Primary Study of Rice AFLP Analysis-Optimization of Reaction Conditions and Analysis of Thermo sensitive Genic Male Sterile Rice Allelic Mutant Lines

AFLP analysis was performed between a pair of thermo-sensitive genic male sterile (TGMS) rice allelic mutant lines (5460S and 5460F). The reaction conditions for rice AFLP assay were optimized. The relative efficiencies for polymorphism detection of RFLP, RAPD and AFLP were compared. The results indicated that the efficiency for polymorphism detection in rice was in the order of AFLP > RAPD > RFLP, and also indicated that AFLP was a powerful DNA molecular marker technique for polymorphism detection, especially in the case of extremely low polymorphism, such as isogenic lines and allehc mutant hnes. Some of the AFLP products between the TGMS rice allehc mutant lines were cloned. Three of them were used as mixed probes to screen BAC library of rice line 5460S. 12 positive clones were screened out. In addition, the advantages and disadvantages of these three molecular marker systems were discussed.     

水稻的AFLP研究初报——反应条件的优化及对温敏核不育等位突变系的分析

通过对５４６０Ｓ和５４６０Ｆ这一对水稻等位突变系的ＡＦＬＰ分析,比较了ＡＦＬＰ与ＲＡＰＤ及ＲＦＬＰ检测ＤＮＡ多态性的相对效率。结果表明,这３种分子标记的ＤＮＡ多态性检出效率依次为ＡＦＬＰ＞ＲＡＰＤ＞ＲＦＬＰ;找出了水稻ＡＦＬＰ分析的最适反应条件;在这对等位突变系之间找到了一些多态性ＡＦＬＰ产物,已完成了对４个多态性ＡＦＬＰ产物的克隆,其中３个为单拷贝顺序;用这３个单拷贝克隆的混合物为探针,对作者自己构建的５４６０Ｓ水稻的ＢＡＣ库进行了筛选,获得了１２个阳性克隆,为今后ＢＡＣ库的筛选打下了基础。此外,对上述３种分子标记各自的优缺点及它们在ＤＮＡ多态性检测中的适用之处进行了分析探讨。     

The isolation of cDNA clones from cucumber (Cucumis sativus L.) floral buds coming from plants differing in sex

In this study, we found flower cDNA clones which may be connected with the development of flower sex in cucumber. Two pairs of nearly-isogenic lines: gynoecious GY3 (FFMMGG) versus hermaphrodite HGY3 (FFmmGG) and monoecious B10 (ffMMGG) versus gynoecious 2gg (ffMMgg) were used for clone isolation. To obtain differentially-expressed clones, we applied the differential screening method. 454 clones from GY3 and 478 from B10 cDNA libraries were isolated. The results of RFLP analysis with 56 cDNA clones showed no clones which cosegregated with sex in cucumber. The 28 cDNA B10 and 33 cDNA GY3 clones isolated using the differential screening method were sequenced. Some of them seem to may play a role in cell differentiation or flower development. Among the 61 identified clones, 14 show high homology to plant proteins, although of unknown function. 11 show high homology to known proteins, and the possible function of some of them is discussed. For 3 clones, no significant similarity was found. The 31 clones displayed high homology to plant cDNA in EST database. The patterns of expression of five differential cDNA clones, 35GY3, 216GY3, 47GY3, 100B10 and 157B10, were analyzed in cucumber flower buds using in situ RT-PCR. The most interesting clone is 35GY3, because of its possible role in the inhibition of the development of male specific elements in the female cucumber flower.     

Contents and recovery of gibberellins in monoecious and gynoecious cucumber plants

Diffusates from seedlings and root exudates from 6-week-old plants of a monoecious line of cucumber, Cucumis sativus L., contained considerably higher levels of gibberellin-(GA-) like substances than did those from plants of an isogenic gynoecious line. Most of the GA-like activity was found in a chromatogram region typical of GA₁ and GA₃; some activity, particularly in root exudates, appeared also at an R_F similar to that of GA₄ and GA₇.

When seedlings were treated with ³H-labeled GA₁, more radioactivity was found in the diffusates from monoecious seedlings than from gynoecious ones. The same was true of biological activity in root diffusates from older plants which had been treated with gibberellin A₄₊₇.

In conjunction with evidence present in literature, these results support the idea that endogenous GAs play a part in the regulation of sex expression in cucumber, relatively high levels favoring the formation of staminate flowers.

