The Frequency of Survivorship in Heterozygous Diploids of Cdc13-1exo1Δ Mutants of S. cerevisiae Is One Survivor Cell in 72 Cells/Generation at 36°C

Telomeres cap ends of eukaryotic chromosomes prevent them from degradation and ensure genomic stability. Cdc13 is an essential telomere recruitment and maintenance protein. A temperature-sensitive point mutation in cdc13 gene leads to telomere impairment, giving rise to cdc13-1 mutants that suffer lethality at enhanced temperatures. Deleting Exo1 gene from these mutants, however, leads to the emergence of temperature-tolerant mutants called survivors. Yeasts are known to exist as either diploids or haploids. These yeast genotypes generate survivors. The frequency of survivorship in the haploid genotype is one cell in 104 cells/generation at 36°C, however, the frequency at which they emerge in their diploid counterparts at the same temperature is not known. In this study, we investigated the frequency of Survivorship in heterozygous diploids of cdc13-1exo1Δ mutants of S. cerevisiae at 36°C. Diploids were constructed by mating haploid strains of opposite mating type cdc13-1 exo1:LEU strains with strains of cdc13-1 exo1:HIS. The crosses were 1296 × 3181, 2561 × 3182, 1296 × 3182 and 2561 × 3181. Genetic markers and phenotypic appearance were considered while mating the mutant cells. Using a stick, a smear of one haploid strain was made on each YEPD plate labelled C2, C8, C9, D1, D14, and D15. A smear of another opposite mating type was made on the previous strain. They were mixed and allowed to mate overnight, before culturing on media lacking Luecine and Histidine (-L and -H). Survivors were generated by culturing these diploids at 36°C. Using SPSS 20.0 software for windows SPSS, 2011, the frequency was determined as one Survivor cell in 72 cells/generation, as their frequency of survivorship averaged 5.9 × 10-5 ± 0.04.

The microarray analysis for gene expression in haploids and diploids derived from twin-seedling rice

In this study, microarray technique was employed to analyze the gene expression at the RNA level between haploids and corresponding diploids derived from a rice twin-seedling line SARII-628. Differ- ent degrees of expression variations were observed in the plant after haploidization. The main results are as follows: (1) after haploidization, the ratio of the sensitive loci was 2.47% of the total loci designed on chip. Those loci were randomly distributed on the 12 pairs of rice chromosomes and the activated loci were more than the silenced ones. (2) Gene clusters on chromosome were observed for 33 se- quences. (3) GoPipe function classification for 575 sensitive loci revealed an involvement in the bio- logical process, cell component and molecular function. (4) RT-PCR generally validated the result from microarray with a coincidence rate of 83.78%. And for the randomly-selected activated or silenced loci in chip analysis, the coincidence rate was up to 91.86%.

Comparative Analysis of the Transcriptome and Metabolome in Leaves of Diploid and Tetraploid Fagopyrum tataricum

Tartary buckwheat(Fagopyrum tataricum)is a dual-purpose medicinal and food crop grown for its high contents of functional compounds and abundant nutrients.Although studies have shown the differences of total flavonoid content in Tartary buckwheat at different ploidy levels,the composition of flavonoid and its regulatory mechanisms are largely unknown.In this study,the leaf metabolome and transcriptome of diploid and tetraploid accessions of Tartary buckwheat were analyzed to gain insight into the impact of polyploidization on comparative secondary metabolite composition and molecular regulatory mechanism.Based on a widely targeted metabolomics analysis,a total of 792 metabolites were identified,including 127 flavonoids.The accumulation of 127 metabolites and expression of 3871 genes differed significantly between diploid and tetraploid Tartary buckwheat.Integrated metabolomics and transcriptome analysis revealed that chromosome doubling up-regulated the expression of upstream genes in the flavonoid biosynthesis pathway to promote the accumulation of flavonoids.The present results contribute to elucidation of the molecular mechanism of phenotypic variation associated with polyploidy in Tartary buckwheat.The findings provide a reference for further studies on phenotypic traits in polyploid Tartary buckwheat,the cloning of crucial regulatory genes,and utilization of genetic engineering technologies in the breeding of Tartary buckwheat.

