Molecular mapping and genetic analysis of a QTL controlling spike formation rate and tiller number in wheat.

Spike formation rate (SR), which is based on maximum tiller number per unit area and spike number per unit area, is an important yield-related trait in wheat. Increasing the spike formation rate reduces growth competition and wastage of photosynthate from ineffective tillers. Unfortunately, research studies involving quantitative trait locus (QTL) mapping for wheat spike formation rate are limited. In the present study, a set of 371 recombinant inbreed line (RIL) population, which were derived from 1BL/LRS wheat-rye translocation lines CN18 and T1208, was analysed by simple sequence repeat (SSR) markers. Genetic analysis showed that a stable and major QTL (QSR.sicau-4D) for spike formation rate was localized to chromosome 4D and explained 18.24% and 24.48% of the observed phenotypic variance in 2015 and 2016, respectively. This QTL was closely linked to SSR marker Xcfd23, and the genetic distance between the flank markers was 3.28cM. Furthermore, QSR.sicau-4D might be a novel pleiotropic QTL, which also controlled maximum tiller number per unit area (QMTN.sicau-4D) and tiller number during pre-winter per unit area (QTNW.sicau-4D). The marker Xcfd23 associated with SR may be utilized in marker-assisted selection in wheat breeding.

Abnormal mitosis induced by wheat-rye 1R monosomic addition lines.

Octoploid triticale were derived from common wheat (Triticum aestivum L. 'Mianyang11') × rye (Secale cereale L. 'Kustro'), and some progeny were obtained by the backcrossing of triticale with 'Mianyang11' followed by self-fertilization. In situ hybridization using rye genomic DNA and repetitive sequences pAs1 and pSc119.2 as probes was used to analyze the mitotic chromosomes of these progeny. Three wheat-rye 1R monosomic addition lines and a wheat line (12FT-1685) containing a 1R and a 1BL.1RS translocation chromosome were identified. Abnormal mitosis was observed in the two lines. During mitosis of a 1R monosomic addition line (3-8-20-1R-2), lagging chromosomes, micronuclei, chromosomal bridges, and the one pole segregation of 1R chromosome were observed. Abnormal mitotic behaviour of chromosomes was also observed in some of the self-progeny plants of lines 12FT-1685 and 3-8-20-1R-2. These progeny contained 1R chromosome or 1R chromosome arm. In addition, 4B chromosomes were absent from one of the progeny of 3-8-20-1R-2. This abnormal mitotic behaviour of chromosomes was not observed in two other 1R monosomic addition lines. These results indicate that a single 1R chromosome added to wheat might cause abnormal mitotic behaviour of both wheat and rye chromosomes and different genetic variations might occurr among the sibling 1R monosomic addition lines.

[Application of polarization fluorescence to the study on the effects of oxidative stress on wheat chloroplast].

In the present paper wheat flag leaves were collected during the tasseling period, and then 1 mmol x L(-1) hydrogen peroxide was added to induce oxidative stress on leaves. In comparison, the detached leaves were also kept under drought or darkness condition for 24 h for the same purpose. Following the preparation of chloroplasts, polarization fluorescence spectroscopic method was utilized to measure fluorescence emission spectra and fluorescence excitation spectra of chloroplasts in the case of VV, VH, HV and HH, where V and H is representative of vertical polarization and horizontal polarization, respectively. Gaussian deconvolution was done on emission spectra, and the fitting data revealed that no matter whether Chla or Chlb molecules were excited upon excitation at 436 nm or 475 nm, the ratio of fluorescence peak area at 684 nm and 720 nm, i. e. A684/ A720, tends to increase slightly after oxidative stress. In addition, some useful information was available from polarization excitation spectra, where it was observed that the treatment of oxidative stress gave rise to higher ratio of excitation peak intensity between 436 nm and 475 nm (E436/E475), indicating that Chla made more contribution to PSII fluorescence emission than Chlb did. Simultaneously, the ratio of 475 nm and 600 nm (E475/E600), representing the energy transfer efficiency from Car to Chlb, was also found to be higher after the detached leaves were treated. In addition, both fluorescence polarization and viscosity were calculated in this paper, and the data showed that oxidative stress should be responsible for higher fluorescence polarization at 680 nm and higher viscosity in microenviroment. The above-mentioned phenomenon is consistent with the lipid peroxidation of unsaturated fatty acids. It also provides a simple and feasible method to study oxidative stress.

