巴西橡胶树SSR反应体系的优化

摘 要： 以巴西橡胶树无性系针选2号为试验材料,对橡胶树SSR-PCR反应的5个主要因素：TaqDNA聚合酶、Mg2+、引物、DNA模板及dNTPs进行优化试验。筛选出了各反应因素的最佳水平,建立了橡胶树SSR-PCR最佳反应体系（20ul）: TaqDNA聚合酶0.8U;Mg2+ 浓度1.5mmol•L-1;引物浓度0.5μmol•L-1;模板DNA 含量50ng; dNTPs浓度 0.25 mmol•L-1。并利用此反应体系,用引物1对14个巴西橡胶树品种进行SSR分析,8%的非变性聚丙稀酰胺凝胶电泳检测显示不同品种间DNA谱带多态性丰富,结果证明,此反应体系是稳定可靠的,这为下一步进行的遗传多样性分析、指纹图谱构建等研究奠定基础     

Development and characterization of a new set of 164 polymorphic EST‐SSR markers for diversity and breeding studies in rubber tree (Hevea brasiliensis Müll. Arg.)

Despite its economic importance and recent genome release, the need for molecular tools for Hevea brasiliensis is high. In the frame of a disease resistance study, EST sequences were retrieved from public database or generated by sequencing SSH libraries. Sequences were trimmed and microsatellite motifs searched using an ad hoc bioinformatic pipeline, and pairs of primers for the amplification of candidate markers were generated. We found a total of 10 499 unigenes from both sources of sequences, and 673 microsatellites motifs were detected using the default parameters of the pipeline. Two hundred sixty‐four primer pairs were tested and 226 (85.6%) successfully amplified. Out of the amplified candidate markers, 164 exhibited polymorphism. Relationships based on dendrograms using simple matching index and diversity statistics based on EST‐SSRs were compared with Genomic SSRs, showing the potentialities of EST‐derived microsatellites for resistance studies but also for population genetics approaches.     

Evaluation of random amplified polymorphic DNA (RAPD) markers in Hevea brasiliensis

The applicability of random amplified polymorphic DNA (RAPD) markers in the cultivated rubber tree, Hevea, was evaluated using 43 decamer oligonucleotide primers in a set of 24 clones selected in different South-East Asian countries. A total of 220 0.35–3.5 kb DNA fragments were amplified, of which 111 were polymorphic. Of these, 80 fragments (RAPD markers) which were repeatable and clearly scorable across all genotypes were used to estimate genetic distances among the clones tested. The estimated genetic distances ranged from 0.05 (RRII 308 and PB 5/51) to 0.75 (RRIC 100 and SCATC 88–13). A mean genetic distance of 0.5 indicates a rather high genetic variability among the tested clones. As expected, because of the breeding history of Hevea, UPGMA cluster analysis and Principal Coordinate Analysis (PCoA) indicated the absence of a distinct geographical grouping. The possible application of RAPD markers for clone identification and also for analysis of genetic relationships among Hevea clones is discussed.     

Microsatellite variability and its use in the characterization of cultivated clones of Hevea brasiliensis

Microsatellite markers were developed and evaluated in Hevea brasiliensis, an important crop species producing natural rubber of commercial utility. Of eight microsatellite markers, four were found to be highly informative, amplifying a total of 19 alleles when evaluated against 27 cultivated Hevea clones/genotypes. Power of discrimination of the microsatellite loci was in the range of 0.62‐0.89, with a mean of 0.76 indicating these microsatellites could be valuable genetic markers for diversity characterization. A combination of four microsatellite markers was successfully used to discriminate uniquely all the 27 Hevea clones and some clone‐specific allelic profiles were generated. Cross‐species amplification of the markers developed in H. brasiliensis had also been demonstrated with two other Hevea species, H. benthamiana and H. spruceana, indicating a high degree of sequence homology at the flanking regions. Sequence analysis of the repeat region at the 3′‐UTR of the hydroxymethylglutaryl‐coenzyme A reductase gene, containing clusters of AG repeats in 15 clones, revealed the existence of two alleles based on the repeat length polymorphisms. Homozygosity as well as heterozygosity for both the alleles had also been detected among the clones. Frequency of homozygotes for the smaller allele (allele‐1) was found to be lower than the larger allele (allele‐2) among the primary clones of H. brasiliensis.     

