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.

[Molecular cytogenetics, fertility, and scab resistance of the intergeneric hybrid F1 and BC1 between Triticum aestivum and Roegneria kamoji].

The reciprocal intergeneric hybrids between common wheat and Roegneria kamoji were successfully obtained by means of embryo culture. Morphology, chromosome pairing behavior at meiosis, fertility, and resistance to scab of the hybrid F1 and BC1 were studied. The results showed that the morphology of the reciprocal intergeneric hybrids F1 between R. kamoji and T. aestivum cv. Chinese Spring were intermediate type between the two parental species. The chromosome configuration at metaphase I (MI) of pollen mother cell (PMC) in reciprocal F1 was 40.33I + 0.78II + 0.03III and 40.40I + 0.79II, respectively. All of the F1 plants showed complete male sterility, and the seeds of BC1 were obtained by backcrossing with Chinese Spring pollen. The somatic chromosome numbers in BC1 plants of (R. kamoji x Chinese Spring) F1 x Chinese Spring ranged from 55 to 63. Many univalents were observed at MI of PMC, which resulted in the sterility of BC1 plants. Similarly, the chromosome numbers in BC1 plants of (Chinese Spring xR. kamoji) F1 x Chinese Spring also ranged from 55 to 63; however, many bivalents at MI of PMC and fertile pollen were observed resulting in partial fruitfulness in some BC1 plants by self-crossing. A plant (2n=63) with 42 wheat chromosomes and 21 R. kamoji chromosomes was obtained from R. kamoji x Chinese Spring cross, which had a chromosome configuration at MI of 26.40I + 18.30II. Because many univalents existed, this plant showed complete male sterility, and BC1 plants were obtained by back-crossing with Chinese Spring as the pollen parent. The chromosome numbers of BC1 ranged from 40 to 59, which contained less alien chromosomes. Although the morphology of the spike in BC1 plants was similar to that of Chinese Spring, these BC1 plants were still sterile. All F1 and most of the BC1 plants showed high resistance to Fusarium graminearum, which indicated that the resistance to scab from R. kmoji can be transferred into wheat.

[Molecular marker analysis of wheat-Roegneria ciliaris additions lines.].

A total of 135 EST, 27 STS, and 253 SSR primer pairs located in 7 homoeologous groups of wheat and barly were used for amplification of 24 possible Wheat-R. ciliaris disomic addition lines. Fifty-five primer pairs could amplified polymorphic bands between common wheat variety "Inayama Komugi" and the Inayama Komugi- R. ciliaris amphiploid, and 31 of the 55 could amplify the specific bands of R. ciliaris in the addition lines. According to the PCR (polymerase Chain Reaction) results, the added R. ciliaris chromosomes in lines 07K02, 07K06, 07K39, 07K201, 07K202, 07K255, and 07K256 belong to homoeologous group 1 of wheat; the added R. ciliaris chromosomes in lines 07K07, 07K08, 07K09, 07K11, 07K14, and 07K17 belong to homoeologous group 2 of wheat; the added R. ciliaris chromosomes in lines 07K15, 07K16, 07K21, and 07K47 belong to homoeologous group 6 of wheat.

Plastidial glutathione reductase from Haynaldia villosa is an enhancer of powdery mildew resistance in wheat (Triticum aestivum).

A full-length cDNA (Hv-GR) whose transcript accumulation increased in response to infection by Blumeria graminis DC.f.sp. tritici (Bgt) was isolated from Haynaldia villosa. Southern analysis revealed a single copy of Hv-GR present in H. villosa. This gene encodes a glutathione reductase (GR) with high similarity to chloroplastic GRs from other plant species. Chloroplastic localization of Hv-GR was confirmed by targeting of the green fluorescent protein (GFP)-Hv-GR fusion protein to chloroplasts of epidermal guard cells. Following inoculation with Bgt, transcript accumulation of Hv-GR increased in a resistant line of wheat, but no significant change was observed in a susceptible line. In vivo function of Hv-GR in converting oxidized glutathione (GSSG) to the reduced form (GSH) was verified through heterologous expression of Hv-GR in a yeast GR-deficient mutant. As expected, overexpression of this gene resulted in increased resistance of the mutant to H(2)O(2), indicating a critical role for Hv-GR in protecting cells against oxidative stress. Moreover, overexpression of Hv-GR in a susceptible wheat variety, Triticum aestivum cv. Yangmai 158, enhanced resistance to powdery mildew and induced transcript accumulation of other pathogenesis-related genes, PR-1a and PR-5, through increasing the foliar GSH/GSSG ratio. Therefore, we concluded that a high ratio of GSH to GSSG is required for wheat defense against Bgt, and that chloroplastic GR enzymes might serve as a redox mediator for NPR1 activation.