[Establishment of wheat-rye addition lines and De Novo powdery mildew resistance gene from chromosome 5R].

The F5 plants derived from octoploid triticale × common wheat were investigated by FISH methods using repetitive DNA sequences pAS1 and pSc119.2 as probes. The disease resistance of these plants was also screened and evaluated in the field. The 1R, 2R, 3R, 4R, 5R, 6R and 7R monosomic addition lines and 1R and 2R disomic addition lines were found. The occurrence frequencies of chromosomes 1R and 4R addition lines were higher than that of chromosomes 2R, 3R, 5R, 6R, and 7R addition lines in the high generation screened. The 5R and 6R monosomic addition lines were immune to powdery mildew. The chromosome 5R in this study might carry new powdery mildew resistance gene(s). In addition, the preferential elimination of chromosome 4B was observed in several plants.

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

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

[Effects of redox state of disulfide bonds on the intrinsic fluorescence and denaturation of Trx-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea].

In the present paper, thioredoxin-fused gibberellin-induced cysteine-rich protein from Gymnadnia conopsea, desigated as Trx-GcGASA and expressed prokaryotically, was purified and identified by using Ni(2+) -NTA affinity chromatography column and SDS-PAGE, and then its intrinsic fluorescence was investigated in the absence and presence of dithiothreitol (DTT), oxidized glutathione (GSSG), peroxide and guanidine hydrochloride (GdnHCl) by means of steady-state fluorescence spectroscopic methods. It was found that (1) at the neutral pH Trx-GcGASA had maximum fluorescence emission at 305 nm following excitation at different wavelengths varying from 250 to 280 nm, which was ascribed to the fluorescence emission from tyrosine residues. (2) The reduction of disulphide bonds lead to the changes in the relative fluorescence intensity between tyrosine and tryptophan residues from 0.7 to 1.8. (3) Both Tyr and Trp residues underwent 12%-21% decrease in fluorescence intensity with the addition of 0.5 mmol x L(-1) GSSG or 5 mmol x L(-1) peroxide. The latter was roughly consistent with the antioxidative activity reported in vivo. (4) No matter whether 1 mmol x L(-1) DTT was absent or present, the fusion protein could not be fully unfolded with lambda(max) < 350 nm following the treatment of 6 mol x L(-1) GdnHCl. (5) Fusion protein Trx-GcGASA experienced GdnHCl-induced denaturation process, and the unfolding equilibrium curve could be well fitted by using two-state model, giving the Gibbs free energy change (deltaG) of 3.7 kJ x mol(-1). However, it was not the case for reduced Trx-GcGASA protein. The aforementioned experimental results will not only provide some guides to investigate the effects of fusion partner Trx on the unfolding thermodynamics, kinetics and refolding process of Trx-GcGASA, but also will be useful for further studies on the strucuture of GA-induced cysteine-rich protein with the help of spectroscopic methods.

Vascular gene transfer and drug delivery in vitro using low-frequency ultrasound and microbubbles.

AIM: To determine the effects of ultrasound exposure in combination with a microbubble contrast agent (SonoVue) on the cellular uptake and delivery of drugs/genes into human umbilical vein endothelial cells (HUVECs) as well as their biological effects on migration. METHODS: HUVECs in suspension were exposed to pulsed ultrasound with a 10% duty cycle in combination with various concentrations of a microbubble contrast agent (SonoVue) using a digital sonifier at a frequency of 20 kHz and an intensity of 3.77 W/cm(2) on the surface of a horn tip. Cell culture inserts were used to determine the cell migration ability. RESULTS: Exposure to pulsed ultrasound resulted in enhanced green fluorescent protein (EGFP) gene transfection efficiencies ranging from 0.2% to 2%. The transfection efficiency of HUVECs was approximately 3-fold higher in the presence of SonoVue than in its absence at the effective exposure time of 6 s. For drug delivery to HUVECs using ultrasound, the delivery efficiencies of a low-molecular-weight model drug (TO-PRO-1, M(W) 645.38) were significantly higher when compared to drug delivery without ultrasound, with a maximum efficiency of approximately 34%. However, the delivery efficiencies of a high-molecular-weight model drug (Dextran-Rhodamine B, M(W) 70,000) were low, with a maximum delivery efficiency of nearly 0.5%, and gene transfection results were similarly poor. The migration ability of HUVECs exposed to ultrasound was also lower than that of the control (no exposure). CONCLUSION: The use of low-frequency and low-energy ultrasound in combination with microbubbles could be a potent physical method of increasing drug/gene delivery efficiency. This technique is a promising nonviral approach that can be used in cardiovascular disease therapy.

