甘蔗与斑茅割手密复合体杂交后代的分子标记鉴定

为有效利用甘蔗野生种质拓宽甘蔗遗传基础,本研究利用甘蔗与斑茅割手密复合体进行杂交,同时应用序列相关扩增多态性(SRAP)和微卫星(SSR)分子标记技术鉴定其后代。两种分子标记鉴定结果相互印证表明:3个杂交组合的后代中,经表型鉴定含斑茅血缘的34个后代为真杂种。该真杂种后代为进一步综合利用斑茅、割手密的优异基因改良甘蔗品种提供了优良的创新种质。     

斑茅割手密复合体(GXAS07-6-1)及其与甘蔗F_1的GISH分析

斑茅割手密复合体(GXAS07-6-1)是广西蔗茅属斑茅和广西甘蔗属割手密的属间杂种,聚集了双亲的优点。本研究利用基于Alu-like的PCR鉴定方法对GXAS07-6-1及甘蔗与GXAS07-6-1的3份F_1材料进行真实性鉴定,基于基因组原位杂交技术(GISH)对父本GXAS07-6-1及其3份F_1染色体组成及核型进行分析。研究结果表明:3份F_1材料为GXAS07-6-1的真杂种;父本GXAS07-6-1的染色体众数为62条,其中30条来自蔗茅属斑茅,32条来自甘蔗属割手密,核型分类属于1B,其染色体按"n+n"方式传递;GXASF_108-2-17、GXASF_108-2-22、GXASF_108-2-32的染色体数目为78~80条,其中69~71条来自甘蔗属,9~11条来自蔗茅属斑茅,3份F_1的核型分类分别属于2B、1B、1B,染色体传递方式均为"n+n"。父本GXAS07-6-1及3份F_1材料中均未发现有染色体的交换或易位现象。甘蔗与斑割复合体杂交,蔗茅属斑茅染色体在亲子间传递过程存在丢失现象。     

基于GISH的甘蔗与斑茅F1染色体遗传与核型分析

斑茅在甘蔗育种的利用是现代甘蔗育种种质创新的热点,育种者期望把斑茅中优异的特性通过杂交渗透到甘蔗中。甘蔗与斑茅的F1是斑茅利用研究的难点也是基础。本研究利用基因组原位杂交技术(GISH)分析甘蔗与斑茅的F1染色体构成和核型,探讨甘蔗与斑茅F1染色体的遗传行为。GISH结果表明,甘蔗与斑茅杂交F1的染色体众数68~69条,其中40条来自甘蔗热带种Badila,28~29条来自海南斑茅,未发现有染色体的交换或易位现象。参试材料大部分染色体都属于中部着丝点(m)的染色体,少数为近中部着丝点(sm),YCE95-41核型属2B型,其余的核型都为1B型。甘蔗与斑茅的染色体按n+n的方式传递给F1,本研究结果为斑茅种质在甘蔗育种中的利用及其杂交后代染色体细胞遗传研究提供参考依据。     

DNA Marker Transmission and Linkage Analysis in Populations Derived from a Sugarcane (Saccharum spp.) x Erianthus arundinaceus Hybrid

Introgression of Erianthus arundinaceus has been the focus of several sugarcane breeding programs in the world, because the species has desirable traits such as high biomass production, vigour, ratooning ability and good resistance to environmental stresses and disease. In this study four genetic maps were constructed for two intergeneric populations. The first population (BC₁) was generated from a cross between an Erianthus/Saccharum hybrid YC96-40 and a commercial sugarcane variety CP84-1198. The second population (BC₂) was generated from a cross between YCE01-116, a progeny of the BC₁ cross and NJ57-416, a commercial sugarcane cultivar. Markers across both populations were generated using 35 AFLP and 23 SSR primer pairs. A total of 756 and 728 polymorphic markers were scored in the BC₁ and BC₂ populations, respectively. In the BC₁ population, a higher proportion of markers was derived from the Erianthus ancestor than those from the Saccharum ancestor Badila. In the BC₂ population, both the number and proportion of markers derived from Erianthus were approximately half of those in the BC₁ population. Linkage analysis led to the construction of 38, 57, 36 and 47 linkage groups (LGs) for YC96-40, CP84-1198, YCE01-116, and NJ57-416, encompassing 116, 174, 97 and 159 markers (including single dose, double dose and bi-parental markers), respectively. These LGs could be further placed into four, five, five and six homology groups (HGs), respectively, based on information from multi-allelic SSR markers and repulsion phase linkages detected between LGs. Analysis of repulsion phase linkage indicated that Erianthus behaved like a true autopolyploid.     