etr1-1在矮牵牛中诱导表达可提高叶片对灰霉病的抗性

【目的】研究etr1-1诱导表达对矮牵牛抗灰霉病的作用。【方法】以转GVG:etr1-1矮牵牛为材料,地塞米松(DEX)处理,接种灰霉病菌,通过调查发病程度和定量半定量基因表达分析揭示etr1-1在矮牵牛抗灰霉病中的作用。【结果】与对照叶片相比,DEX处理的矮牵牛叶片表现出延缓衰老和减轻病害的症状。接种病原菌后,DEX处理的叶片发病率增加比例为0,对照叶片为66.77%;DEX处理的叶片平均病斑扩展速率为4.69 mm·d-1,对照叶片为6.29 mm·d-1。qRT-PCR和semi-qRT-PCR基因表达分析表明,DEX处理的叶片etr1-1与Bcact表达呈相反趋势。在DEX处理叶片中,DEX诱导了叶片etr1-1表达,随着诱导时间延长,基因相对表达量上升,诱导后第3天达到最大,无菌水接种叶片的表达量是病原菌接种叶片的10.71倍。DEX处理的叶片,Bcact在接种后第1天表达量最大;对照叶片,Bcact在接种后第2天表达量最大;其中处理叶片Bcact的最大表达量比对照叶片降低了20倍。在DEX处理的叶片上随着etr1-1表达增强,Bcact表达量下降。DEX处理的叶片,CP10仅在接种后第3天轻微表达;对照叶片上,随接种时间延长,CP10表达量上升,接种后第3天达到最大,处理最大表达量比对照降低了23.6倍。随接种时间延长,DEX处理的叶片和对照叶片,ACO表达均先升高后降低,在第2天达到最大,处理叶片上最大表达量仅为6.75,比对照低7.33倍。DEX处理的叶片,乙烯途径基因ETR2、ERS1、EIL1和EIN2表达受到抑制。ERS1和ETR2在DEX处理和对照叶片上均有表达,并在接种后第3天达到最大,但处理的最大表达量比对照低51倍。DEX处理的叶片,接种后第1—2天EIN2表达被抑制,第3天表达上升,在对照叶片上,EIN2表达量随接种时间而增加,第3天达到最大,DEX处理的叶片EIN2最大表达量比对照低31.58倍;DEX处理的叶片和对照叶片,EIL1与EIN2有相似的表达趋势,且DEX处理的叶片EIL1最大表达量低于对照两倍。AOC和COI1在JA生物合成和信号转导途径中起重要作用。DEX处理的叶片,AOC和COI1表达均被减弱,AOC在接种后第1—2天被抑制,第3天表达,对照叶片上,随接种时间延长AOC表达量增加,在第3天达到最大,处理叶片AOC最大表达量比对照低12.96倍;COI1在DEX处理和对照叶片上均有表达,且在第3天表达量最大,DEX处理叶片COI1最大表达量比对照低6.14倍。病原菌侵染植物时,ERFs会被乙烯或JA调控以激活病程相关基因表达。经DEX处理的叶片ERF4表达先升高后降低,第2天表达量最大;对照叶片随接种时间延长ERF4表达量上升,在第3天达到最大;处理最大表达量比对照低2.96倍。无论处理还是对照,ERF8表达量都随接种时间延长而上升,在第3天达到最大,但处理的最大表达量比对照低3.52倍。接种病原菌后,DEX处理的叶片上,所有病程相关基因表达微弱,最大表达量均低于10;而对照叶片上,所有病程相关基因均有表达,并在接种后第3天表达量最大。其中以PR1表达量最大为10 521.11,其次是EX-CHI和OSM,分别为184.95和184.96,Defense1和AC-CHI表达量最小为23.39和14.58。【结论】etr1-1诱导表达通过延缓灰霉病菌引起的叶片衰老,从而提高转基因矮牵牛对灰霉病的抗性。

人二倍体细胞2BS株制备三价脊髓灰质炎活疫苗免疫原性研究

在我国多年来口服脊髓灰质炎活疫苗(OPV),都是分型先后服用,故仍有部分儿童不能及时获得免疫。1982年观察了三价疫苗同时服用的抗体应答情况,效果令人满意。胡希民等报告了同样的结果。1985年国家批准正式生产三价疫苗(TOPV),使用效果也是满意的,简化了免疫程序。

Comparison Between a Tetraploid Turnip and Its Diploid Progenitor(Brassica rapa L.):The Adaptation to Salinity Stress

Polyploidy is pursued in plant breeding programs due mainly to its ability to yield larger vegetative or reproductive organs. In controlled growth chamber experiments, a tetraploid turnip （cv. Aijiaohuang, 4n） and its diploid progenitor （cv. Aijiaohuang, 2n） were evaluated for their tolerance to salinity stress via investigations on a group of physiological parameters. The results indicate that the tetraploid turnip exhibit better adaptation to a high concentration salt medium （200 mmol L-1）, as evidenced by a less-affected germination rate and a healthier morphological appearance at the seedling stage. Furthermore, an extension of salinity stress up to a certain period of time at the 5-7-leaf stage shows differences between the tetraploid turnip and its diploid progenitor. The former had a higher K＋/Na＋ ratio in the roots, higher glutathione concentration and antioxidant activities in the leaves, and smaller reductions in photosynthetic capacity in terms of leaf chlorophyll content. Studies on the differences between an autopolyploid and its respective relative, from which the autopolyploid originated, in terms of their tolerance to salinity and/or other abiotic stresses, have remained rather limited. The comparison is interesting due to a homogenous genetic background.

