Isozyme gene markers in the dioecious species Asparagus officinalis L.

Summary Extracts from phylloclads of Asparagus officinails were electrophoretically analyzed for isozyme polymorphism. Fourteen enzyme systems were examined using four buffer systems: seven enzymes (acid phosphatase, catalase, glutamate-oxaloacetate transaminase, isocitrate dehydrogenase, malate dehydrogenase, peroxidase, and 6-phosphogluconate dehydrogenase) exhibited clear and consistent banding patterns. Isozyme polymorphism was studied in seven pairs of male and female doubled haploids and in their male F₁s. Segregation of polymorphic loci was examined in the backcross progenies and was found to be consistent with a simple Mendelian inheritance in all cases, except for three anodical peroxidases, where two factors have been hypothesized. No linkage could be found between isozyme markers that were segregating in the same cross, but association was demonstrated between one malate dehydrogenase locus and the sex determining genes. The availability of isozyme markers may be useful in breeding and, in particular, the localization of one malate dehydrogenase locus on the sex chromosomes may be helpful in mapping the sex genes.     

芦笋皂苷的抗肿瘤作用研究进展

芦笋是一种常见蔬菜,富含多种营养物质,在多种疾病的预防和治疗中发挥良好的药理效应。芦笋中的甾体皂苷是其生物活性的主要表现物质,现已从芦笋中分离出的皂苷单体有19种。本文概述了它们的来源及结构,对其中已被报道的几种皂苷单体在肿瘤预防和治疗方面的作用、机理及研究进展加以综述,为进一步分离新的芦笋皂苷单体及其对肿瘤的预防和治疗提供参考。     

A genetic map of Asparagus officinalis based on integrated RFLP, RAPD and AFLP molecular markers

 An integrated genetic map of the dioecious species Asparagus officinalis L. has been constructed on the basis of RFLP, RAPD, AFLP and isoenzyme markers. The segregation analysis of the polymorphic markers was carried out on the progeny of five different crosses between male and female doubled-haploid clones generated by anther culture. A total of 274 markers have been organized to ten linkage groups spanning 721.4 cM. Since the haploid chromosome number of asparagus is ten, the established linkage groups probably represent the different chromosomes; however, the only group associated with a specific chromosome is the one which includes sex, whose determinant genes have been located on chromosome 5. A total of 33 molecular markers (13 RFLPs, 18 AFLPs, 2 RAPDs and 1 isoenzyme) have been located on this chromosome. The closest marker to the sex determinant is the AFLP SV marker at 3.2 cM. Received: 26 March 1998 / Accepted: 30 April 1998     

几种因素对白芦笋试管苗生根的影响

白芦笋的不同筛选株系之间生根能力存有差异;培养温度是影响生根率的关键因素,高温[≥(25±1)℃]不利生根,发根的适宜培养温度为(20±1)℃;低光照强度(≤25mmol·m-2·s-1)对生根不利,适宜的光合光量子通量密度(PPFD)为40μmol·m-2·s-1;PP333、嘧啶醇不能提高试管苗生根率,且浓度过高还会降低生根率和抑制植株生长。     

RP-HPLC法测定芦笋中黄酮类化合物芦丁的含量

以芦丁标准品为对照,利用反相高效液相色谱法对芦笋中黄酮类化合物芦丁的含量进行定量测定。采用Agilent Eclipse XDB-C18色谱柱,柱温25℃,流动相由甲醇-水-磷酸(55∶44.5∶0.5)组成,流速为0.7 mL/min,检测波长390 nm。结果表明黄酮类化合物中各组分基线分离良好;进样量在0.07~0.7μg/20μL范围内,峰面积A与进样浓度C呈良好的线性关系,回归方程为A=1 5176C-10.388,相关系数R2=0.999 4;加样回收率为101.051%,RSD=3.306%;以保留时间和峰面积作精密度试验,RSD分别为0.199%和1.24%。该方法样品处理简单,准确度高,精密度好,适合于芦笋中芦丁含量的测定。     

Development of sex-linked PCR markers for gender identification in Actinidia

 Two sex-linked random amplified polymorphic DNA (RAPD) markers identified from Actinidia chinensis were converted into sequence-characterised amplified regions (SCARs) for the large-scale screening of Actinidia breeding populations. Initial SCAR primers converted one RAPD (SmX) into a dominant marker, but the other (SmY), which was potentially more useful because of its linkage to the male determining ‘Y’ locus, failed to retain polymorphism. This difficulty was overcome by cloning and sequencing the alternate ‘allele’ from female plants, and then designing ‘allele’-specific primers that utilised nucleotide differences between the sexes. Using a quick squash-blot method of DNA extraction, the SCAR primers were tested in 120 A. chinensis plants to determine their gender. The system is now in use for large-scale screening of seedling populations in the Actinidia breeding programme. The sex-linked SCAR primers also functioned with plants from some other geographically separate accessions of A. chinensis and with plants in the closely related polyploid species A. deliciosa, but did not amplify a sex-linked band in more distantly related species of Actinidia. Received: 27 December 1997 / Accepted: 5 March 1998     

