Pear genomes display significant genetic diversity and provide novel insights into the fruit quality traits differentiation

The pear(Pyrus spp.)is well known for diverse flavors,textures,and global horticultural importance.However,the genetic diversity responsible for its extensive phenotypic variations remains largely unexplored.Here,we de novo assembled and annotated the genomes of the maternal(PsbM)and paternal(PsbF)lines of the hybrid‘Yuluxiang'pear and constructed the pear pangenome of 1.15 Gb by combining these two genomes with five previously published pear genomes representing cultivated and wild germplasm.Using the constructed pangenome,we identified 21224 gene PAVs(Presence-absence variation)and 1158812 SNPs(Single Nucleotide Polymorphism)in the non-reference genome that were absent in the PsbM reference genome.Compared with SNP markers,PAV-based analysis provides additional insights into the pear population structure.In addition,some genes associated with pear fruit quality traits have differential occurrence frequencies and differential gene expression between Asian and European populations.Moreover,our analysis of the pear pangenome revealed a mutated SNP and an insertion in the promoter region of the gene PsbMGH3.1 potentially enhance sepal shedding in‘Xuehuali'which is vital for pear quality.PsbMGH3.1 may play a role in the IAA pathway,contributing to a distinct low-auxin phenotype observed in plants by heterologously overexpressing this gene.This research helps capture the genetic diversity of pear populations and provides genomic resources for accelerating breeding.

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。

Chromosomal Karyotypes of Fragaria Plants

[Objective]This study was to analyze the karyotypes of wild diploid strawberry species,so as to provide basis for revealing the ori- gins,evolvement and genetic breeding of strawberry. [Method] Four diploid strawberry species including Fragaria gracilis A. Los,Fragaria nilgerrensis Schidl,Fragaria pentaphylla A. Los. and Fragaria vesca L. were used as experimental materials to analyze their chromosomal kary- otypes. [Result]The karyotype formula for strawberry species tested was as follows： 2n =2x =14 =10m ＋2sm ＋2m＊ for F. gracilis A. Los, belonging to type 1B; 2n =2x =14 =14m for F. nilgerrensis Schidl,belonging to type 1A; 2n =2x =14 =12m ＋2sm for F. pentaphylla A. Loz. , belonging to type 1A; 2n =2x =14 =14m for F. vesca L. ,belonging to type 1A. Karyotype analysis showed that karyotypic asymmetry degree of the four species was F. gracilis A. Los. F. pentaphylla A. Loz. F. vesca L. F. nilgerrensis Schidl. [Conclusion] The evolution order of these four species was probably F. gracilis A. Los. ,F. pentaphylla A. Loz. ,F. vesca L. ,F. nilgerrensis Schidl.

Induction of Gynogenesis of Silurus astus Using Cold Shock

[ Objective] The cold shock method was used to induce diploid gynogenesis of Silurus astus. [ Mthod ] The Silurus astus sperms were irradiated by ultraviolet, then conducted for fertilization. The different cold shock starting time and duration were set to observe diploid gynogenesis of Silurus astus. [ Result] Under such inducing condition that ultraviolet irradiated 15 min,cold shock started 5 rain after fertilization, and cold shock duartion was 40 rain,the survival rate of diploid Silurus astus reached the highest（8.5% ）. [ Conclusion] The experiment laid foundation for culturing new excellent Silurus astus variety and accumulated original files tor further study of gynogenesis development mechanism.

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.

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。

The Ploidy of Yeast and Its Identification Method

[Objective] The aim was to indentify haploid and diploid yeast.[Method] The characteristics of haploid and diploid yeast and their relation were analyzed firstly,and then haploid and diploid yeast were indentified through the methylene blue staining,safranine staining of ascospore and special low temperature treatment test.[Result] After methylene blue staining,the differences of haploid and diploid yeast in activity and size could be observed clearly;the ascospores of diploid strains after sporulating became red after safranine staining,and diploid thalluses were blue,while there were only blue thalluses for haploid strains;after special low temperature treatment,the colony of diploid was obviously larger than that of haploid on the plate.[Conclusion] Haploid and diploid yeast had great differences in morphology,heredity and physiology and could be identified well by means of aided test.

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.

STUDY ON EMBRYONIC DEVELOPMENT AND EARLY GROWTH OF TRIPLOID AND GYNOGENETIC DIPLOID LEFT-EYED FLOUNDER, PARALICHTHYS OLIVACEUS (T. et S.)