Genome evolution trend of common carp (Cyprinus carpio L.) as revealed by the analysis of microsatellite loci in a gynogentic family

Genome evolution arises from two main ways of duplication and reduction. Fish specific genome duplication （FSGD） may have occurred before the radiation of the teleosts. Common carp （Cyprinus carpio L.） has been considered to be a tetraploid species, because of its chromosome numbers （2n=100） and its high DNA content. Using 69 microsatellite primer pairs, the variations were studied to better understand the genome evolution （genome duplication and diploidization） of common carp from a gynogenetic family. About 48% of primer pairs were estimated to amplify duplicates based on the number of PCR amplification per individual. Segregation patterns in the family suggested a partially duplicated genome structure and disomic inheritance. This indicates that the common carp is tetraploid and polyploidy occurred by allotetraploidy. Two primer pairs （HLJ021 and HLJ332） were estimated to amplify reduction based on the number of PCR amplification per individual. One allele in HLJ002 locus and HLJ332 locus was clearly lost in the gynogenetic family and the same as in six wild populations. Segregation patterns in the family suggested a partially diplodization genome structure. A hypothesis transition （dynamic） and equilibrium （static） were proposed to explain the common carp genome evolution between genome duplication and diploidization.

Current advances in genome sequencing of common wheat and its ancestral species

Common wheat is an important and widely cultivated food crop throughout the world.Much progress has been made in regard to wheat genome sequencing in the last decade.Starting from the sequencing of single chromosomes/chromosome arms whole genome sequences of common wheat and its diploid and tetraploid ancestors have been decoded along with the development of sequencing and assembling technologies. In this review, we give a brief summary on international progress in wheat genome sequencing, and mainly focus on reviewing the effort and contributions made by Chinese scientists.

Analysis of three types of triterpenoids in tetraploid white birches(Betula platyphylla Suk.) and selection of plus trees

Betulin, oleanolic acid, and betulinic acid are naturally occurring pentacyclic triterpenoids that have significant medicinal value. Considerable amounts of these triterpenoids are available in the outer bark of white birch. In this study, we used ultrasound-assisted extraction （UAE） to extract triterpenoids from birch bark rapidly and with high efficiency. Using high performance liquid chro- matography （HPLC）, three types of triterpenoids were separated and detected. We examined the differences among triterpenoids extracted from diploid versus tetra- ploid white birch. Then, we used factor analysis to screen out tetraploid white birches with comprehensively excel- lent performance. The results indicate that the optimum conditions for extraction include the use of ethanol as an extraction solvent, a solid-to-liquid ratio of 0.1 g/10 ml, ultrasonic power set at 100 W, a temperature of 60 ℃ and an extraction time of 15 min. A reversed-phase C18 col- umn （4.6 mm × 250 mm × 5 μm） with a column tem- perature of 30 ℃ and the mobile phase composed of A （acetonitrile） and B （0.1% aqueous phosphoric acid, v/v） at a flow rate of 0.5 ml/min were used, and the detection wavelength was 195 nm. No significant difference wasobserved between diploid and tetraploid white birch in terms of the content of three types of triterpenoids （at a confidence level of 0.05）. As triterpenoid content, height, and DBH （diameter at breast height） are strongly interre- lated, we used factor analysis to evaluate all individuals, and we screened out six plus trees with excellent com- prehensive characters.