芦笋茎叶游离氨基酸的提取及含量测定

通过对芦笋茎叶游离氨基酸提取工艺中温度、提取时间、料液比、乙酸浓度等影响因素的试验分析,确定芦笋茎叶游离氨基酸测定过程中的最佳提取条件为：提取温度60℃;料液比为1：35;提取时间为2．5h;并对芦笋茎叶游离氨基酸含量进行了测定,确定芦笋茎叶游离氨基酸含量为76．54mg／100g。     

Identification of two markers linked to the sex locus in dioecious <Emphasis Type=Italic>Asparagus officinalis</Emphasis> plants

One hundred decamer primers of random-amplified polymorphic DNA were tested on dioecious Asparagus officinalis plants to identify sex-linked molecular markers. One primer (S368) produced two markers (S368-928 and S368-1178) in female plants. These two DNA markers were identified in 30 male and female plants, respectively, and a S368-928 marker was proved to be linked to the female sex locus. The female-linked S368-928 marker was sequenced and specific primers were synthesized to generate a 928 bp marker of sequence characterized amplified regions (SCAR) in female plants, SCAR₉₂₈. SCAR₉₂₈ could be used to correctly screen homozygous mm female plants of A. officinalis. However, results of Southern blot analysis suggest that the hybridization pattern of S368-928 was presented in both sex plants. This text was submitted by the authors in English.     

The MADS box gene AOM1 is expressed in reproductive meristems and flowers of the dioecious species Asparagus officinalis

MADS box genes are implicated in different steps of plant development. Some of them are expressed in vegetative organs. Most of them, however, are expressed in flower tissues and are involved in different phases of flower development. Here we describe the isolation and characterization of an Asparagus officinalis MADS box gene, AOM1. The deduced AOM1 protein shows the highest degree of similarity with FBP2 of Petunia hybrida and AGL9 (SEP3), AGL2 (SEP1) and AGL4 (SEP2) of Arabidopsis thaliana. In situ hybridization analyses, however, show that the expression profile of AOM1 is different from that of these genes: AOM1 is expressed not only in flower organs but also in inflorescence and flower meristems. These data indicate a possible function of AOM1 during flower development as well as in earlier stages of the flowering process. Asparagus officinalis is a dioecious species which bears male and female flowers on different individuals. AOM1, which is expressed very early during the process of flowering and has a similar expression profile in male and female flowers, does not seems to be involved in asparagus sex differentiation. Received: 3 July 2000 / Revision accepted: 4 August 2000     

石刁柏胚性细胞诱导过程中的内源激素和多胺含量变化

用高效液相色谱法分析石刁柏愈伤组织胚性细胞诱导过程中不同时期内源激素和多胺含量的结果表明,在胚性细胞诱导过程中,Put和GA3一直呈上升趋势,胚性细胞出现时,IAA、Put和GA3含量都达到最高水平,显示高含量的IAA以及高比例的Pu“（Spd＋Spm）可能有利于胚性细胞的形成。     

正交试验优选超声提取芦笋总皂苷的工艺

目的:优选超声提取芦笋总皂苷的最佳工艺。方法:以高氯酸作为显色剂,用紫外分光光度法测定芦笋中总皂苷的含量,并以提取率为评价指标,采用单因素实验和正交试验优选最佳提取工艺。结果:芦笋总皂苷超声提取的最佳工艺为乙醇浓度70%,料液比1:15(W/V),超声时间50 min,超声温度40℃。结论:该提取工艺可行,为芦笋总皂苷的进一步研究提供了依据。     

Improved callus formation and plant regeneration for shed microspore culture in asparagus (Asparagus officinalis L.)

