:4 progenies using the flanking markers CIT02g-13/CIT02g-18, respectively. Through marker analysis using 30 chromosome 2G-specific markers located in the Pm6 region, the identified recombinants were divided into 14 haplotypes. Pm resistance evaluation of these haplotypes enabled us to narrow Pm6 to a 0.9 Mb physical region of 2BL, flanked by markers CIT02g-20 and CIT02g-18. Six wheat varieties containing Pm6 were identified from a natural population, and they showed increased Pm resistance. This implied Pm6 is still effective, especially when used in combination with other Pm resistance genes.

Dissection and cytological mapping of chromosome arm 4VS by the development of wheat-Haynaldia villosa structural aberration library.

KEY MESSAGE: A cytological map of Haynaldia villosa chromosome arm 4VS was constructed to facilitate the identification and utilization of beneficial genes on 4VS. Induction of wheat-alien chromosomal structure aberrations not only provides new germplasm for wheat improvement, but also allows assignment of favorable genes to define physical regions. Especially, the translocation or introgression lines carrying alien chromosomal fragments with different sizes are useful for breeding and alien gene mapping. Chromosome arm 4VS of Haynaldia villosa (L.) Schur (syn. Dasypyrum villosum (L.) P. Candargy) confers resistances to eyespot and wheat yellow mosaic virus (WYMV). In this research, we used both irradiation and the pairing homoeologous gene (Ph) mutant to induce chromosomal aberrations or translocations. By using the two approaches, a structural aberration library of chromosome arm 4VS was constructed. In this library, there are 57 homozygous structural aberrations, in which, 39 were induced by the Triticum aestivum cv. Chinese Spring (CS) ph1b mutant (CS ph1b) and 18 were induced by irradiation. The aberrations included four types, i.e., terminal translocation, interstitial translocation, deletion and complex structural aberration. The 4VS cytological map was constructed by amplification in the developed homozygous aberrations using 199 4VS-specific markers, which could be allocated into 39 bins on 4VS. These bins were further assigned to their corresponding physical regions of chromosome arm 4DS based on BLASTn search of the marker sequences against the reference sequence of Aegilops tauschii Cosson. The developed genetic stocks and cytological map provide genetic stocks for wheat breeding as well as alien gene tagging.

Characterization of the Heavy-Metal-Associated Isoprenylated Plant Protein (HIPP) Gene Family from Triticeae Species.

Heavy-metal-associated (HMA) isoprenylated plant proteins (HIPPs) only exist in vascular plants. They play important roles in responses to biotic/abiotic stresses, heavy-metal homeostasis, and detoxification. However, research on the distribution, diversification, and function of HIPPs in Triticeae species is limited. In this study, a total of 278 HIPPs were identified from a database from five Triticeae species, and 13 were cloned from Haynaldia villosa. These genes were classified into five groups by phylogenetic analysis. Most HIPPs had one HMA domain, while 51 from Clade I had two, and all HIPPs had good collinear relationships between species or subgenomes. In silico expression profiling revealed that 44 of the 114 wheat HIPPs were dominantly expressed in roots, 43 were upregulated under biotic stresses, and 29 were upregulated upon drought or heat treatment. Subcellular localization analysis of the cloned HIPPs from H. villosa showed that they were expressed on the plasma membrane. HIPP1-V was upregulated in H. villosa after Cd treatment, and transgenic wheat plants overexpressing HIPP1-V showed enhanced Cd tolerance, as shown by the recovery of seed-germination and root-growth inhibition by supplementary Cd. This research provides a genome-wide overview of the Triticeae HIPP genes and proved that HIPP1-V positively regulates Cd tolerance in common wheat.