Molecular contribution to selection of intergeneric hybrids between sugarcane and the wild species Erianthus arundinaceus.

Erianthus arundinaceus has great potential as a germplasm source for better ratoonability, vigour, tolerance to environmental stresses, and disease resistance in sugarcane. Many unsuccessful attempts have been made to introduce these characters into modern sugarcane cultivars. We report on significant progress made since molecular tools were implemented. Sequence-tagged PCR, revealing size variation in the 5S rDNA cluster, was performed on intact leaf tissue to identify genuine hybrids six weeks after germination. This early screening of seedlings avoids the loss of genuine hybrids due to competition with selfed progeny. Of 96 crosses made involving female Saccharum officinarum or sugarcane cultivars (Saccharum spp.) and male E. arundinaceus, 26 were fertile producing 1328 seedlings. Thirty-seven genuine hybrids were unequivocally identified but only 19 have survived. Genuine hybrids were produced from only three crosses, all involving S. officinarum as the female parent. Chromosome elimination was observed in all seven hybrids analyzed using genomic in situ hybridization (GISH). Very little cross-hybridization was observed between the genomes of the two species after GISH, confirming recent molecular studies which showed that E. arundinaceus is quite distant from the genus Saccharum. The major limitation in the introgression of E. arundinaceus resides now in the apparent sterility of the hybrids.     

Identification and characterisation of sugarcane intergeneric hybrids,Saccharum officinarum x Erianthus arundinaceus,with molecular markers and DNA in situ hybridisation

Molecular markers were used to characterise sugarcane intergeneric hybrids between S. officinarum and E. arundinaceus. Very simple diagnostic tools for hybrid identification among the progeny were derived from isozyme electrophoresis and a sequence-tagged PCR. Two enzyme systems (GOT and MDH B) and PCR amplification revealing spacer-size variation in the 5s-rDNA cluster were found most convenient. Specific characterisation of the two genomic components was possible using RFLP and in situ hybridisation. The strong molecular differentiation between S. officinarum and E. arundinaceus allows the identification of numerous Erianthus-specific RFLP bands in the hybrids. Genomic DNA in situ hybridisation allows for the differentiation of the chromosomes contributed by S. officinarum and E. arundinaceus in chromosome preparations of the hybrids. In situ hybridisation with the 18s-5.8s-25s rDNA probe highlights the basic chromosome numbers in the two parental species. The potential of these techniques to monitor the Erianthus genome during the introgression process is discussed.     

中国斑茅种质资源分子多态性分析

采用ISSR分子标记技术,对采自我国8省区的30个斑茅[Erianthus arundinaceus (Retz.) Jesw.]无性系的分子多态性进行了分析, 共扩增出87条多态带, 平均每个引物扩增的多态带数为 5.1条, 多态性条带比率(PPB)为 64.89%,多态信息量(PIC)为0.58,说明我国斑茅具有较丰富的遗传多样性.根据研究结果进行聚类和主成分分析,可将30个斑茅无性系归为4大类,同一地区的斑茅无性系基本聚在同一类,呈现出一定的地域性分布规律.     