     

The M locus and ethylene-controlled sex determination in andromonoecious cucumber plants.

Sex determination in cucumber (Cucumis sativus L.) plants is genetically controlled by the F and M loci. These loci interact to produce three different sexual phenotypes: gynoecious (M-F-), monoecious (M-ff), and andromonoecious (mmff). Gynoecious cucumber plants produce more ethylene than do monoecious plants. We found that the levels of ethylene production and the accumulation of CS-ACS2 mRNA in andromonoecious cucumber plants did not differ from those in monoecious plants and were lower than the levels measured in gynoecious plants. Ethylene inhibited stamen development in gynoecious cucumbers but not in andromonoecious ones. Furthermore, ethylene caused substantial increases in the accumulation of CS-ETR2, CS-ERS, and CS-ACS2 mRNA in monoecious and gynoecious cucumber plants, but not in andromonoecious one. In addition, the inhibitory effect of ethylene on hypocotyl elongation in andromonoecious cucumber plants was less than that in monoecious and gynoecious plants. These results suggest that ethylene responses in andromonoecious cucumber plants are reduced from those in monoecious and gynoecious plants. This is the first evidence that ethylene signals may influence the product of the M locus and thus inhibit stamen development in cucumber. The andromonoecious line provides novel material for studying the function of the M locus during sex determination in flowering cucumbers.     

Isolation of a partial sequence of a putative nucleotide sugar epimerase, which may involve in stamen development in cucumber (Cucumis sativus L.)

Sex determination is the most widely studied subject in cucumber. The sex of cucumber plants can be monoecious, hermaphrodite, gynoecious, androecious, or andromonoecious. Besides environmental factors, three major genes, F/f, M/m, and A/a mainly govern the sex types in cucumber. Regardless of their sex all floral buds are bisexual at the early bud stage. A stage specific arrest of either stamen or carpel leads to unisexual flower development. The possible downstream product of the interaction of the sex determining genes that may directly allow the growth or selectively arrest stamen or pistil is not yet identified. Therefore, in the current study, we performed suppression subtractive hybridization using floral buds from nearly isogenic gynoecious and hermaphrodite cucumber plants and identified for the first time a cDNA homologous to nucleotide sugar epimerase. The expression level of the isolated putative nucleotide sugar epimerase is weak in female floral buds but strong in bisexual and male flowers. The weak level of the putative nucleotide sugar epimerase may be an indication for its improper functioning, which may influence stamen development in cucumber plants.     

棉花纤维发生相关AFLP标记

以徐州142及其无绒无絮（无纤维）突变体(fl)为材料,利用AFLP标记技术对该近等基因系进行DNA多态性分析。在64对引物产生的6360多条扩增带中,发现1条差异带（CF1）稳定出现在有纤维和无纤维之间。进一步用徐州142无绒无絮突变系与纤维发育正常的渝棉一号的杂交后代F2、F3分离群体以及几个陆地棉品种作材料验证。结果表明,差异带（CF1）与纤维的产生呈共分离,该差异带是与棉花纤维发生相关的分子标记。进而对CF1进行回收、克隆和序列测定,CF1长205bp,通过6个开放阅读框推断的氨基酸序列,与酚羟化酶α-亚基、外表皮蛋白C、NADH脱氢酶亚基1、NADH－CoQ氧化还原酶、2－氧代－铁氧化蛋白－氧化还原酶以及假拟14．5kD蛋白（hypotheitcal 14.5kD protein）有较高的相似性。     

Sexual differentiation in cucumber: The effects of abscisic acid and other growth regulators on various sex genotypes

AbA, ethephon and gibberellin were applied to cucumber plantsof monoecious, gynoecious, andromoneocious and hermaphroditeinbred lines, as well as to F₁ (gynoecious?monoecious) plants.Exogenous AbA enhanced the male tendency in monoecious cucumberplants and the female tendency in gynoecious plants, irrespectiveof light regime. Exogenous ethephon treatments increased thefemale tendency in monoecious plants, and decreased it in gynoeciousones. These effects were influenced by day length. ExogenousAbA counteracted the effect of gibberellin (A₄₊₇) treatmentin gynoecious plants, but had no such effect in monoecious ones. In addition to its differential effect on sexual differentiation,AbA stimulated flower development in gynoecious plants and inhibitedit in monoecious plants. These responses to AbA are discussedin the light of previously reported effects of plant growthregulators on various sex types of cucumber. The present resultsare being integrated into an updated working hypothesis on sexcontrol in cucumbers. (Received August 30, 1976; )     