[Study on the effect of C-terminal acidic protein on the prokaryotic expression of alpha-thionin by FTIR microspectroscopy].

Fourier transform infrared (FTIR) microspectroscopy was used to investigate the effects of C-terminal acidic protein on the secondary structure of wheat alpha-thionin in the absence of signal peptide during the prokaryotic expression process. SDS-PAGE analysis revealed that the presence of acidic protein gave rise to the formation of inclusion body, however, the absence of acidic protein greatly enhanced the solubility of the heterogenous protein expressed in E. coli BL21(DE3) with the induction of 1 mmol x L(-1) IPTG at 37 degrees C. Difference spectra in amide I region were obtained by subtraction between the spectra of intact cells containing S and Sc, which corresponds to the absence and presence of C-terminal acidic proteins, respectively. The second derivative of the difference spectra measured 2 h after induction showed one principal component at approximately 1 630 cm(-1), while no significant peak appeared at the same peak position when the spectra before induction were compared. Combined with SDS-PAGE of recombinant protein, the authors presumed that the peak absorption at approximately 1 630 cm(-1) is most likey to be assigned to protein aggregate within inclusion body. Gaussian curve-fitting was done on the Fourier self-deconvolution spectra within amide I region of intact cells containing S and Sc. The experimental data revealed that the relative content of aggregate absorption at (1 629 +/- 1) cm(-1) gradually increased with induction time, which is consistent with the results of SDS-PAGE. Simutaneously, the formation of aggregate gave rise to the increase of alpha-helix, as well as the decrease of beta-turn and random coil in the case of Sc. It was not the case for S, however, where random coil experienced the increase in the relative average fractions, while beta-turn and beta-sheet at (1 629 +/- 1) cm(-1) behaved in different ways. The above mentioned phenomenon indicated that beta-sheet and random coil are most likely to transform into aggregate and alpha-helix with the introduction of C-terminal acidic protein.

[Identification, characterization and utilization of simple sequence repeat markers derived from Salvia miltiorrhiza expressed sequence tags].

Despite Salvia miltiorrhiza being one of the most important medicine plants in China, there is a limited availability of genomic resources, especially of the expressed sequence tag-based markers. In this study, we selected and characterized functional markers in S. miltiorrhiza, which consisted of 4,192 non-redundant expressed sequence tags (ESTs) from 10,288 identified S. miltiorrhiza ESTs in dbEST data bank. Among them, 159 simple sequence repeats (SSR) were detected, which amounted to 3.79% of the non-redundant starting sequence population. This incidence was equivalent to one EST-SSR in every 12.74 kb of S. miltiorrhiza ESTs. Among the different motifs ranging from 1 bp to 6 bp, di-nucleotide repeat motif was the most abundant (77, 48.43%), followed by tri-nucleotide (41, 25.79%), hexa-nucleotide (23, 14.47%), penta-nucleotide (12, 7.55%) and tetra-nucleotide (6, 3.77%). In 47 identified motif types, the detected frequency above 5% were GA/CT (16.35%), AG/TC (15.09%), TCA/AGT (10.69%), AT/TA (6.29%), GAAAAG/CAAAAC (6.29%) and TA/AT (5.03%). Based on flank sequence of detected SSR, a total of 83 EST-SSR primer pairs were designed and tested for the amplification efficiency, polymorphism and transferability in thirteen S. mihiorrhiza samples and other ten species from the genus Salvia. The results showed that 72 primer pairs were successfully amplified in S. miltiorrhiza samples to yield and 279 loci with an average of 3.88 loci per primer pair. The cross-transferability of S. miltiorrhiza EST-SSR markers to other ten Salvia plants was very high, ranging from 60% to 100% with an average of 85%. Further analysis of the genetic similarity based on the polymorphic bands showed the EST-SSR could detect the genetic diversity on different levels among the whole test samples and distinguish the S. miltiorrhiza from other Salvia plants effectively. It is expected that the potential markers described here would add to the repertoire of DNA markers needed for genetic analysis, linkage mapping and comparative genomics studies in S. miltiorrhiza and related Salvia genus plants.

cDNA cloning and overexpression of ribosomal protein S19 gene (RPS19) from the Giant Panda.