[Discrimination of the Triticum aestivum-T. timopheevii introgression lines using PCR-based molecular markers].

In order to determine the fragment size of Triticum timopheevii chromosome segments introduced into wheat background and physically map the Pm6 gene, a total of 72 primers (including SSR and STS primers) were used to analyze the eight introduced introgression lines containing Pm6 gene. Referring to the available mapping information of the analyzed markers on chromosome 2B, Pm6 was physically located in distal part of the long arm of chromosome 2B at the region of Bin 2BL-6. The introgressed fragment sizes of the chromosome 2G in different introgression lines was determined to be as follow (from short to long): IGV1-465

[Distribution of 45S rDNA sequence on chromosomes of Triticum aes-tivum and its relative species].

Fluorescent in situ hybridization (FISH) and sequential C-banding and FISH techniques were used in locating 45S rDNA sequence on specific chromosomes of Triticum monococcum, T. dicoccoides, T. aestivum, Hordeum vulgare, Haynaldia villosa, T. durum-Haynaldia villosa amphiploid, Avena fatus, and Roegneria kamoji. High polymorphism between common wheat and its relatives on 45S rDNA sequence distribution was detected. All the chromosomal secondary constriction regions of these species had 45S rDNA loci. Some non-satellite chromosomes had additional 45S rDNA loci. A 45S rDNA locus was located on the terminal region of the short arm of R. kamoji chromosome 1Rk#1.

[Plant transient expression system in functional genomics].

With the development of structural and functional genomics, nowadays specific plant genome and transcriptome sequences can be cloned much easier and faster. Next step is to identify the functions of different genes and regulating elements to unravel the genetic mechanisms behind plant growth and development. Expression and its regulation are the language and dynamic property of genetic material, so expression and regulation analysis of target genes and sequences in plant cell is the basis for function study. Besides stable genetic transformation, plant transient expression system gains broad application in recent years, and its combination with other new technologies as gene shuffling, VIGS and RNAi plays a more and more important role in plant functional genomics.

[Transformation of powdery mildew resistance-related genes of wheat].

Anthocyanin synthesis regulation gene C1-Lc was used as the reporter gene to optimize the parameters of gene-gun transformation protocol through counting of red spots on wheat calli after transient expression. Wheat Beclin1 like gene TaTBL and thiosulfate sulfutransferase gene TaTST proved to have an increased expression level after induction of wheat powdery mildew fungus (Erysiphe graminis f.sp. tritici Em. Marchal.). These two resistance-related genes were constructed into expression vectors driven by the strong ubi promoter and used to perform genetic transformation on wheat cv Yangmai158 immature embryo-derived calli through particle bombardment. After two rounds of herbicide bialaphos selection and regeneration, herbicide-resistance plants were obtained, which were subsequently subjected to PCR analysis. Five TaTBL transgenic plants and six TaTST transgenic plants were identified. Pathogen inoculation of detached leaves showed that the introduction of exogenous gene increased wheat resistance level by delaying the development of powdery mildew symptoms.

Screening and applying wheat microsatellite markers to trace individual Haynaldia villosa chromosomes.

Wheat (Triticum aestivum L.) microsatellite markers were screened for detecting Haynaldia villosa L. chromosomes introduced into wheat background. Two hundred and seventy six primer pairs mapped on 7 homeologous groups of wheat were used to amplify the gDNA of T. aestivum and H. villosa. The results showed that 148 of 276 microsatellite primers amplified polymorphic bands between common wheat cv. Chinese Spring and H. villosa. Primers wmc49 (1BS), wmc25 (2BS), gdm36 (3DS), gdm145 (4AL), wmc233 (5DS), wmc256 (6AL) and gwm344 (7BL) produced a specific polymorphic DNA fragment on chromosome 1V to 7V of H. villosa, respectively. In addition, gwm469 (6DS) detected a specific band on 2V; gdm107 (2DS) amplified a specific band on 6V. These microsatellite markers were effective in identifying individual H. villosa chromosomes in other T. aestivum-H. villosa chromosome addition, substitution and translocation lines involved in different H. villosa accessions and wheat backgrounds. Therefore, these chromosome-specific microsatellite markers could be used as molecular markers for detection of chromosomes of H. villosa in common wheat.