Conversion of AFLP bands into high-throughput DNA markers

The conversion of AFLP bands into polymorphic sequence-tagged-site (STS) markers is necessary for high-throughput genotype scoring. Technical hurdles that must be overcome arise from genome complexity (particularly sequence duplication), from the low-molecular-weight nature of the AFLP bands and from the location of the polymorphism within the AFLP band. We generated six STS markers from ten AFLP bands (four AFLPs were from co-dominant pairs of bands) in soybean (Glycine max). The markers were all linked to one of two loci, rhg1 on linkage group G and Rhg4 on linkage group A2, that confer resistance to the soybean cyst nematode (Heterodera glycines I.). When the polymorphic AFLP band sequence contained a duplicated sequence or could not be converted to a locus-specific STS marker, direct sequencing of BAC clones anchored to a physical map generated locus-specific flanking sequences at the polymorphic locus. When the polymorphism was adjacent to the restriction site used in the AFLP analysis, single primer extension was performed to reconstruct the polymorphism. The six converted AFLP markers represented 996 bp of sequence from alleles of each of two cultivars and identified eight insertions or deletions, two microsatellites and eight single-nucleotide polymorphisms (SNPs). The polymorphic sequences were used to design a non-electrophoretic, fluorometric assay (based on the TaqMan technology) and/or develop electrophoretic STS markers for high-throughput genotype determination during marker-assisted breeding for resistance to cyst nematode. We conclude that the converted AFLP markers contained polymorphism at a 10- to 20-fold higher frequency than expected for adapted soybean cultivars and that the efficiency of AFLP band conversion to STS can be improved using BAC libraries and physical maps. The method provides an efficient tool for SNP and STS discovery suitable for marker-assisted breeding and genomics.     

Variation of DNA fingerprints among accessions within maize inbred lines and implications for identification of essentially derived varieties: II. Genetic and technical sources of variation in AFLP data and comparison with SSR data

Accuracy and reproducibility of genetic distances (GDs) based on molecular markers are crucial issues for identification of essentially derived varieties (EDVs). Our objectives were to investigate (1) the amount of variation for amplified fragment length polymorphism (AFLP) markers found among different accessions within maize inbreds and doubled haploid (DH) lines, (2) the proportion attributable to genetic and technical components and marker system specific sources, (3) its effect on GDs between maize lines and implications for identification of EDVs, and (4) the comparison to published SSR data from the same plant materials. Two to five accessions from nine inbred lines and five DH lines were taken from different sources of maintenance breeding or drawn as independent samples from the same seed lot. Each of the 41 accessions was genotyped with 20 AFLP primer combinations revealing 988 AFLP markers. Map positions were available for 605 AFLPs covering all maize chromosomes. On average, six (0.6%) AFLP bands were polymorphic between different accessions of the same line. GDs between two accessions of the same line averaged 0.013 for inbreds and 0.006 for DH lines. The correlation of GDs based on AFLPs and SSRs was tight (r = 0.97**) across all 946 pairs of accessions but decreased (r = 0.55**) for 43 pairs of accessions originating from the same line. On the basis of our results, we recommend specific EDV thresholds for marker systems with different degree of polymorphism. In addition, precautions should be taken to warrant a high level of homogeneity for DNA markers within maize lines before applying for plant variety protection.     

AFLP targeting of the 1-cM region conferring the vrs1 gene for six-rowed spike in barley, Hordeum vulgare L.

Spike morphology is a key characteristic in the study of barley domestication, yield, and use. Multiple alleles at the vrs1 locus control the development and fertility of the lateral spikelets of barley. We developed five amplified fragment length polymorphism (AFLP) markers tightly linked to the vrs1 locus using well-characterized near-isogenic lines as plant materials. The AFLP markers were integrated into three different maps, in which 'Azumamugi' was used as the maternal parent. Of the three maps, Hordeum vulgare L. 'Azumamugi' x H. vulgare 'Golden Promise' showed recombination of the AFLP markers and the vrs1 locus (closest, 0.05 cM), providing the best mapping population for positional cloning of alleles at the vrs1 locus. Conversion of AFLP bands into polymorphic sequence-tagged sites (STSs) is necessary for further high-throughput genotype scoring and for bacterial artificial chromosome (BAC) library screening. We cloned and sequenced the five AFLP bands and synthesized primer pairs. PCR amplification generated DNAs of the same size from all four parental lines for each marker. Restriction endonuclease treatment of e40m36-1110/AccIII, e34m13-260/Psp1406I, e52m32-270/FokI, and e31m26-520/MnlI revealed fragment length polymorphisms between 'Azumamugi' and all the two-rowed parents. Allelism between the AFLPs and corresponding STS markers was confirmed genetically, indicating the usefulness of the STSs as genetic markers.     