斑茅两个看家基因片段的克隆及其在基因芯片中的应用

根据已发表的同源基因序列,利用RT-PCR技术分离了斑茅(Erianthus arundinaceus)的GAPDH和APRT两个看家基因片段,用它们作为cDNA芯片阳性参照,以未经聚乙二醇(PEG)胁迫处理的斑茅叶片为对照,和PEG胁迫的4组材料同cDNA芯片进行杂交分析。杂交结果显示,GAPDH杂交的Cy5与Cy3平均信噪比(Signal/Noise,S/N)分别为56.12和60.8,APRT杂交的Cy5与Cy3平均信噪比分别为51.06和47.25,信噪比均很高;同时两个看家基因的杂交都显示出极强的信号,其中GAPDH的杂交信号值大于10000,APRT也在8000以上,杂交结果可靠。分析了PEG胁迫4个时段BADH与两个看家基因的表达,BADH的表达有明显变化,而看家基因表达均较稳定。上述结果表明所克隆的两个看家基因在斑茅中表达量高,且PEG胁迫下表达较为稳定,是基因芯片理想的阳性参照。     

Optimisation of a Microsatellite Enrichment Technique in Saccharum spp.

The creation of enriched microsatellite libraries can ensure an abundant supply of microsatellite sequences at a considerably reduced cost. We report here the modification to an existing enrichment protocol and the optimisation of this technique for use on specific plant species, in this case, sugarcane. Using this optimised protocol, we achieved a 40% increase in the level of enrichment.     

Studies on trehalase in Saccharum spp. leaf and storage tissues

Trehalase properties and behavior were examined in commercialsugarcane varieties (predominandy S. officinarum), pure S. spontaneumclones, and pure S. robustum clones propagated in sand culture.The standard commercial variety, P. R. 980, contained trehalasein all tissues except the leaf sheath, widi greatest activitylocalized in expanding internodes and spindle leaves. OptimumpH was 6.0 and the Michaelis constant was 6.3 ? 10^–4 Mfor trehalose. Foliar experiments based on leaf rank, variableillumination, and diurnal sugar and enzyme changes gave no evidenceof trehalase involvement in sugar transport processes. Activitylevels ranged from 4- to 10-fold higher in S. spontaneum clonesthan in commercial varieties. Present evidence suggests a trehalaserole in sugar utilization rather than sugar storage which favoredthe evolutionary survival of primitive Saccharum forms. ¹Present address: Biophysics Program, Washington State University,Pullman, Washington 99163, U.S.A. (Received July 4, 1972; )     

甘蔗野生种滇蔗茅种质创新利用研究 Ⅰ. 甘蔗与滇蔗茅远缘杂交F1群体构建与SSR分子标记鉴定

利用光周期调控技术诱导甘蔗热带种路打士开花,并与滇蔗茅云南95-19进行远缘杂交,培育实生苗76株,大田移栽成活62株,成活率81.6%;选用4对引物对获得的62份杂交后代进行SSR分子标记鉴定,结果表明,62份杂交后代全部为真杂种,所选SSR引物对滇蔗茅后代群体具有较高的鉴定效率。该杂交组合后代杂种真实率为100%,是目前数量最大的滇蔗茅F1群体。     

Intergeneric and intrageneric conjugal transfer of plasmid-encoded antibiotic resistance determinants in Leuconostoc spp.

Transfer of the broad-host-range resistance plasmids pIP501 and pAM beta 1 from Streptococcus faecalis to Leuconostoc dextranicum and Leuconostoc cremoris occurred between cells that were immobilized on nitrocellulose filters in the presence of DNase. Transfer of pIP501 to Leuconostoc spp. also occurred when Streptococcus sanguis and Streptococcus lactis were used as donors. In addition, transfer of pIP501 and pAM beta 1 was observed from L. cremoris and L. dextranicum transconjugants to S. sanguis and S. faecalis. Expression of the pAM beta 1 erythromycin and pIP501 erythromycin and chloramphenicol resistance determinants was essentially equivalent in donors and transconjugants. Frequencies of transfer generally ranged from 10(-4) to 10(-7) transconjugants per input donor cell. Intrageneric transfer of pIP501 and pAM beta 1 occurred between L. cremoris and L. dextranicum strains in the same approximate range. These data further extend the host range of pIP501 and pAM beta 1 and demonstrate another example of gene transfer in the genus Leuconostoc.     