[Analysis of the functions of wheat resistance-related genes by a transient expression system].

Transient expression system was used to analyze the functions of three resistance- related genes: TaTBL, TaPK1 and TaTST. Target genes were constructed into plant expression vectors and transformed into leaf epidermal cells of a powdery mildew-susceptible wheat variety by gene gun. GUS gene was co-transformed with target gene to mark the transformed cells. After transformation, leaf surface was inoculated with powdery mildew conidiospores. Forty eight hours after inoculation, penetration of the fungus and formation of haustoria in transformed cells were observed to evaluate the effects of the target gene's products on the invasion of powdery mildew. The results implied that all these three genes, when transiently expressed in leaf epidermal cells of susceptible wheat variety, could partly inhibit the penetration of conidiospores and formation of haustoria, and to some extent increase the resistance of cells to powdery mildew.

[Inheritance analysis and molecular marker selection of genes for wheat spindle streak mosaic disease resistance].

Three wheat spindle streak mosaic viruses (WSSMV) resistant cultivars ('Yining Xiaomai', 'Xu87-633', and 'Xifeng') and one susceptible cultivar ('Zhen9523') were used as parents of 3 crosses in this experiment. WSSMV resistance of the parents, F1, and F2 was evaluated under field condition. Based on the segregation ratios of resistant and susceptible plants in F, and F2 populations, it was deduced that the resistance to WSSMV was dominant and the inheritable factors controlling WSSMV resistance were encoded by the nuclear genome. WSSMV resistances in 'Yining Xiaomai' and 'Xifeng' were controlled by two pairs of alleles, which showed complementary effects. However the resistance in 'Xu-87633' was controlled by a single dominant gene. 266 pairs of SSR primers located on 21 wheat chromosomes were used for polymorphic analysis of the two resistant and the susceptible parents 'Yining Xiaomai' and 'Zhen9523', and 108 of them amplified polymorphic DNA products. By Bulk Segregant Analysis of resistant and susceptible pools, one pair of primer located on chromosome arm 2DS, Xgwm261, were found being linked to WSSMV resistance. The 224 F2 individuals were then amplified with marker Xgwm261. The statistic genetic distance between Xgwm261 and the resistance locus was calculated to be 22.9 cM using the software Mapmaker 3.0.

[Development and identification of a set of Triticum aestivum-Thinopyrum bessarabicum disomic alien addition lines].

In order to transfer the genes for salt tolerance and disease resistance from Thinopyrum bessarabicum into wheat, the hybrid progenies between T. aestivum cv. Chinese Spring and T. aestivum cv. Chinese Spring-amphiploid Th. bessarabicum were screened. A set of T. aestivum-Th. bessarabicum disomic addition lines was developed with the assistance of mitotic chromosome C-banding and genomic in situ hybridization (GISH), as well as GISH on meiotic M I chromosome preparations. The results indicated that all the wheat chromosomes in the lines remained unchanged karyotypically, while the added Th. bessarabicum chromosomes paired regularly in meiosis. The developed disomic addition lines were designated temporarily as DAJ1, DAJ2, DAJ3, DAJ4, DAJ5, DAJ6 and DAJ7 respectively. Determination of the homoelogous groups of the added Th. bessarabicum chromosomes and localization of the genes for salt tolerance and disease resistance are undergoing.

Cloning, mapping and protein expression of wheat thaumatin protein gene (TaTLP1).

