Molecular characterization of S-RNase genes and S-genotypes in the Japanese apricot (Prunus mume Sieb. et Zucc.)

Three partial S-RNase genes, MSRN-1, MSRN-2, and MSRN-3, in the Japanese apricot (Prunus mume Sieb. et Zucc.) were isolated from the three cultivars Nankou, Gyokuei, and Kairyouuchidaume, respectively. The structural characteristics revealed that S-RNase genes from the Japanese apricot were in the T2/SRNase-type S-RNase family with five conserved regions (C1, C2, C3, RC4, and C5) and one variable region (RHV) as reported in the other rosaceous plants. In the phylogenetic tree of T2/S SRNase-type RNases, three S-RNase genes of the Japanese apricot did not form a species-specific subgroup but the Prunus subfamily did. At least seven S-allelic genes were present in the Japanese apricot, and S-genotypes of six representative cultivars, including Nankou, Gyokuei, Kairyouuchidaume, Baigou, Kagajizou, and Oushuku were first established in this study as S ₁ S ₇, S ₂ S ₆, S ₃ S ₄, S ₃ S ₆, S ₃ S ₆ and S ₁ S ₅, respectively. An extended elucidation of the S-genotype would contribute to a more efficient breeding program of the Japanese apricot. Received: 5 September 2000 / Revision accepted: 22 December 2000     

Comparative proteomic analysis of pistil abortion in Japanese apricot (Prunus mume Sieb. et Zucc)

The phenomenon of pistil abortion widely occurs in Japanese apricot and has seriously affected the yield in production. We used a combination of two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) approaches to identify the differentially expressed proteome between perfect and imperfect flower buds in Japanese apricot. More than 400 highly reproducible protein spots (P<0.05) were detected and 27 protein spots showed a greater than two-fold difference in their expression values. The proteins identified were classified into eight functional classifications and ten process categories, according to the Gene Ontology (GO). Acetyl-CoA produced by ATP citrate lyase (ACL) as a structural substance during formation of the cell wall could regulate pistil abortion in Japanese apricot. S-adenosylmethionine (SAM), xyloglucan endotransglucosylase/hydrolases (XTHs) and caffeoyl-CoA-O-methyl transferase (CCoAOMT) could promote cell wall formation in perfect flower buds of Japanese apricot, greatly contributing to pistil development. Spermidine hydroxycinnamoyl transferase (SHT) may be involved in the O-methylation of spermidine conjugates and could contribute to abnormal floral development. The identification of such differentially expressed proteins provides new targets for future studies that will assess their physiological roles and significance in pistil abortion.     

miR169 and PmRGL2 synergistically regulate the NF-Y complex to activate dormancy release in Japanese apricot (Prunus mume Sieb. et Zucc.)

Plant Molecular Biology - This study is the first to demonstrate that GA4-induced dormancy release is associated with the NF-Y complex, which interacts with gibberellin inhibitor RGL2 in Japanese...     

梅REMAP分子标记技术体系的优化及其应用

通过分析构成梅REMAP分子标记技术体系的主要成分适宜浓度、SSR引物选择性碱基的添加以及REMAP-PCR扩增产物检测的非变性PAGE最佳浓度,建立了适合梅REMAP分子标记的技术体系,并对24个梅品种的遗传多样性进行了分析.结果表明:(1)适宜梅REMAP-PCR的反应体系为:25.0 μL总体积中,基因组DNA 40.0 ng、2.5 mmol/ L MgCl2 1.5 μL、2.5 mmol/L dNTPs 2.0 μL、10×PCR Buffer(TaKaRa) 2.5 μL、Taq DNA聚合酶1.5 U、10 μmol/L LTR引物1.0 μL、10 μmol/L SSR引物1.0 μL,且SSR引物的3′端加碱基可明显提高REMAP的扩增效果,但碱基数量无明显影响.(2)5.0%非变性PAGE电泳条带数量多,扩增范围广(300～2 000 bp),且各品种扩增产物均可显示,能够清晰地反映品种间的多态性.(3)24个梅品种的遗传多样性分析显示,花梅的绿萼型及朱砂型分别聚在一起,这与形态学分类的结果一致,证明建立的REMAP技术体系的有效性和可行性.     

超低温保存的梅花花药萌发率和授粉后的结实能力

试验比较梅花花粉和花药超低温保存后的花粉萌发率、授粉结实率和座果率的结果表明:(1)超低温保存花药用室温、自来水冲洗和温水浴3种方法化冻后的花粉萌发率差异不显著。(2)花药超低温保存1h后的花粉萌发率与新鲜花粉差异不显著。(3)超低温保存1个月和1年的花粉和花药都有授粉结实能力。     

果梅完全花与不完全花的差异蛋白分析

应用双向电泳技术对果梅完全花与不完全花的蛋白质组分进行了比较分析。经专业分析软件（PDQuest）对电泳图谱分析表明两者的蛋白分布相似,在完全花中发现了1个特异蛋白、1个上调蛋白、21个下调蛋白,在不完全花中发现2个特异蛋白,这些蛋白差异点可能与雌蕊的败育有关。应用质谱技术对3个特异点及5个差异大的蛋白点进行分析,得到的肽段数据与蛋白质数据库比对发现其中一个蛋白（28．2kD,pI4．53）与光敏色素B有关。     

Genome-Wide Characterization and Linkage Mapping of Simple Sequence Repeats in Mei (Prunus mume Sieb. et Zucc.)