 To establish an efficient asparagus microspore culture system, experiments were conducted to investigate the effects of medium components, period of cold pretreatment for flower buds, and period of anther co-culture on culture response. All factors affected the frequency of asparagus microspore division and the yields of microspore-derived calli. The best results were obtained by pretreating genotype G459 flower buds at 4  °C for 7–9 days, co-culturing anthers with shed microspores for 14 days, and including 6% sucrose, 2 mg l^–1α-naphthaleneacetic acid and 1 mg l^–1 N⁶-benzylaminopurine in the culture medium. After 4 days of culture, most shed microspores contained starch-like bodies and died. The 2% of shed microspores lacking these structures divided to produce microcalli. For the best treatments in the different experiments, about 140 calli per 100 anthers were recovered. Cultured on four different regeneration media, 19.6–21% and 3.9–8.0% of microspore-derived calli produced shoots and embryos, respectively, and ultimately plantlets, among which 49% were haploid, 34% diploid, 4% triploid and 11% tetraploid. Received: 3 September 1998 / Revision received: 25 November 1998 / Accepted: 5 December 1998     

外源腐胺对石刁柏愈伤组织胚性能力的影响

石刁柏胚性愈伤组织继代过程中,添加浓度为10 mg·L^-1的外源腐胺能有效地保持愈伤组织的胚胎发生能力,并减少愈伤组织的褐化,但不能提高愈伤组织的体细胞胚的诱导率.腐胺处理过的胚性愈伤组织的内源腐胺含量明显提高.这可能是外源腐胺保持细胞胚性、降低褐化程度的原因.     

Sequence-tagged sites (STS) for studies of molecular epidemiology of scleroderris canker of conifers

 Scleroderris canker is a very damaging disease of conifers caused by a fungal pathogen, Gremmeniella abietina var ‘abietina’. This fungal pathogen is now known to comprise a number of distinct races and biotypes. In North America, two races, an indigenous North American race and an introduced European race, are present. In Europe, three distinct biotypes have been reported within the European race: one in the Alps, another in Fennoscandia, and a third that overlaps with the first two. We used random amplified microsatellites (RAMS) and DNA sequencing with arbitrary primer pairs (SWAPP) to design five PCR primer pairs flanking polymorphic regions of the genome of the European race of G. abietina. Length polymorphisms produced by repeats of basic units in microsatellites were distinguished by electrophoresis of PCR products in agarose gels, and point mutations were identified by low-ionic-strength single-strand conformation polymorphisms (LIS-SSCP). Some primers generated private alleles in the European biotype and the psychrophilic Alpine and Fennoscandian biotypes, i.e., alleles that were fixed within the two groups but polymorphic between them. Conversely, one pair of primers amplified at least 3, 4, and 7 alleles in the Fennoscandian, Alpine, and European biotypes, respectively. The Alpine and Fennoscandian biotypes, although geographically separated, were genetically more closely related to one another than to European biotype, which has an overlapping distribution. However, both Alpine and Fennoscandian biotypes have similar ecotypic adaptation. The evolution of these biotypes could be explained by their geographic separation following the end of the last glaciation. Received: 17 January 1998 / Accepted: 3 March 1998     

Regeneration of haploid plants from isolated microspores of asparagus (Asparagus officinalis L.)

High percentages of micro-calli and micro-derived embryos were produced from isolated asparagus microspores at late uninucleate stage on MS liquid medium supplemented with 1.0 mg l^–1 2,4-D and 0.5 mg l^–1 BA. Two types of calli, namely compact callus (CC) and loose callus (LC), were found. Plantlets were regenerated via organogenesis, when these calli were transferred onto MS solid medium supplemented with 1.0 mg l^–1 BA and 0.2 mg l^–1 IBA 6 weeks. Embryos were produced from liquid cultured microspores, or from solid cultured micro-calli. The frequencies of haploid plant production from organogenesis and embryogenesis were compared. Effects of plant growth regulators on callus production, plantlet regeneration, and haploid plant production were tested. The combination of BA 1.0 mg l^–1 and IBA 0.2 mg l^–1 resulted the highest precentage of haploid plant production (7.7% from CC, 4.3% from LC).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IBA 3-indolybutyric acid - BA 6-binzyladinine - NAA naphtalene acetic acid - MS Murashige and Skoog     

AFLP and STS tagging of Lr19, a gene conferring resistance to leaf rust in wheat

Amplified fragment length polymorphism (AFLP) markers were used to enrich the map of the wheat chromosomal region containing the Thinopyrum-derived Lr19 leaf rust resistance gene. The region closest to Lr19 was targeted through the use of deletion and recombinant lines of the translocated segment. One of the AFLP bands thus identified was converted into a sequence-tagged-site (STS) marker. This assay generated a 130-bp PCR fragment in all Lr19-carrying lines tested, except for one deletion mutant, while non-carrier template failed to amplify any product. This sequence represents the first marker to map on the distal side of Lr19 on chromosome 7el₁. The conversion process of AFLP fragments to STS markers was technically difficult, mainly because of the presence of contaminating fragments. Various approaches were taken to reduce the frequency of false positives and to identify the correct clone. We were able to formulate a general verification strategy prior to clone sequencing. Various other factors causing problems with converting AFLP bands to an STS assays are also discussed. Received: 15 September 2000 / Accepted: 5 January 2001     