巴西橡胶树小橡胶粒子蛋白（SRPP）基因启动子的克隆及其功能分析

摘要：小橡胶粒子蛋白（SRPP）是巴西橡胶树中参与橡胶生物合成的重要蛋白因子之一。SRPP的氨基酸序列与橡胶延长因子（REF）和菜豆胁迫相关蛋白（PVSRP）的同源性分别为72%和68%。为了进一步研究SRPP基因表达及其调控的机制,作者采用接头连接介导的PCR散步法（ligation-mediated PCR）,克隆了SRPP基因的启动子。序列分析表明,SRPP基因启动子中与光诱导相关的顺式元件占39%,可能属于光诱导型启动子,因此,SRPP基因的表达可能受光信号的调控。此外,SRPP基因启动子还具有热激响应元件（HSE）、干旱胁迫响应元件（MBS）、防卫和胁迫响应元件（TC-rich repeats）、脱落酸响应元件（ABRE）、赤霉素响应元件（GARE）和激发子响应元件（EIRE,W box）等,这表明橡胶树SRPP基因不仅参与调控橡胶生物合成,而且可能是橡胶树中响应逆境信号的抗性基因,在橡胶树抵御逆境胁迫的生理过程中发挥重要作用。     

Simple sequence repeat markers for botanical varieties of cultivated peanut (Arachis hypogaea L.)

Cultivated peanut (Arachis hypogaea L.) consists of six botanical varieties. Identification of DNA markers associated with botanical varieties would be useful in plant genotyping, germplasm management, and evolutionary studies. We have developed 130 simple sequence repeat (SSR) markers in peanut, 38 of which were used in this study because of their ability in detecting genetic polymorphism among 24 peanut accessions. Eight SSR markers were found useful to classify botanical varieties. Among them, six SSR markers were specific to botanical varieties fastigiata and vulgaris, one to botanical varieties hypogaea and hirsuta, and one to botanical varieties peruviana, and aequatoriana. Also, three of them derived from peanut expressed sequence tags (ESTs) were associated with putative genes. As botanical varieties have different morphological traits and belong to different subspecies in A. hypogaea, these markers might be associated with genes involved in the expression of morphological traits. The results also suggested that SSRs (also called microsatellites) might play a role in shaping evolution of cultivated peanut. Multiplex PCR of botanical variety-specific markers could be applied to facilitate efficient genotyping of the peanut lines.     

巴西橡胶树排胶机制研究进展

橡胶树是世界上商业用天然橡胶最主要的来源,排胶是天然橡胶生产的最后关键环节,为了安全高效的获取胶乳产量,橡胶树排胶机制的研究一直受到人们的重视。橡胶树中的乳管是天然橡胶合成和储存的组织,人们通过割胶收集从乳管中流出的胶乳用作提炼天然橡胶。胶乳的排出速度及排胶持续时间是决定天然橡胶产量的重要因素,是排胶动力和排胶阻力共同作用的结果。笔者对与排胶相关的乳管细胞学基础、排胶主要初动力的乳管膨压、排胶继动力的乳管胶乳水分稀释效应以及乳管排胶阻力的形成及其调控机制等研究进行了归纳,主要分析了乳管伤口堵塞物的形成和影响排胶阻力的多种因素,指出乳管堵塞在胶乳的停排中起重要作用。针对目前排胶机制的研究进展缓慢这一现状,我们提出有必要从以下几点展开深入研究：（1）乳管膨压形成相关的树皮木质素合成和调控;（2）水分运输相关的水通道蛋白基因表达和活性分析;（3）停排相关的乳管伤口末端堵塞物的形成和调节。这些研究将为全面解析橡胶树排胶机制奠定基础,也可为分子辅助育种-选育排胶通畅的种质提供依据。     

Development of genomic and EST-SSR markers in radish (Raphanus sativus L.)

