起始密码子区域相关序列多态性的开发及在苜蓿遗传多样性上的应用

目前广泛使用的基于PCR基础的分子标记多为扩增非编码区域,或是随机基因组中扩增,在QTL定位中得到的位点一般与目标性状基因距离较远,我们开发了一个新的基于启动子序列目的基因型分子标记技术——启动子区域相关序列多态性（SCRP）,试图使标记能够更为准确的反映不同品种的遗传基础。它利用启动子位置保守一致序列 （“Kozak”序列） 作为其上游引物,利用内含子富含“AATT”的特性,作为核心序列设计下游引物,上下游引物均为18bp,引物间通过组合配对的方式作为扩增引物对。设计了14条上游引物和10条下游引物,共140对引物组合,对34个苜蓿品种进行扩增,研究了34个苜蓿的遗传多样性。每个PCR反应产生3~16个50~2000bp的条带,结果表明该标记简单、可靠、具有较高多态性,并且扩增区域为一种目的基因型分子标记,在种质资源研究中具有重要价值。     

Conserved Region Amplification Polymorphism (CoRAP), a Novel Marker Technique for Plant Genotyping in <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

Salvia miltiorrhiza (SM), a widely popular Chinese herb, is grown in various regions in China. Identifying SMs grown in different provinces of China is difficult, and therefore genotyping these collections would be highly valuable. Based on the techniques of sequence-related amplified polymorphism and target region amplified polymorphism, a novel PCR-based molecular marker technique called conserved region amplification polymorphism (CoRAP) is reported in this study to genotype SMs. The CoRAP technique is based on the use of two primers: fixed and arbitrary primers. The former is derived from target EST sequences deposited in Genbank; while, the core sequence (CACGC) of the latter is a conserved region found in most introns. In the present study, we utilized CoRAP to genotype SMs from different geographical origins. PCR amplification is performed for 30 cycles at an annealing temperature of 52°C. Each PCR reaction has generated as many as 30–50 fragments of 50 to 1,000 bp in size. The successful DNA genotyping of SMs by CoRAP was achieved. This new genotyping method is rapid, efficient, and reproducible.     

Start Codon Targeted (SCoT) Polymorphism: A Simple,Novel DNA Marker Technique for Generating Gene-Targeted Markers in Plants

Random amplified polymorphic DNA (RAPD) markers have been used for numerous applications in plant molecular genetics research despite having disadvantages of poor reproducibility and not generally being associated with gene regions. A novel method for generating plant DNA markers was developed based on the short conserved region flanking the ATG start codon in plant genes. This method uses single 18-mer primers in single primer polymerase chain reaction (PCR) and an annealing temperature of 50°C. PCR amplicons are resolved using standard agarose gel electrophoresis. This method was validated in rice using a genetically diverse set of genotypes and a backcross population. Reproducibility was evaluated by using duplicate samples and conducting PCR on different days. Start codon targeted (SCoT) markers were generally reproducible but exceptions indicated that primer length and annealing temperature are not the sole factors determining reproducibility. SCoT marker PCR amplification profiles indicated dominant marker like RAPD markers. We propose that this method could be used in conjunction with these markers for applications such as genetic analysis, bulked segregant analysis, and quantitative trait loci mapping, especially in laboratories with a preference for agarose gel electrophoresis.     

Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in Brassica

We developed a simple marker technique called sequence-related amplified polymorphism (SRAP) aimed for the amplification of open reading frames (ORFs). It is based on two-primer amplification. The primers are 17 or 18 nucleotides long and consist of the following elements. Core sequences, which are 13 to 14 bases long, where the first 10 or 11 bases starting at the 5′ end, are sequences of no specific constitution (”filler” sequences), followed by the sequence CCGG in the forward primer and AATT in the reverse primer. The core is followed by three selective nucleotides at the 3′ end. The filler sequences of the forward and reverse primers must be different from each other and can be 10 or 11 bases long. For the first five cycles the annealing temperature is set at 35°C. The following 35 cycles are run at 50°C. The amplified DNA fragments are separated by denaturing acrylamide gels and detected by autoradiography. We tested the marker technique in a series of recombinant inbred and doubled-haploid lines of Brassica oleracea L. After sequencing, approximately 45% of the gel-isolated bands matched known genes in the Genbank database. Twenty percent of the SRAP markers were co-dominant, which was demonstrated by sequencing. Construction of a linkage map revealed an even distribution of the SRAP markers in nine major linkage groups, not differing in this regard to AFLP markers. We successfully tagged the glucosinolate desaturation gene BoGLS-ALK with these markers. SRAPs were also easily amplified in other crops such as potato, rice, lettuce, Chinese cabbage (Brassica rapa L.), rapeseed (Brassica napus L.), garlic, apple, citrus, and celery. We also amplified cDNA isolated from different tissues of Chinese cabbage, allowing the fingerprinting of these sequences. Received: 3 November 2000 / Accepted 24 November 2000     