To understand wheat powdery mildew resistance mechanism, reverse-transcription polymerase chain reaction (RT-PCR) and cDNA library screening were performed to isolate the full-length cDNA of wheat thaumatin protein gene from wheat-Haynaldia villosa 6VS/6AL translocation line. The putative amino acid sequence of this gene consists of 173 amino acid residues, and is an acid polypeptide. It was highly homologous to thaumatin proteins isolated from other plants, so it is designated as TaTLP1 (GenBank accession number: AF384146). Northern blot analysis of TaTLP1 showed that the transcription difference obviously existed between resistant wheat-Haynaldia villosa 6VS/6AL translocation line and susceptible "Yangmai 5". The result of western blot showed that the protein expression product of TaTLP1 gene in wheat seedling leaf was a soluble cytoplasm protein, whose expression was induced by fungus Erysiph graminis and apparently related to disease resistance of the 6VS/6AL translocation line. Southern blot indicated that the TaTLP1 gene had 1-2 copies in wheat genome, and had been localized on the specific region of 7B and 7D chromosomes in wheat.

[Screening for resistance gene candidate from a genomic TAC library of Triticum aestivum-Haynaldia villosa translocation line 6VS/6AL by pooled PCR].

A pair of degenerate primers were designed based on NBS (nucleotide binding site, NBS) domain of resistance(R) gene and used to perform PCR with cDNA from the translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa. A clone (N7) characterized with NBS was obtained by sequencing analysis. Two specific primers were designed from the N7 sequence and used to screen a genomic TAC (transformation-competent artificial chromosome, TAC) library of 6VS/6AL consisting of ca. 2 x 10(6) clones. The library was stored as clone pools in twenty-two 96-well plates, each well containing approximately 1000 TAC clones. TAC plasmids were prepared from all the 2112 pools. Using a pooled PCR screening procedure, a positive TAC clone having a 40 kb insert was obtained. The positive clone was confirmed by Southern hybridization with the NBS fragment as a probe. The results indicate that the pooled PCR method is effective for screening of genomic libraries having large number of clones.

Molecular cloning, characterization and mapping of a rhodanese like gene in wheat.

To isolate genes related to resistance to Erysiphe graminis (Blumeria graminis) DC. f. sp. tritici in wheat (Triticum aestivum L.), differential display analysis was conducted for mRNA extracted from seedlings of a wheat-Haynaldia villosa 6VS/6AL translocation line 92R137 that contains a powdery mildew resistance gene Pm21. A full-length cDNA sequence named TaTST (Triticum aestivum thiosulfate sulfurtransferase) homologous to the thiosulfate sulfurtransferase (rhodanese) in Datisca glomerata was isolated. Northern blot showed that the expression of TaTST was enhanced after infection with Erysiphe graminis. TaTST was mapped on the short arm of 6B chromosomes of wheat through Southern blot and GSP-PCR using Chinese Spring nullisomic/tetrasomic lines and ditelosomic lines. There is a homologue of TaTST on 6VS too.

[Development of Triticum aestivum-Leymus racemosus translocation lines NAU601 and NAU618 and their test-cross analysis with double ditelosomic].

Two Triticum aestivum-Leymus racemosus disomic translocation lines were selected and identified from irradiated progenies of T. aestivum-L. racemosus addition lines Lr. 2 and Lr. 7 by somotic chromosome C-banding and fluorescence in situ hybridization by using L. racemosus genomic DNA as probe. NAU618 (MS142-3), 2n = 44, is a disomic addition translocation line with a pair of translocated chromosome consisting of 5/6 length of L. racemosus chromosome Lr. 7 (including centromere) and 1/3 distal part of wheat chromosome 1A short arm. The alien segment is about 4/5 of the length of translocated chromosome. NAU601 (MS101-4), 2n = 42, is a disomic translocation lines with a pair of translocated chromosome consisting of 4B short arm, interstitial 1/3 part of 4BL and almost Lr. 2S, the alien segment is a half of the long arm of translocated chromosome. The results of test-cross analysis with T. aestivum c. v Chinese Spring double ditelosomic combine meiotic chromosomes C-banding indicated that the translocated chromosomes in these two translocation lines were involved in chromosome 1A and 4B respectively. Their resistance to wheat scab was identified using single floret inoculation in three successive years and showed that the resistance of NAU618(MS142-3) was similar to that of resistant check variety Sumai 3, and NAU601(MS101-4) was lower than Sumai 3 but much higher than susceptible parent Chinese Spring.

[Structural changes of 4V chromosome of Haynaldia villosa induced by gametocidal chromosome 3C of Aegilops triuncialis].