Because of its popularity as an ornamental plant in East Asia, mei (Prunus mume Sieb. et Zucc.) has received increasing attention in genetic and genomic research with the recent shotgun sequencing of its genome. Here, we performed the genome-wide characterization of simple sequence repeats (SSRs) in the mei genome and detected a total of 188,149 SSRs occurring at a frequency of 794 SSR/Mb. Mononucleotide repeats were the most common type of SSR in genomic regions, followed by di- and tetranucleotide repeats. Most of the SSRs in coding sequences (CDS) were composed of tri- or hexanucleotide repeat motifs, but mononucleotide repeats were always the most common in intergenic regions. Genome-wide comparison of SSR patterns among the mei, strawberry (Fragaria vesca), and apple (Malus×domestica) genomes showed mei to have the highest density of SSRs, slightly higher than that of strawberry (608 SSR/Mb) and almost twice as high as that of apple (398 SSR/Mb). Mononucleotide repeats were the dominant SSR motifs in the three Rosaceae species. Using 144 SSR markers, we constructed a 670 cM-long linkage map of mei delimited into eight linkage groups (LGs), with an average marker distance of 5 cM. Seventy one scaffolds covering about 27.9% of the assembled mei genome were anchored to the genetic map, depending on which the macro-colinearity between the mei genome and Prunus T×E reference map was identified. The framework map of mei constructed provides a first step into subsequent high-resolution genetic mapping and marker-assisted selection for this ornamental species.     

Genetic relationships among fruiting-mei (Prunus mume Sieb. et Zucc.) cultivars evaluated with AFLP and SNP markers.

Genetic relationships among 50 fruiting-mei (Prunus mume Sieb. et Zucc.) cultivars from China and Japan were investigated, using 767 amplified fragment length polymorphism (AFLP) and 103 single nucleotide polymorphism (SNP) markers. The polymorphism among the cultivars was found to be 69.77%, based on EcoR I + Mse I AFLP primer pairs. The sequence alignment of 11 group sequences, derived from 50 samples, yielded 103 SNPs; the total length of genomic sequences was 3683 bp. Among these SNPs, 73 were heterozygous in the loci of different cultivars. The SNP distribution was 58% transition, 40% transversion, and 2% InDels. There was also 1 trinucleotide deletion. AFLP and SNP markers allowed us to evaluate the genetic diversity of these 50 fruiting-mei cultivars. The 2 derived cladograms did display some differences: all cultivars formed 2 subclusters (1A and 1B) in the cladogram based on AFLP polymorphisms, and formed 3 subclusters (2A, 2B, and 2C) in the cladogram based on SNP polymorphisms; and, in the cladogram based on AFLP polymorphisms, most cultivars from the Guangdong to Fujian provinces (G-F) in China, from the Yunnan, Hunan, and Sichuan provinces (Y-S-H) in China, and from Japan grouped in cluster 1A, and 18 (78.26%) of 23 cultivars from Jiangsu to Zhejiang provinces in China (J-Z) grouped in cluster 1B. The results demonstrate that mei cultivars from Japan are clustered with cultivars from China, and support the hypothesis that mei in Japan were introduced from China. Cultivars from the J-Z region of China have more genetic similarities. Cultivars from the G-F and Y-S-H regions have fewer genetic similarities and suggest more germplasm exchanges in the past.     

Genetic relatedness and genetic diversity of ornamental mei (Prunus mume Sieb. et Zucc.) as analysed by AFLP markers

The genetic diversity and genetic relatedness of mei (Prunus mume; 2n = 16) were studied using amplified fragment length polymorphism (AFLP) markers. Eight EcoRI–PstI AFLP primer combinations were applied to 121 distinct genotypes of mei cultivars and related species. A total of 508 AFLP product bands were produced, of which 382 were polymorphic. The unweighted pair group method with arithmetic averages analysis was carried out based on these AFLP markers. From this analysis, “Qugeng Mei,” “Yan Mei,” “Chaodou Mei,” and mei cultivars were seen to share the same P. mume genetic stem. The AFLP data were able to clearly discriminate P. mume from other species in the genus Prunus, with P. armeniaca aligning as its closest related species. Two major groups and nine subgroups of mei flower were identified, and there was a strong coincidence of these AFLP-based groupings with the respective morphological characters of the accessions. The genetic diversity of mei accessions was greatest in the Yunnan Province and decreased toward Eastern China and Japan, so supporting the hypothesis that the southwest of China represents the genetic diversity center of the species.     