Radish (Raphanus sativus L.) belongs to Brassicaceae family and is a close relative of Brassica. This species shows a wide morphological diversity, and is an important vegetable especially in Asia. However, molecular research of radish is behind compared to that of Brassica. For example, reports on SSR (simple sequence repeat) markers are limited. Here, we designed 417 radish SSR markers from SSR-enriched genomic libraries and the cDNA data. Of the 256 SSR markers succeeded in PCR, 130 showed clear polymorphisms between two radish lines; a rat-tail radish and a Japanese cultivar, ‘Harufuku’. As a test case for evaluation of the present SSRs, we conducted two studies. First, we selected 16 SSRs to calculate polymorphism information contents (PICs) using 16 radish cultivars and four other Brassicaceae species. These markers detected 3–15 alleles (average = 9.6). PIC values ranged from 0.54 to 0.92 (average = 0.78). Second, part of the present SSRs were tested for mapping using our previously-examined mapping population. The map spanned 672.7 cM with nine linkage groups (LGs). The 21 radish SSR markers were distributed throughout the LGs. The SSR markers developed here would be informative and useful for genetic analysis in radish and its related species.     

Isolation and characterization of microsatellite markers from guineagrass (Panicum maximum) for genetic diversity estimate and cross-species amplification

Guineagrass ( Panicum maximum Jacq.) is one of the major forage grasses in tropical and semitropical regions, largely apomictic and predominantly exist as tetraploid. Non-availability of polymorphic molecular markers has been a major limitation in its characterization and improvement. We report isolation and characterization of microsatellites in P. maximum and cross-species results with other five Panicum species. Based on microsatellite-motifs, 15 functional and polymorphic simple sequence repeat (SSR) primer-pairs were designed, validated and employed in estimating genetic relationship among 34 guineagrass accessions. Thirteen primer-pairs amplified single locus and remaining two generated more than two loci with an average of 3.57 bands per locus amounts to 63 bands with 34 guineagrass accessions. Average expected heterozygosity ( H _E) of 0.35 (maximum 0.97) and observed heterozygosity ( H _O) of 0.37 (maximum 0.91) established the efficiency of developed markers for discriminating guineagrass accessions. Dice's similarity coefficients-based unweighted pair group with arithmetic average method-clustering supported with high bootstrap values (≥40) indicated its significance and distinguished all accessions except IG97-93 and IG97-6. Utility of these new SSR loci in genetic diversity study of P. maximum and other cross–amplified species is discussed.     

The breeding system of rubber (Hevea brasiliensis): an evaluation of controlled hand pollination methods

Summary Controlled hand pollinated pistils of rubber were observed using fluorescence microscopy to assess the efficiency of the universally-employed method for the production of progeny for plant breeding. The controlled hand pollination method conducted in the morning resulted in the deposition of a mean of 15.6 pollen grains on the stigma, with no stray pollination observed. Over 36% of the pistils had the potential to set fruit.Pollinations conducted in the afternoon at the normal time of anthesis, had double the fruit set potential of morning pollinations as measured by penetration of ovules by pollen tubes. Pollinator efficiency also varied, with excessive damage to stigmas resulting in reduced pollen germination and tube growth. There were differences between clones in both female and male fertility, in the proportion of pistils with more than three carpels and in the production of small abnormal stigmas. There was no difference in pollen tube growth following self- or cross-pollination, indicating that the self-sterility mechanism of rubber operates in the ovary. Pollen could be stored for 5 days at 5°C and 75% RH with a 22% loss of fertility.     

Chromosome doubling of pear haploid plants and homozygosity assessment using isozyme and microsatellite markers

The improvement of perennial fruit trees through traditional breedingmethods is a long-term effort because of their long generation time. Theproduction of haploid and doubled haploid plants should offer newpossibilities for genetic studies and breeding work. In this study, haploidclones of pear were treated in vitro by oryzalin for chromosomedoubling; the level of ploidy was assessed by flow cytometry. Oryzalinappeared to be an efficient agent for chromosome doubling, the optimalconcentrations range from 200 to 300 M. For homozygosityassessment, analyses of isozyme markers were carried out, together withmicrosatellite markers PCR-amplified with primers initially developed forapple. The use of isozyme markers confirmed homozygosity of all thedoubled haploid clones except for one. The microsatellite markers can beused earlier than isozymes for checking homozygosity during theprogramme of haploid and doubled haploid clones production. Truedoubled haploid clones of pear were obtained in less than one year andtheir acclimatisation in greenhouse has already started.     