Ploidy Level and DNA Content of Erianthus arundinaceus as Determined by Flow Cytometry and the Association with Biological Characteristics

Erianthus arundinaceus is not only an important germplasm resource for sugarcane breeding but also a potential bioenergy plant. Making clear the distribution of the chromosome ploidy of wild E. arundinaceus in china is the premise of the research and utilization of this species. Therefore, the objectives of this study were to determine the ploidy level and DNA content of the 55 E. arundinaceus accessions using flow cytometry and to identify the correlation between ploidy and phenotypic traits. Among the 55 accessions, four tetraploids and 51 hexaploids were identified. The four tetraploids originated from Mengma Yunnan, Shuangjiang Yunnan, Gaozhou Guangdong and Chengle Sichuan. The mean DNA content was 4.82 pg/2C for the tetraploid and 7.30 pg/2C for the hexaploid plants. The ploidy was negatively correlated with cellulose content and positively correlated (P<0.05) with plant height, stem diameter, leaf width, dry weight per plant, fresh weight per plant and hemicellulose content. However, ploidy was not correlated with leaf length, tiller number and the ratio of dry weight and fresh weight. This study will be useful for revealing the distribution of the ploidy of wild E. arundinaceus in Chin, traits markers analysis, and utilization of this species, such as cultivar improvement and sugarcane breeding in the future.     

Prions in Saccharomyces and Podospora spp.: Protein-Based Inheritance

Genetic evidence showed two non-Mendelian genetic elements of Saccharomyces cerevisiae, called [URE3] and [PSI], to be prions of Ure2p and Sup35p, respectively. [URE3] makes cells derepressed for nitrogen catabolism, while [PSI] elevates the efficiency of weak suppressor tRNAs. The same approach led to identification of the non-Mendelian element [Het-s] of the filamentous fungus Podospora anserina, as a prion of the het-s protein. The prion form of the het-s protein is required for heterokaryon incompatibility, a normal fungal function, suggesting that other normal cellular functions may be controlled by prions. [URE3] and [PSI] involve a self-propagating aggregation of Ure2p and Sup35p, respectively. In vitro, Ure2p and Sup35p form amyloid, a filamentous protein structure, high in β-sheet with a characteristic green birefringent staining by the dye Congo Red. Amyloid deposits are a cardinal feature of Alzheimer’s disease, non-insulin-dependent diabetes mellitus, the transmissible spongiform encephalopathies, and many other diseases. The prion domain of Ure2p consists of Asn-rich residues 1 to 80, but two nonoverlapping fragments of the molecule can, when overproduced, induce the de nova appearance of [URE3]. The prion domain of Sup35 consists of residues 1 to 114, also rich in Asn and Gln residues. While runs of Asn and Gln are important for [URE3] and [PSI], no such structures are found in PrP or the Het-s protein. Either elevated or depressed levels of the chaperone Hsp104 interfere with propagation of [PSI]. Both [URE3] and [PSI] are cured by growth of cells in millimolar guanidine HCl. [URE3] is also cured by overexpression of fragments of Ure2p or fusion proteins including parts of Ure2p.     

Application of Isoelectric Focusing to the Taxonomic Identification of Meloidogyne spp.

Meloidogyne incognita, M. arenaria, M. hapla, and M. javanica were distinguishable from each other by isoelectric focusing (IEF) of nematode egg proteins. Proteins extracted from larvae and adults of Hoplolaimus columbus and from eggs of Heterodera glycines had distinctive profiles, also. Protein profiles from eggs, preparasitic larvae and egg-laying adults of M. incognita showed differences. It was necessary to compare samples run at the same time to ensure reliability.     