黄瓜植株性别表现与3种氧化酶同工酶的关系

采用同工酶电泳技术分析了二叶期纯雌株和雌雄株黄瓜（Ｃｕｃｕｍｉｓ ｓａｔｉｖｕｓ Ｌ．）子叶和真叶过氧化物酶、多酚氧化酶和超氧化物歧化酶同工酶,结果发现：给株比雌雄株酶活性强、酶带数量多,这种差异酶带大多与雌性或雌雄性别紧密相关,经检验可以作为黄瓜雌性株早期鉴定的生化标记,尤其以真叶中多酚氧化酶同工酶Ｒｆ０．２８７表现稳定,鉴定成功率高。等电聚焦电泳垂直平板聚丙烯酰胺凝胶电泳分辨效果好。     

Association of vitamin D receptor gene polymorphism with the urine calcium level in nephrolithiasis patients

Abstract

Association of vitamin D receptor (VDR) gene polymorphism with the urine calcium level in nephrolithiasis patients from the published reports are still conflicting. This study was conducted to evaluate the relationship between VDR BsmI (rs1544410), Fok1 (rs2228570), TaqI (rs731236) and ApaI (rs7975232) gene polymorphism and urine calcium level in nephrolithiasis patients using meta-analysis method. The association studies were identified from PubMed, and Cochrane Library on 1 April 2014, and eligible investigations were included and synthesized using meta-analysis method. Four reports were recruited into this meta-analysis for the association of VDR BsmI, Fok1, TaqI and ApaI gene polymorphism with urine calcium level in nephrolithiasis patients. In this meta-analysis, VDR BsmI B allele and BB genotype, Fok1 f allele and ff genotype, TaqI, and ApaI gene polymorphism were not associated with urine calcium level in nephrolithiasis patients. However, the BsmI bb genotype and Fok1 FF genotype were associated with the urine calcium level in nephrolithiasis patients. In conclusion, VDR BsmI bb genotype and Fok1 FF genotype were associated with the urine calcium level in nephrolithiasis patients. However, more studies should be conducted to confirm it.     

黄瓜茎端ABA/GA_4比值与花芽性别分化的关系(英文)

在同一环境条件下,单雌性株茎端ABA含量显著高于雌雄同株的,而GA_4水平却正好相反。 较高的ABA/GA_4比值有利于黄瓜雌性分化,反之则有利于雄性分化。促进黄瓜雄性分化的生长物质GA_3和AgNO_3会引起黄瓜茎端ABA/GA_4比值的下降,而促进其雌性分化的生长物质乙烯利(Ethrel)则会引起ABA/GA_4比值的上升。     

Involvement of abscisic acid in the regulation of sex expression in the cucumber

The interaction between abscisic acid (ABA), gibberellin (GA)and ethephon on sex determination in cucumber (Cucumis sativusL.) flowers was examined. ABA promoted the female tendency ofgynoecious plants, but did not change the sex expression ofmonoecious ones. When ABA was applied together with GA₄₊₇ thepromoting activity of the GA on male flower formation in thegynoecious line was reduced. ABA also inhibited tendril appearanceand internode length, characteristic of GA treatments. A combinedABA and ethephon treatment resulted in a synergistic activityinhibiting growth and increasing the period of female flowerappearance in the monoecious line. It is suggested that ABAparticipates in the sex regulation of the cucumber by inhibitingGA activity. (Received March 8, 1974; )     

不同生态环境下雌雄同株黄瓜单性结实性遗传的比较

以两个单性结实性不同的雌雄同株黄瓜自交系构建的4世代群体为试材,采用植物数量性状主基因+多基因混合遗传模型联合分析方法,对南京江宁和河北昌黎两地雌雄同株黄瓜单性结实性遗传进行了比较研究.结果表明：不同生态环境下,雌雄同株黄瓜单性结实性遗传均符合E-1-1模型,受2对加性-显性-上位性主基因+加性-显性多基因控制,存在基因型与环境互作效应.但是,不同环境条件下F₁的遗传倾向和遗传参数不同,F₂的主基因遗传率为42.1%～97.5%,其遗传差异主要由黄瓜结果期两地日照和温度差异所致.强单性结实黄瓜品种选育以双亲均为强单性结实材料为宜,杂种后代宜在不同生态条件下选择鉴定.     