Chromosome 3C of Aegilops triuncialis was discovered with ability to be transferred preferentially in the case of its monosomic status in wheat background, whereas, those gametes without 3C would result in chromosome structural changes including deletions and translocations. In the present study, Triticum aestivum-Haynaldia villosa substitution line 4V(4D) developed in our laboratory, was crossed to T. aestivum c.v. Norin 26-Aegilops triuncialis 3C addition line, and the hybrids F1 were then backcrossed with common wheat in order to induce structural changes of 4V. Both chromosome C-banding and genomic in situ hybridization was applied to search such chromosome variations. In this case, total genomic DNA of Haynaldia villosa was labelled by Biotin-11-dUTP as probes and total genomic DNA of Chinese Spring as the block. Moreover, several chromosome changes within common wheat such as isochromosome 1BL.1BL(B39-2) and others were also revealed. The result indicated that two translocation lines T4VL.3AS(A47-10-3) and T4VS.4DL(A47-25-4), two telocentric chromosome lines A47-7-2(4VS) and A47-32-2(4VL), and two isochromosomes including 4VS.4VS(A47-23) and 4VL.4VL(A412-5-4) were identified from BC1F2 or BC1F3. This result indicated that gametocidal chromosome 3C of Aegilop triuncialis could effectively induce structural changes of both chromosome 4V of Haynaldia villosa and chromosomes of wheat.

[Homoeologous grouping of R. kamoji chromosomes introduced in wheat using RFLP molecular marks].

Twenty six DNA probes from seven homoeologous groups of triticeae were screened to reveal the RFLP between 45 wheat-R. kamoji derivatives and their parents R. kamoji, Chinese Spring, Yangmai 5. The result showed that the introduced R. kamoji chromosomes in 16 wheat-R. kamoji alien chromosome lines including additions, substitution or putative translocations were grouped into homoeologous group 1, 3, 5, 6 and 7. Alien chromosome pairs could be readily transmitted into the descendants. The added chromosomes in K139, K141, K214, K218, K219 and K224 disomic addition lines were grouped into homoeologous group 1, but the added chromosomes in K214 and K218 were different from K219 and K224 which originated from different genomes of R. kamoji. Ditelosomic addition line K147 might involve a R. kamoji chromosome long arm homoeologous to group 1 of wheat, and the added R. kamoji 1 L chromosomes in K139, K141 and K147 probably derived from different three genomes of R. kamoji. U chromosome of R. kamoji. showed homology to wheat homoeologous group 1. Homoeologous group 1 chromosome of R. kamoji, particularly its long arm is related to genes for scab resistance. Result also demonstrated a possible rearrangement occurred between homoeologous group 1 and group 6 of R. kamoji. Two R. kamoji chromosomes introduced in K203 were grouped to homoeologous group 1 and 6, respectively. In K166, the introgressed R. kamoji chromosome involved the short arm of group 5. Another alien chromosome line K177 was revealed as to be with introduced chromosome involving group 5L, 6S and 7SL of R. kamoji. Results also confirmed the homoeology between S, H and Y genomes of R. kamoji.

[Construction and analysis of near-isogenic line derived from wheat cultivar sumai 3].

Wheat scab can cause significant yield lost and quality decrease as well as toxicoses in animals and humans. Sumai 3, a resistant cultivatr to scab, is widely used in the wheat breeding of scab resistance. To study the inheritance of resistance to scab in Sumai 3, the highly susceptible cultivar Chuan 980 was crossed with Sumai 3 and backcrossed with Sumai 3 as a recurrent parent for seven times, thus a near-isogenic line S016 susceptible to scab was developed. S016 was evaluated in five regions and a period of two years for resistance to spread of scab in spike. Results showed that S016 was as highly susceptible to wheat scab as one of the parent Chuan 980. Molecular markers (RFLP, RAPD) were screened to identify chromosome regions of negative element of resistance gene (s) in S016. The DNA polymorphism between the near-isogenic lines was showed using six restrict enzymes (EcoRI, EcoRV, HindIII, Dra I, BamH I, Xba I) and 85 probes located in wheat chromosome 2D. Among the amplified bands of 450 10-mer random primers, OPH191400 and OPH191200 were perhaps linked to negative regulator element. Some reported probes linking to scab resistance gene(s) did not show any polymorphism between this near-isogenic line and their generation.