The early effects of chromosomal manipulation of eggs and sperm on the yields of triploid and gynogenetic diploid larvae of Paralichthys olivaceus were investigated. Triploidy was achieved by cold shocking fertilized eggs at 0-2℃ for 45 minutes duration 5 minutes after fertilization, and the induced triploidy rates were 31.2%-50% and the relative hatching rates were 53.3%-99%. Gynogenetic diploids were obtained when eggs were inseminated with irradiated sperm and cold shocked at 0-2℃ for 45 minutes duration 5 minutes after fertilization. The induced gynogenetic diploid rates and the relative hatching rates were 94%-96% and 48.5%-68.5% respectively. The embryonic development of the triploid experimental group and of the gynogenetic diploid experimental group was delayed at first compared with the control group. But from the gastrula stage, it was not delayed anymore. There were no significant differences in the growth of the triploid experimental group larvae and the control group larvae, and in the growth of the gynogenetic diploid experimental group larvae and the control group larvae according to Student’s t test (α=0.05). The relationship between the early growth of the triploid experimental group larvae and that of gynogenetic diploid experimental group larvae was also studied.

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.

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.

Comparison of Taste Components between Triploid and Diploid Oyster

The main taste components of triploid and diploid oyster (Crassostrea gigas) were compared. Free amino acids, inosine monophosphate, succinate, trimethylamine oxide and betaine in fresh and boiled extractives were analyzed. The protein, lipids, glycogen, moisture and ash, which may affect the flavour, were evaluated. In boiled extractives, the amino acids were 394.1 mg(100 g) -1 in diploid and 183.5 mg(100 g) -1 in triploid. However, in fresh oyster extractives, they were 320.0 mg(100 g) -1 and 147.3 mg(100 g) -1 respectively. The inosine monophosphate in triploid was 44% more than that in diploid, and a little difference existed in the content of trimethylamine oxide between them. The contents of these taste components were the basis for taste flavour pattern determination.

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.

Seasonal Variation of the Glycogen Enzyme Activity in Diploid and Triploid Pacific Oyster Gonad During Sexual Maturation

The glycogen content and the activities of two key enzymes in glycogen metabolism, glycogen phosphorylase and gly- cogen synthetase, in the gonad of diploid and triploid Pacific oysters (Crassostrea gigas) were compared during maturation. The glycogen content in the gonad of diploids decreased with gametogenesis (by 85.7%), but the glycogen content in the gonad of trip- loids did not vary significantly. Activity of glycogen phosphorylase (GP) in the gonad of diploids decreased with gametogenesis (by 55.5%), while GP activity of triploids did not vary significantly during maturation. Activity of glycogen synthetase (GS) in the gonad of diploids increased slightly with gametogenesis, reaching a peak in June. Activity of GS declined sharply from June to July, which might be due to gonad spawning. GS activity of triploid oysters in spawning time (July and August) was significantly higher than that in other months, which might be explained with a ‘compensating’ mechanism for the higher glycogen content in triploids.

Microsatellite-Centromere Mapping in Japanese Scallop(Patinopecten yessoensis) Through Half-Tetrad Analysis in Gynogenetic Diploid Families

Gene-centromere mapping is an essential prerequisite for understanding the composition and structure of genomes. Half-tetrad analysis is a powerful tool for mapping genes and understanding chromosomal behavior during meiosis. The Japanese scallop(Patinopecten yessoensis), a cold-tolerant species inhabiting the northwestern Pacific coast, is a commercially important marine bivalve in Asian countries. In this study, inheritance of 32 informative microsatellite loci was examined in 70-h D-shaped larvae of three induced meiogynogenetic diploid families of P. yessoensis for centromere mapping using half-tetrad analysis. The ratio of gynogenetic diploids was proven to be 100%, 100% and 96% in the three families, respectively. Inheritance analysis in the control crosses showed that 51 of the 53 genotypic ratios observed were in accordance with Mendelian expectations at the 5% level after Bonferroni correction. Seven of the 32 microsatellite loci showed the existence of null alleles in control crosses. The second division segregation frequency(y) of the microsatellite loci ranged from 0.07 to 0.85 with a mean of 0.38, suggesting the existence of positive interference after a single chiasma formation in some chromosomes in the scallop. Microsatellite-centromere distances ranged from 4 c M to 42 c M under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards the assembly of genetic maps in the commercially important scallop species.

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.