High-density genetic map construction and identification of a locus controlling weeping trait in an ornamental woody plant (Prunus mume Sieb. et Zucc)

High-density genetic map is a valuable tool for fine mapping locus controlling a specific trait especially for perennial woody plants. In this study, we firstly constructed a high-density genetic map of mei (Prunus mume) using SLAF markers, developed by specific locus amplified fragment sequencing (SLAF-seq). The linkage map contains 8,007 markers, with a mean marker distance of 0.195 cM, making it the densest genetic map for the genus Prunus. Though weeping trees are used worldwide as landscape plants, little is known about weeping controlling gene(s) (Pl). To test the utility of the high-density genetic map, we did fine-scale mapping of this important ornamental trait. In total, three statistic methods were performed progressively based on the result of inheritance analysis. Quantitative trait loci (QTL) analysis initially revealed that a locus on linkage group 7 was strongly responsible for weeping trait. Mutmap-like strategy and extreme linkage analysis were then applied to fine map this locus within 1.14 cM. Bioinformatics analysis of the locus identified some candidate genes. The successful localization of weeping trait strongly indicates that the high-density map constructed using SLAF markers is a worthy reference for mapping important traits for woody plants.     

气调和乙烯对梅果叶绿素和内源激素含量的影响

本文研究了在 ( 2 5± 1 )℃下 ,气调包装和乙烯吸收剂处理对采后青梅果实叶绿素含量、内源激素IAA ,GA3,ABA含量和乙烯释放量的影响及它们之间的关系。结果表明 :气调包装果实叶绿素含量最高 ,其次是乙烯吸收剂处理的 ;各处理中气调包装果实的乙烯释放量始终很低 ,GA3含量较高 ,IAA和ABA含量则较低 ;对照果实的则相反 ,乙烯释放量很高 ,IAA和ABA含量较高 ,而GA3含量较低。乙烯吸收剂处理的处于二者之间。气调包装可以维持果实较高的GA3水平 ,降低ABA含量 ,保持较高的GA3/ABA值 ,抑制IAA和乙烯的生成 ,延缓梅果叶绿素的降解。     

Pistil abortion in Japanese apricot (<Emphasis Type="Italic">Prunus mume</Emphasis> Sieb. et Zucc.): isolation and functional analysis of <Emphasis Type="Italic">PmCCoAOMT</Emphasis> gene

The abnormal pistils widely occur in Japanese apricot (Prunus mume Sieb. et Zucc) and seriously affect the fruit production. In this study, a CCoAOMT homologue, PmCCoAOMT, was cloned in Japanese apricot and the bioinformatics software analyzed the structural characteristics. The PmCCoAOMT protein was detected to be located in the cell cytoplasm by onion transient expression experiment. Analysis of the real-time PCR data showed that PmCCoAOMT gene expressed in the prophase development of pistil and the expression level in ‘Daqiandi’ was higher than ‘Longyan.’ The expression level in ‘Longyan’ was higher than ‘Daqiandi’ in the late period development of pistil, and the expression level of perfect flower (perfect pistil) was higher than imperfect flower (pistil deformity and no pistil). Compared with the control, the over-expression of PmCCoAOMT transgenic tobacco lines showed bigger flowers, darker petals. The lignin monomer composition in transgenic tobacco lines was also measured, and the results showed that transgenic tobacco lines had a higher S (Syringyl)/G (Guaiacyl) ratio (22.3 %) than control lines (11.8 %). Also, the perfect flower buds contained more S/G ratio (92.62 %) than imperfect flower buds (83.55 %) in ‘Daqiandi.’ Our results indicated that the PmCCoAOMT gene might have function in lignin accumulation, which contributed to pistil development in Japanese apricot.     

Cytochromes P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin. Cloning, functional expression in Pichia pastoris, and substrate specificity of the isolated recombinant enzymes

The first committed steps in the biosynthesis of the two cyanogenic glucosides linamarin and lotaustralin in cassava are the conversion of L-valine and L-isoleucine, respectively, to the corresponding oximes. Two full-length cDNA clones that encode cytochromes P-450 catalyzing these reactions have been isolated. The two cassava cytochromes P-450 are 85% identical, share 54% sequence identity to CYP79A1 from sorghum, and have been assigned CYP79D1 and CYP79D2. Functional expression has been achieved using the methylotrophic yeast, Pichia pastoris. The amount of CYP79D1 isolated from 1 liter of P. pastoris culture exceeds the amounts that putatively could be isolated from 22,000 grown-up cassava plants. Each cytochrome P-450 metabolizes L-valine as well as L-isoleucine consistent with the co-occurrence of linamarin and lotaustralin in cassava. CYP79D1 was isolated from P. pastoris. Reconstitution in lipid micelles showed that CYP79D1 has a higher k(c) value with L-valine as substrate than with L-isoleucine, which is consistent with linamarin being the major cyanogenic glucoside in cassava. Both CYP79D1 and CYP79D2 are present in the genome of cassava cultivar MCol22 in agreement with cassava being allotetraploid. CYP79D1 and CYP79D2 are actively transcribed, and production of acyanogenic cassava plants would therefore require down-regulation of both genes.