RPS19 is a component of the 40S small ribosomal subunit encoded by RPS19 gene. The cDNA of RPS19 was cloned successfully for the first time from the Giant Panda using RT-PCR technology. It was also sequenced, analyzed preliminarily, and expressed in Escherichia coli. The length of cDNA fragment cloned is 469 bp, and it contains an open-reading frame of 438 bp encoding 145 amino acids. Alignment analysis indicates that the nucleotide sequence and the deduced amino acid sequence share a high homology with those of Homo sapiens, Mus musculus, and Rattus norvegicus by 95.89%, 92.47%, and 93.61%, and 100.00%, 99.31%, and 99.31%, respectively. Topology prediction shows that there is one cAMP- and cGMP-dependent protein kinase phosphorylation site, four protein kinase C phosphorylation sites, one casein kinase II phosphorylation site, one tyrosine kinase phosphorylation site, three N-myristoylation sites, one amidation site, and one ribosomal protein S19e signature in the RPS19 protein of the Giant Panda (Ailuropoda melanoleuca). The RPS19 gene was overexpressed in E. coli and expression confirmed by western blotting. The results indicated that the RPS19 gene can be readily expressed in E. coli, and the N-terminally GST-tagged protein was a 42 kDa polypeptide, in good agreement with the predicted molecular weight. The protein obtained could be purified and its function studied.

cDNA, genomic sequence and overexpression of crystallin alpha-B Gene (CRYAB) of the Giant Panda.

AlphaB-crystallin, a small heat-shock protein, has been shown to prevent the aggregation of other proteins under various stress conditions. Here we have cloned the cDNA and the genomic sequence of CRYAB gene from the Giant Panda (Ailuropoda melanoleuca) using RT-PCR technology and Touchdown-PCR, respectively. The length of cDNA fragment cloned contains an open reading frame of 528bp encoding 175 amino acids and the length of the genomic sequence is 3189bp, containing three exons and two introns. Alignment analysis indicated that the nucleotide sequence and the deduced amino acid sequence are highly conserved to other four species studied, including Homo sapiens, Mus musculus, Rattus norvegicus and Bos taurus. The homologies for nucleotide sequences of Giant Panda CRYAB to that of these species are 93.9%, 91.5%, 91.5% and 95.3%, respectively, and the homologies for amino acid sequences are 98.3%, 97.1%,97.7% and 99.4%, respectively. Topology prediction shows that there are only four Casein kinase II phosphorylation sites in the CRYAB protein of the Giant Panda. The cDNA of CRYAB was transfected into E. coli, and the CRYAB fused with the N-terminally His-tagged protein gave rise to the accumulation of an expected 24KDa polypeptide, which accorded with the predicted protein. The expression product obtained could be used for purification and study of its function further.

[Development and application of a genome specific marker for Secale africanum].

Genome in situ hybridization (GISH) analysis of wheat-Secale africanum amphiploid revealed that the S. africanum genome displayed significant divergence to the Secale cereale genome. It is thus valuable to deploy genes from S. africanum. We performed the PCR analysis on S. africanum, wheat-S. afticanum amphiploid, T. eastivum cv. Anyuepaideng and other genetic stocks by 100 ISSR primers. A specific segment of 561 bp, named pSaUBC815561, was obtained from S. africanum using primer UBC815. This segment was not amplified from the control wheat lines. Primer UBC815 also am-plified fragments from wild species of genus Secale, including S. vavilovii, S. sylvestre, and other cultivated ryes. Based on the sequence of pSaUBC815561, a pair of special primers U815-F and U815-R was designed and was used to amplify the DNA of wheat related species in Triticeae aimed at validating the specificity of pSaUBC815561. PCR analysis indicated that this specific DNA fragment was amplified not only from a set of Chinese Spring wheat-Imperial rye chromosome addition lines but also from certain wheat-rye introgression lines. Therefore, pSaUBC815561 can be used as a specific marker for detection of chromosomes of Secale genome in wheat.

Discrimination of repetitive sequences polymorphism in Secale cereale by genomic in situ hybridization-banding.

Genomic in situ hybridization banding (GISH-banding), a technique slightly modified from conventional GISH, was used to probe the Chinese native rye (Secale cereale L.) DNA, and enabled us to visualize the individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat (Triticum aestivum L.) background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S. cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattern of Secale vavilovii was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromatin is discussed.

Variations of tandem repeat, regulatory element, and promoter regions revealed by wheat-rye amphiploids.