Natural Homologous Triploidization and DNA Methylation in SARII-628,a Twin-seedling Line of Rice (Oryza sativa)

A total of five pairs of diploidotriploid twin-seedlings （a diploid seedling and a triploid seedling emerged from a grain） were selected out from 4500 pairs of seedlings from SARII-628, a twin-seedling rice line. SSR analysis indicated that no difference between the diploid seedling and corresponding triploid seedling in a twin-seedling was found at the 310 loci, indicating that there was no obvious change in DNA primary structure. A modified AFLP technique ＇MSAP （methylation-sensitive AFLP）＇ was used to analyze methylation mutation. Although no methylation mutation was noted among the five diploids, 29 methylation mutation loci were found from the corresponding triploids. This suggested that methylation mutation happened rapidly on Mogeneration after natural homologous triploidization. The mutations were classified into 10 types, including 3 increased types, 3 decreased types and 4 undecided types of methylation-degrees. The bands of 22 loci were sequenced and then those sequences were searched through website. The result showed that the methylation mutation involved into the whole rice genome and the 12 pairs of chromosomes. The mutation trend was site-related and there were different mutation loci for different triploids, which foretold that SARII-628 would have different evolution fates after natural homologous triploidization.

PRELIMINARY STUDY ON CHROMOSOMES OF INDUCED TRIPLOID IN THE RED SEA BREAM, PAGROSOMUS MAJOR

Triploidy red sea bream were induced by cold shock techniques (0 3℃) in Qingdao in April 1994, May 1995 and May 1996. Normal diploidy and triploidy chromosome metaphases were produced by chromosome spreads from the gastrula. Counts of 104 chromosome metaphases of normal diploid showed each of them consisted of 2 acrocentric (st) and 46 telocentric (t) chromosomes. Based on the relative lengths and arm ratios, the 48 chromosomes were matched into 24 pairs. Counts of 107 chromosome metaphases of induced triploid showed that each metaphase consisted of 3 acrocentric (st) and 69 telocentric (t) chromosomes. The 72 chromosomes were easily matched into three sets of chromosomes, based on the relative lengths and arm ratios.

Molecular genetic analysis of phosphomannomutase genes in Triticum monococcum

In higher plants, phosphomannomutase(PMM) is essential for synthesizing the antioxidant ascorbic acid through the Smirnoff–Wheeler pathway. Previously, we characterized six PMM genes(Ta PMM-A1, A2, B1, B2, D1 and D2) in common wheat(Triticum aestivum, AABBDD).Here, we report a molecular genetic analysis of PMM genes in Triticum monococcum(AmAm), a diploid wheat species whose Amgenome is closely related to the A genome of common wheat. Two distinct PMM genes, Tm PMM-1 and Tm PMM-2, were found in T. monococcum. The coding region of Tm PMM-1 was intact and highly conserved. In contrast, two main Tm PMM-2 alleles were identified, with Tm PMM-2a possessing an intact coding sequence and Tm PMM-2b being a pseudogene. The transcript level of Tm PMM-2a was much higher than that of Tm PMM-2b, and a bacterially expressed Tm PMM-2a recombinant protein displayed relatively high PMM activity. In general, the total transcript level of PMM was substantially higher in accessions carrying Tm PMM-1 and Tm PMM-2a than those harboring Tm PMM-1 and Tm PMM-2b. However, total PMM protein and activity levels did not differ drastically between the two genotypes. This work provides new information on PMM genes in T. monococcum and expands our understanding on Triticeae PMM genes, which may aid further functional and applied studies of PMM in crop plants.

Sequencing of a Cultivated Diploid Cotton Genome-Gossypium arboreum

Sequencing the genomes of crop species and model systems contributes significantly to our understanding of the organization,structure and function of plant genomes.In a `white paper' published in 2007,the cotton community set forth a strategic plan for sequencing the AD genome of cultivated upland cotton that initially targets less complex diploid genomes.This strategy banks on the high

Comparative Embryological Studies on Infertility of Interspecific Hybridizations Between Oryza sativa with Different Ploidy Levels and O. officinalis

As maternal parents, diploid （L202-2x） and autotetraploid （L202-4x） of Oryza sativa cv. L2O2 were crossed with O. officinalis. Embryo development and fertilization in these two crosses were comparatively studied. There were no mature hybrid seeds obtained because all the hybridized spikelets died 30 days after pollination. The main reasons for no seed set were abnormal fertilization and development of the embryos and endosperms in the interspecific hybrids. There were doublefertilization, egg cell single-fertilization and non-fertilization in these crosses. Although 59.45% and 54.87% of hybrid embryos produced in the crosses of L202-2x/O. officinalis and L202-4x/O. officinalis, respectively, hybrid embryos ceased to develop or degenerated and plenty of free endosperm nuclei were in disaggregating state without developing cellular endosperms three days after pollination. Besides, some embryological differences in these two crosses were found, that is, the rate of double-fertilization and total rate of doubleand single-fertilization in L202-2x/O. officinalis were higher than those in L202-4x/O. officinalis. The embryo and endosperm of hybrids developed more slowly, and embryos and free endosperm nuclei were more severely degenerated in L202-4x/O. officinalis than in L202-2x/O. officinalis. Five days after pollination, a few of embryos in L202-2x/O. officinalis developed into pear-shaped ones, however, embryos in L202-4x/O. officinalis were all degenerated. Therefore, it is more difficult to obtain interspecific hybrids by wide crosses between autotetraploid of O. sativa and O. officinalis.