Ultrahigh-Density Linkage Map for Cultivated Cucumber (Cucumis sativus L.) Using a Single-Nucleotide Polymorphism Genotyping Array

Genotyping arrays are tools for high-throughput genotyping, which is beneficial in constructing saturated genetic maps and therefore high-resolution mapping of complex traits. Since the report of the first cucumber genome draft, genetic maps have been constructed mainly based on simple-sequence repeats (SSRs) or on combinations of SSRs and sequence-related amplified polymorphism (SRAP). In this study, we developed the first cucumber genotyping array consisting of 32,864 single-nucleotide polymorphisms (SNPs). These markers cover the cucumber genome with a median interval of ~2 Kb and have expected genotype calls in parents/F₁ hybridizations as a training set. The training set was validated with Fluidigm technology and showed 96% concordance with the genotype calls in the parents/F₁ hybridizations. Application of the genotyping array was illustrated by constructing a 598.7 cM genetic map based on a ‘9930’ × ‘Gy14’ recombinant inbred line (RIL) population comprised of 11,156 SNPs. Marker collinearity between the genetic map and reference genomes of the two parents was estimated at R² = 0.97. We also used the array-derived genetic map to investigate chromosomal rearrangements, regional recombination rate, and specific regions with segregation distortions. Finally, 82% of the linkage-map bins were polymorphic in other cucumber variants, suggesting that the array can be applied for genotyping in other lines. The genotyping array presented here, together with the genotype calls of the parents/F₁ hybridizations as a training set, should be a powerful tool in future studies with high-throughput cucumber genotyping. An ultrahigh-density linkage map constructed by this genotyping array on RIL population may be invaluable for assembly improvement, and for mapping important cucumber QTLs.     

Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon

Three different types of molecular markers, RAPD, AFLP and RFLP were used to measure genetic diversity among six genotypes of Cucumis melo L. Each line represented a different melon genotype: Piel de Sapo, Ogen, PI161375, PI414723, Agrestis and C105. A number of polymorphic RAPD, AFLP and RFLP bands were scored on all materials and the genetic similarity measured. Clustering analysis performed with the three types of markers separated the genotypes into two main groups: (1) the sweet type, cultivated melons and (2) the exotic type, not cultivated melons. While the data obtained suggest that all three types of markers are equally informative, AFLPs showed the highest efficiency in detecting polymorphism. Received: 30 December 1999 / Accepted: 24 January 2000     

Development of AFLP markers in barley

To investigate the application of amplified fragment length polymorphism (AFLP) markers in barley, 96 primer combinations were used to generate AFLP patterns with two barley lines, L94 and Vada. With seven primer combinations, only a few intense bands were obtained, probably derived from repeated sequences. With the majority of the remaining 89 primer combinations, on average about 120 amplification products were generated, and the polymorphism rate between the two lines was generally over 18%. Based on the number of amplified products and the polymorphism rate, the 48 best primer combinations were selected and tested on 16 barley lines, again including L94 and Vada. Using a subset of 24 primer combinations 2188 clearly visible bands within the range from 80 to 510 bp were generated; 55% of these showed some degree of polymorphism among the 16 lines. L94 versus Vada showed the highest polymorphism rate (29%) and Proctor versus Nudinka yielded the lowest (12%). The polymorphism rates per primer combination showed little dependence on the barley lines used. Hence the most efficient and informative primer combinations identified for a given pair of lines turned out to be highly efficient when applied to others. Generally, more than 100 common markers (possibly locus specific) among populations or crosses were easily identified by comparing 48 AFLP profiles of the parent lines. The existence of such a large number of markers common to populations will facilitate the merging of molecular marker data and other genetic data into one integrated genetic map of barley. Received: 28 October 1996 / Accepted: 27 November 1996