To better understand the evolution of allopolyploids, 4 different combinations between wheat (Triticum aestivum L.) and rye (Secale cereale L.) including 12 F1 hybrids and 12 derived amphiploids were analyzed and compared with their direct parental plants by PCR analysis using 150 wheat SSR (single sequence repeat) markers and by FISH analysis using a rye-specific repetitive sequence (pSc200) as a probe. Nine SSR markers amplified rye-specific fragments whose sizes ranged from 471 bp to 1089 bp. These fragments contain regulatory elements and (or) promoters. Some of these fragments were amplified from all 24 progenies, while others were amplified from a subset of the progenies. The disappearance of rye-specific fragments from some progenies was caused by sequence elimination or DNA modification. Marker Xgwm320 amplified a new fragment (403 bp), a rye-specific tandem repeat, from some of the progenies. Twenty-eight SSR markers displayed microsatellite variation in progenies derived from 'Chinese Spring' x 'Jinzhou-heimai', but none of the 150 SSR markers displayed microsatellite variation in the progenies derived from the other three combinations. FISH signals of pSc200 were eliminated from one telomere/subtelomere of 4 chromosomes of 'Kustro' during allopolyploidization and expanded in amphiploids derived from 'Chinese Spring' x 'AR106BONE'. Thus, allopolyploidization in wheat-rye can be accompanied by rapid variation of tandem repeats, regulatory elements, and promoter regions. The alterations of repetitive sequence pSc200 indicate coordination between the constituent genomes of the newly formed amphiploids. Different genetic backgrounds of parents appear to affect genome changes during allopolyploidization.

[Analysis of St-chromosome-containing triticeae polyploids using specific molecular markers].

In this study, random amplified polymorphic DNA (RAPD) analysis was performed on Pseudoroegneria spicata, Aegilops ventricosa, Secale cereale cv. Jingzhou Rye, Dasypyrum villosum and other 11 triticeae materials using 200 random 10-based primers. A 542 bp specific RAPD fragment (Accession No. DQ992032), named OPH11542, was obtained from Ps. Spicata. Based on the sequence of OPH11542, a pair of sequence characterized amplified region PCR (SCAR-PCR) primers was designed and used to amplify the materials of triticeae. The result demonstrated that 3 specific DNA segments of 542 bp, 742 bp (DQ992033)and 743 bp (EF014218) respectively, were obtained from St chromosome, however, these 3 segments were not appear in materials not contain St chromosome. Sequence similarity analysis revealed that these 3 segments were new repeated DNA sequences. SCAR-PCR was also performed on 15 materials containing St chromosome, and the result showed that OPH11742 or OPH11743 was always found in materials containing StY chromosome, whereas OPH11542 in materials containing StH chromosome. These results indicated that, chromosome recombination or modification often occurred in St chromosome in the course of combination of St and other chromosome to form a polypolid, and OPH11542, OPH11742 and OPH11743 could be used as molecular markers for the detection of St chromosome.

[Cloning and expression of Gymnadenia conopsea GcSec61beta gene encoding endosplasmic reticulum membrane translocation channel protein].

Protein translocation channel in endosplasmic reticulum (ER) of eukaryotes is composed of several subunits of Sec61 complex, which is essential for protein secretion. In the present study, we cloned a full-length cDNA fragment of 621 bp coding 107 amino acids from a psychrophile and endangered plant Gymnadenia conopsea, which grows in high land. Sequence analysis revealed that the gene was highly homologous to the member Sec61beta of ER protein transporter channel, which was thus designated as GcSec61beta. Phylogenetic tree shows that the GcSec61beta was closely related to the corresponding genes from Arabidopsis thaliana and Oryza sativa. Results of semi-quantitative RT-PCR showed that the expression of GcSec61beta was high both in leaves and the bud, and also induced by low temperature treatment. The sequence of the GcSec61beta was introduced into pET28a vector and transformed to E. coli strain BL21. The growth of E. coli was slowed down but the cold resistance was increased by the expression of GcSec61beta, which provides a new function of GcSec61beta protein.

Study on powdery mildew resistance transfer from S. cereale L.cv. Weiling rye into wheat.