Sequence-tagged-site (STS) markers of arbitrary genes: the utility of black spruce-derived STS primers in other conifers

 Sequence-tagged-site primers, previously developed based upon black spruce (Picea mariana) cDNA sequences, were tested for their ability to direct specific amplification in two individuals of each of 12 additional conifer species. Nearly all (95–97%) of the primers functioned well in congeneric trials, while a lower proportion (21–33%) scored positively in other Pinaceae genera. Outside of the Pinaceae, amplification of homologous products was not achieved. Products from the various species often differed in size from their homologs in black spruce. In one case a large difference in size was due to the lack of an intron in a jack pine product while in several other cases the differences were due to the presence or absence of large direct repeats in the DNA sequences. Length polymorphism was occasionally evident between the two individuals examined of a given species. We investigated marker polymorphism in detail in a panel of 15 white spruce (Picea glauca) trees. Allelic segregation among haploid megagametophytes was revealed directly at 16 loci by standard agarose-gel electrophoresis without any additional manipulation of amplification products. Polymorphisms observed at 12 of these loci were exclusively co-dominant. For this subset of 12 loci, the average number of alleles was 3.2 and the average observed heterozygosity was 0.37. Received: 10 April 1998 / Accepted: 22 April 1998     

The physical map of the chloroplast DNA from Asparagus officinalis L.

The genus Asparagus consists of 100–300 species of both dioecious and hermaphrodite plants. Since there are diploid, tetraploid, and hexaploid plants in this genus, RFLP (restriction fragment length polymorphism) analysis of chloroplast DNA (ctDNA) is suitable for examining the phylogenetic relationships. We have constructed a physical map of the ctDNA of garden asparagus (A. officinalis L. cv Mary Washington 500 W) using five restriction endonucleases, namely, BamHI, PstI, SalI, HindIII, and XhoI. Asparagus ctDNA was digested with restriction enzymes and cloned into plasmid and phage vectors, and a clone bank was constructed that covered 70% of the genome. A physical map was constructed by Southern hybridization of total DNA from asparagus with homologous and heterologous probes. The asparagus ctDNA was about 155 kb long and it contained two inverted repeats (23kb each) separated by a large single-copy region (90kb) and a small single-copy region (19kb). Fifteen genes, encoding photosynthesis-related proteins, rDNAs, and tRNAs, were localized on the physical map of asparagus ctDNA. Comparing the length and the gene order of asparagus ctDNA with that of other plants, we found that asparagus ctDNA was similar to tobacco ctDNA but different from rice ctDNA. The restriction patterns of the ctDNAs from several varieties of A. officinalis and three species of Asparagus were analyzed. The restriction patterns of the varieties of A. officinalis were very similar, but polymorphisms were detected among the three species of Asparagus.     

Fast and reliable genotype validation using microsatellite markers in Arabidopsis thaliana

 In this paper we show how rogue genotypes in the parental stocks or contaminants among the crossed progeny of Arabidopsis thaliana can be readily identified and excluded from the breeding process using microsatellite markers derived from a small quantity of intact leaf tissue which has been alkali-treated. This method is fast and cost effective as it does not require DNA extraction, is highly reliable, and is less damaging to small plants where only limited quantities of plant tissue are available. Furthermore, a large number of samples can be processed in 1 day, facilitating the identification process prior to selfing or crossing the plants. In addition, the procedure could potentially be automated since no centrifugation is required. Received: 2 April 1998 / Accepted: 31 May 1998     

The structure of the chloroplast genome in members of the genus Asparagus

 In a previous study we constructed a physical map of the chloroplast DNA (ctDNA) of garden asparagus (Asparagus officinalis L. cv ‘Mary Washington 500W’; Lee et al. 1996). In the present study we have constructed and compared HindIII and XhoI restriction maps of the ctDNAs of eight species of Asparagus: namely, A. officinalis, A. schoberioides, A. cochinchinensis, A. plumosus, A. falcatus, A. sprengeri, A. virgatus and A. asparagoides. The ctDNA of A. officinalis has 32 and 23 sites that are recognized by HindIII and XhoI, respectively. Taking the physical map of the ctDNA of A. officinalis as a standard, we found that the ctDNAs of A. falcatus, A. sprengeri, and A. asparagoides each had one additional HindIII site and lacked one XhoI site. We also detected two relatively large deletions of nucleotides in the ctDNA from A. cochinchinensis by sequencing analysis. Both of these deletions were located in a non-coding region between the ndhC and trnV genes and were 95 bp and 347 bp in length, respectively. The regions around the deletions exhibited strong homology, and short direct-repeat sequences were detected at the borders of the deletions, an indication that these deletions were the result of intramolecular recombination mediated by the direct repeats. Received: 16 June 1997 / Accepted: 17 July 1997