Genic microsatellite markers for discrimination of spinach cultivars

A set of simple sequence repeat (SSR or microsatellite) markers was used to discriminate a collection of 33 Spinacia oleracea hybrid cultivars from seven different breeding stations all over the world. All SSR markers were genic microsatellite markers located in coding or non-coding regions of genes of known function. Cluster analysis based on 13 of the SSR markers showed that the spinach hybrids grouped into three clusters. The first two clusters consisted of European spinach types, which were well discriminated according to their origin from different breeding stations. The third cluster was a mixture of Asian as well as European types of spinach. Subclusters in this group did not reflect differences in morphology, earliness or company origin. The data show that genic microsatellites are a powerful tool for discrimination of spinach cultivars.     

Development of simple sequence repeat (SSR) markers in Eucalyptus from amplified inter-simple sequence repeats (ISSR)

Eucalyptus spp. are widely used in exotic plantations. Since many of these trees are derived from vegetative propagation, the routine identification of clones has become increasingly important. The most widely used molecular based method for fingerprinting these clones is by random amplified polymorphic DNAs (RAPDs). Although this technique is useful, its results are not very repeatable, especially between laboratories. The aim of this study was to develop microsatellite markers that are highly repeatable, and to investigate their value in Eucalyptus fingerprinting. Typically, this process involves the expensive procedure of constructing an enriched genomic library. However, we used an intersimple sequence repeat (ISSR) polymerase chain reaction (PCR)‐based enrichment technique for microsatellite‐rich regions. With this relatively inexpensive method, microsatellite‐rich regions were amplified directly from genomic DNA, after which PCR products were cloned and sequenced. From these microsatellite‐rich sequences, primer sets were constructed to amplify mono‐, di‐, tri‐, hexa‐and nona‐nucleotide repeats. These markers were all inherited in a Mendelian fashion in the progeny of a test cross between two Eucalyptus grandis trees. The primer sets developed were also able to amplify the corresponding microsatellite loci from five different Eucalyptus spp., namely E. grandis, E. nitens, E. globulus, E. camaldulensis and E. urophylla.     

橡胶树中碱性转化酶基因HbNIN7的克隆与表达分析

蔗糖主要由植物光合作用产生,其水解依赖于转化酶和蔗糖合成酶。其中,转化酶催化蔗糖不可逆分解为葡萄糖与果糖。本研究基于橡胶树的基因组和转录组数据库,采用RT-PCR技术克隆得到一个中碱性转化酶基因HbNIN7基因,预测编码683个氨基酸。同源与亚细胞定位分析表明HbNIN7属于α类中碱性转化酶,定位于线粒体。通过原核表达获得其重组蛋白,酶活性分析表明,HbNIN7重组蛋白最适酶活pH值7.2;最适酶活温度45℃;最大反应速率32.69 mmol hexose·mg^-1protein·h^-1;Km值25.04 mmol/L。利用荧光定量方法分析基因的表达特征,结果显示:HbNIN7基因在雄花中表达水平最高,且在雄花发育的不同阶段差异显著,说明Hb NIN7很可能参与橡胶树花组织中糖利用及糖信号调控。     

Classification of Southeast Asian mints (Mentha spp.) based on simple sequence repeat markers

Mentha is a complex genus encompassing many species as a consequence of their interspecific hybridization and polyploidy. Southeast Asian mints have been poorly distinguished though they are widely used for culinary and medical purposes. In this study, we have analyzed Southeast Asian mints and known varieties as well as a related Lamiaceae species (Nepeta sp.) using simple sequence repeat (SSR) markers and leaf morphology. Two types of mints were clearly distinguished based on their venation pattern and leaf shape index. We developed 12 SSR markers that allowed good amplification in the Mentha and another Lamiaceae species. In the SSR-based phylogram, the Mentha lines could be delimited into groups I–VI. The Southeast Asian mints divided into groups I and II, and the phylogram separated most of the available species, with groups I and II containing the known species M. × cordifolia and M. arvensis, respectively. The separation of the two groups was supported by a population structure analysis. The SSR markers developed in this study enabled the simultaneous classification of mints and will help improve our understanding of the genetic composition of known mint varieties and as yet unclassified Southeast Asian mints.     

SSR标记开发及其在植物中的应用

本文简要介绍了SSR标记开发方法及SSR标记在通用性、遗传多样性与种质鉴定、遗传连锁图谱构建、基因定位与克隆等研究中的应用,对SSR标记应用存在的问题提出了看法,并对其应用前景作了展望,为今后SSR标记在植物上的应用提供参考。