Acute motor axonal neuropathy following anti-rabies human diploid cell vaccine:A rare case and review

Rationale:Guillain Barre syndrome(GBS)is an acute neurological illness leading to quadriparesis with respiratory involvement.It can be triggered by infections,vaccinations,surgery,trauma,transplantation and drugs.Anti-rabies cell culture vaccines introduced to overcome the high rate of neurological complications associated with tissue based rabies vaccine,can be very rarely associated with GBS.Patient concerns:A 50-year-old female presented with acute severe upper back pain evolving into pure motor quadriparesis following administration of human diploid cell vaccine for rabies.Diagnosis:Acute motor axonal neuropathy variant of GBS following anti-rabies human diploid cell vaccine.Interventions:Intravenous high dose steroids.Outcomes:Patient recovered completely within 1 month.Lessons:Although anti-rabies cell culture vaccines are highly immunogenic and safe,they are rarely associated with GBS.Clinicians should be aware of this link because prompt diagnosis and treatment can result in complete recovery and avoid complications.

Microsatellite Analysis of Genetic Diversity Between Loach with Different Levels of Ploidy

We used microsatellite markers to investigate the genetic parameters of three different polyploidy populations of Misgurnus anguillicaudatus from Honghu City, Hubei Province. Polyacrylamide gel electrophoresis banding patterns of diploid（2n）, triploid（3n） and tetraploid loaches（4n） were analyzed with Pop Gen software. A total of 68 alleles were obtained from seven microsatellite loci and the polymorphism information content（PIC） indices were all above 0.5. The average expected mean heterozygosity values（He） were 0.8420, 0.7186 and 0.8521; the average observed mean heterozygosity values（Ho） were 0.9674, 0.9785 and 0.8928; and the HardyWeinberg P values were 0.3078, 0.3151 and 0.3762, for diploid, triploid and tetraploid individuals, respectively. The results indicated that the three populations were highly polymorphic, with no deviations from Hardy-Weinberg equilibrium observed at all the seven microsatellite loci. This indicated a high level of genetic diversity within the populations. A cluster analysis diagram showed that the shortest genetic distance was between diploid and tetraploid loaches and they shared a close phylogenetic relationship. The triploid and tetraploid individuals had the most distant phylogenetic relationship.

SAR-3——a resource of diploid true breeding hybrid rice

In 1988, we found strain 9003 from compositehybridization of indica and japonica. 9003 pos-sesses characteristics of twin seedlings withfrequency of 15-20% of the investigated popu-lation. The highest frequency was 45.68%.Afterwards, we discovered that some twinseedlings had variabilities in chromosome set.As to a pair of twin plants, it may be 2n-1n,2n-3n, 3n-3n or 1n-1n. The changes in chro-mosome set also occurred in the plants devel-oped from mono-embryo seeds. All of thesevariants nearly account for 1% in our con-trolled experiment. We named the triploidplants SAR-3 and found that SAR-3 could be aresource of diploid true breeding hybrid rice.

Toward a Genetically-anchored Physical Map of the Cotton Genomes

We are using a high-density(1-cM)molecularmap of the cotton genome based on RFLP,SSR,and EST markers as a foundation fordevelopment of a robust BAC-based physicalmap.The ’overgo’approach is providing anefficient means by which to accomplishhybridization-based anchoring of genetically-

Current Status of Cotton Genome Resource in India and Initiative on Utilization of Gene Pool through Molecular Technologies

The genus Gossypium is one of the largest havingnearly 50 species of which two are tetraploidcultivars and two are diploid cultivars with 2n =52 and 26 chromosome respectively and the restare wild to semi-wild at both these ploidy levels.The tetraploids(AD)are supposed to containthe Asiatic cotton(A)and American diploid(D)species genomes.

Gossypium Germplasm Resources

The genetic variability residing in diploid andtetraploid species of the Gossypium genusrepresents a large,under-utilized resource incurrent cotton improvement efforts.Geneticdiversity in elite germplasm is reported to benarrow.Actual diversity on the land isnarrower,due to preferential mass planting ofsuccessful cultivars and breeding techniques