Weiling rye (S. cereale L.cv.), a Chinese dwarf rye, confers high powdery mildew (Erysiphe gramininis f.sp.tritici) in China. My8443, a wheat cultivars infecting seriously powdery mildew disease, was used as the female parent and Weiling rye was used as the donor of powdery mildew resistance in the study. A new wheat-rye translocation line,named No.147,was developed from BC2F6 progenies of wheat cultivars My8443 and Weiling rye to transfer the resistance from Weiling rye to common wheat. The powdery mildew resistance of No.147 and its parents were investigated in seedling and adult stages by artificially inoculating the mixture of advanced pathogenic races in room and field and the single pathogenic race in room. Improved Giemsa C-banding technique and genomic in situ hybridization (GISH,Genomic in situ hybridization) were used to identify wheat and rye chromosomes. Acid polyacylamide gel electrophoresis(APAGE) separation of endosperm gliadin and simple sequence repeat(SSR) PCR amplification of 11 SCM-Secale cereale markers also were employed for 1RS confirmation in the study. The results showed that No.147 was a new 1BL/1RS wheat-rye chromosome translocation with high powdery mildew resistance derived from Weiling rye. The reason on the formation of the new wheat-rye chromosome translocation was analyzed. The utilizations of resistance gene resource derived from Chinese Weiling rye and the new 1BL/1RS translocation line in wheat genetics and breeding improvement were discussed in the paper.

[Identification and phylogenetic application of unique nucleotide sequence of nad7 intron2 in Rhodiola (Crassulaceae) species].

Genetic characterization of 9 populations of Rhodiola crenulata, R. fastigiata and R. sachalinensis (Crassulaceae) species from Sichuan and Jilin Provinces of China, was investigated using the conserved primer of nad7 intron 2. All PCR products about 800 bp long were shorter than other Crassulaceae plants, which were used as molecular markers to identify the Rhodiola species. The sequence of the products indicated that total exon of 53 bp and intron of 738 bp exhibit only 9 nucleotide variations. Blasting the nad7 sequences to GenBank and the phylogenetic analysis showed that the sequence of Rhodiola species was clusted independently, and the length was smaller than all the registered sequences of higher plants. The result suggests that the Rhiodola species had a unique sequence in this gene region, which might be related to the special growth condition.

[Variation of Rye-Specific repetitive DNA pSc119.1 among sister T1RS.1BL translocations].

Specific primers were designed according to the rye-specific repetitive sequence pSc119.1 and were used for polymerase chain reaction (PCR) analysis using the genomic DNA of two sets of sister T1RS.1BL translocations, CN12, CN17, CN18, 96 I 176-1 and 96 I 176-3 as templates. The results indicated that the target fragments were amplified from CN12, CN17, and CN18. A target and a non-target fragment were produced from 96 I 176-1. However, no products were obtained from 96 I 176-3. Southern blot analysis indicated that the elimination of pSc119.1 did not occur in line 96 I 176-3. Three target fragments were cloned from CN12, CN17, and CN18 respectively through recovering. For each target fragment, ten clones were selected randomly for sequencing. Variation of the sequence pSc119.1 was observed in all of the three wheat lines and line CN18 had the most obvious variation. Most of the 30 sequences had 94% or 95% similarity with the sequence pSc119.1 published and the variation of bases of these sequences. Most variations of most bases arose from transition, and a few of them were transversion. Furthermore, there was great coherence among these changed bases in type and site. The evolution process of progenies of wide hybrids may be continuous. For each set of sister 1RS.1BL translocation, difference of some traits was observed among the wheat lines or cultivars. The difference was probably related to the variation of the repetitive DNA. This research provides some useful information for studying on mechanism of epigenetics.

[Development of Dasypyrum genome specific marker by using wheat microsatellites].

One hundred and two SSR primer pairs, distributed in chromosome 1A to 7A, 1B to 7B, 1D to 7D of Triticum aestivum, were investigated on Dasypyrum breviaristatum, D.villosum, wheat-Dasypyrum amphiploids and its derivatives, with the control of common wheat Chinese Spring and elite wheat cultivars. A specific polymorphic DNA fragment of about 400 bp (415 bp-long by sequenced, named Xgwm301/415) amplified by primer pair Xgwm301 was obtained in all lines containing Dasypyrum chromosomes, but there were not the case in the tested common wheat. Furthermore, PCR analysis was performed on a set of T. aestivum-Dasypyrum addition, the result showed that all the seven pairs of villosum chromosomes contain Xgwm301/415. Therefore, Xgwm301/415 is a genome-specific polymorphic DNA segment for genera of Dasypyrum, and it could be used as a molecular marker for detection of chromosomes of Dasypyrum in wheat.