Single Origin of Sex Chromosomes and Multiple Origins of B Chromosomes in Fish Genus Characidium

Chromosome painting with DNA probes obtained from supernumerary (B) and sex chromosomes in three species of fish genus Characidium (C. gomesi, C. pterostictum and C. oiticicai) showed a close resemblance in repetitive DNA content between B and sex chromosomes in C. gomesi and C. pterostictum. This suggests an intraspecific origin for B chromosomes in these two species, probably deriving from sex chromosomes. In C. oiticicai, however, a DNA probe obtained from its B chromosome hybridized with the B but not with the A chromosomes, suggesting that the B chromosome in this species could have arisen interspecifically, although this hypothesis needs further investigation. A molecular phylogenetic analysis performed on nine Characidium species, with two mtDNA genes, showed that the presence of heteromorphic sex chromosomes in these species is a derived condition, and that their origin could have been unique, a conclusion also supported by interspecific chromosome painting with a CgW probe derived from the W chromosome in C. gomesi. Summing up, our results indicate that whereas heteromorphic sex chromosomes in the genus Characidium appear to have had a common and unique origin, B chromosomes may have had independent origins in different species. Our results also show that molecular phylogenetic analysis is an excellent complement for cytogenetic studies by unveiling the direction of evolutionary chromosome changes.     

Characterization of Genomic Inheritance of Intergeneric Hybrids between Ascocenda and Phalaenopsis Cultivars by GISH,PCR-RFLP and RFLP

BackgroundThe intergeneric hybrids between Ascocenda John De Biase ‘Blue’ and Phalaenopsis Chih Shang''s Stripes have been generated to introduce the blue color into the Phalaenopsis germplasm in prior study. In order to confirm the inheritance in hybrid progenies, genomic in situ hybridization (GISH) and restriction fragment length polymorphism (RFLP) analysis were conducted to confirm the intergeneric hybridization status.Methods/ResultsGISH analysis showed the presence of both maternal and paternal chromosomes in the cells of the putative hybrids indicating that the putative hybrid seedlings were intergeneric hybrids of the two parents. Furthermore, twenty-seven putative hybrids were randomly selected for DNA analysis, and the external transcribed spacer (ETS) regions of nrDNA were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and RFLP analyses to identify the putative hybrids. RFLP analysis showed that the examined seedlings were intergeneric hybrids of the two parents. However, PCR-RFLP analysis showed bias to maternal genotype.ConclusionsBoth GISH and RFLP analyses are effective detection technology to identify the intergeneric hybridization status of putative hybrids. Furthermore, the use of PCR-RFLP analysis to identify the inheritance of putative hybrids should be carefully evaluated.     

Pairing Competition between Identical and Homologous Chromosomes in Autotetraploid Rye. I. Submetacentric Chromosomes

Meiotic pairing preferences between identical and homologous but not identical chromosomes were analyzed in ten induced tetraploid/diploid chimaeral rye plants (Secale cereale) heterozygous for telomeric heerochromatin C-bands in both arms of chromosome 1R. These plants were the progeny of two crosses between only one plant of cv. Petkus, used as male, and two plants of the inbred lines E and R, respectively. Different pairing preferences for chromosome 1R were found: (1) between plants, (2) between chromosome arms within the same plant and (3) between bivalents and multivalents within the same plant. The possible influence in the preferences of several factors such as differences in C-heterochromatin content in the chromosomes analyzed, specific genetic control and independence in pairing behavior between both arms and partner exchange is discussed.     

Oogenesis and the Chromosomes of the Parthenogenic Root-knot Nematode Meloidogyne incognita

220 populations of Meloidogyne incognita and related forms from 46 countries reproduced by mitotic parthenogenesis (apomixis). Determination of somatic chromosome numbers from oogonia and oocytes revealed the existence of a predominant, possibly triploid race A with 3n = 40 to 46 and a rare, diploid race B with 2n = 32 to 36 chromosomes. There is no correlation between cytological races and the four recognized host races of this species. The characteristic behavior of prophase I chromosomes of maturing oocytes, which results in a prolonged prophase stage, is a unifying feature of all forms of M. incognita and supports monophyletic evolution, distinct from that of other Meloidogyne species. Extensive chromosomal polymorphism detected among populations can be helpful in elucidating the cytological pathway of evolution of the species.