白菜的EST标记及其对油菜的通用性

根据白菜的表达序列标签,设计了28对引物。在对引物、dNTP、MgCl2的浓度及退火温度等参数进行测试后,建立了合适的PCR反应体系。在此反应体系下,以构建EST的白菜自交系A的DNA为模板,对设计的引物进行了筛选,发现有18对引物能对白菜DNA扩增出产物。用筛选出来的引物分别对17个白菜类品种进行PCR扩增,用琼脂糖凝胶电泳分析其产物的多态性,发现10对引物有多态性,这占了筛选引物的55.6%。为检测白菜EST标记的通用性,进一步利用设计的引物对不同油菜品种的DNA进行PCR扩增。在检测的28对引物中,共有24对引物能扩增出产物,占引物总数的85.7%,显示多态性的引物为18对,占引物总数的64.3%.。在对白菜DNA能扩增出产物的18对引物中,对油菜完全可用,且有13对引物产生多态性。而在那些对白菜未扩增出产物的10对引物中,也有6对能扩增出产物,其中5对显示多态性。文章研究结果证明,通过EST建立分子标记是可行的,而且这种标记对近缘物种是可通用的。     

Amplification and detection of polymorphic sequence-tagged sites in<Emphasis Type="Italic">Lathyrus sativus</Emphasis>

A simple procedure was developed to convertLathyrus sativus defence-related expressed sequence tags (ESTs) into mappable genetic markers by using PCR. Twenty-nine STS primer pairs were generated on the basis of sequence information from anL. sativus cDNA library. These primers were used to screen for polymorphisms between 2L. sativus accessions, ATC 80878 and ATC 80407, resistant and susceptible, respectively, toMycosphaerella pinodes infection. All 29 primer pairs amplified PCR products in both accessions, 11 of which amplified multiple RAPD-like products. The remaining 18 primer pairs amplified single monomorphic products. Following cloning, sequencing, and database searches, 17 of 18 PCR products were confirmed to have amplified the targeted genome region. Ten of these 17 STS primer pairs revealed polymorphisms between ATC 80878 and ATC 80407 when PCR products were digested with a range of restriction endonucleases. These results suggest that the STS-based PCR analysis will be useful for generating informative molecular markers inL. sativus for future genome mapping experiments.     

Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.     

Multilocus markers for mouse genome analysis: PCR amplification based on single primers of arbitrary nucleotide sequence

Polymerase chain reaction (PCR) based on single primers of arbitrary nucleotide sequence provides a powerful marker system for genome analysis because each primer amplifies multiple products, and cloning, sequencing, and hybridization are not required. We have evaluated this typing system for the mouse by identifying optimal PCR conditions; characterizing effects of GC content, primer length, and multiplexed primers; demonstrating considerable variation among a panel of inbred strains; and establishing linkage for several products. Mg²⁺, primer, template, and annealing conditions were identified that optimized the number and resolution of amplified products. Primers with 40% GC content failed to amplify products readily, primers with 50% GC content resulted in reasonable amplification, and primers with 60% GC content gave the largest number of well-resolved products. Longer primers did not necessarily amplify more products than shorter primers of the same proportional GC content. Multiplexed primers yielded more products than either primer alone and usually revealed novel variants. A strain survey showed that most strains could be readily distinguished with a modest number of primers. Finally, linkage for seven products was established on five chromosomes. These characteristics establish single primer PCR as a powerful method for mouse genome analysis.     

Biologically-generated primer for PCR: PCR primer of unknown sequence.

We describe a method for producing specific PCR primers directly from PCR product, bypassing the usual need to know the primer sequence. Lack of abundance of primers derived from a PCR product is compensated for by the incorporation of an arbitrary 5'TAG sequence which acts as a surrogate template target for the bulk amplification phase. We use the technique to amplify clonospecific rearranged immunoglobulin genes, which have applications as markers of lymphoid neoplasms for tracing the success of therapy. The principle may have wider application wherever conserved and variable regions of DNA are juxtaposed.     

Characterization of CR1 repeat random PCR markers for mapping the chicken genome

Polymerase chain reaction (PCR) primers complementary to portions of the chicken repetitive element CR1 have been used previously to generate useful markers on the chicken genome linkage map. To understand better the genetic basis for this technique and to convert CR1–PCR loci to markers useful in physical genome mapping, five polymorphic CR1–PCR-generated DNAs were cloned and partially sequenced. Inverse PCR was then employed to clone the corresponding region of the genomes of both the Jungle Fowl (JF) and White Leghorn (WL) parental DNA templates. Our results demonstrate that some of the CR1–PCR-generated DNAs arise from priming at an endogenous CR1 element, whereas others are due to chance complementarity between the CR1–PCR primer in use and random annealing sites in the genome, unrelated to a demonstrable CR1 element. In all five instances, it was possible to identify the sequence difference between the JF and WL parental DNAs that gave rise to the initial polymorphism and design allele-specific PCR primer sets that uniquely detect that polymorphism. In four of the five instances, the polymorphism was a one or two basepair sequence difference within the primer annealing site, but in the fifth case the responsible difference was outside, but very close to, the annealing site. In all instances the allele-specific PCR for the sequence polymorphism mapped identically with the corresponding CR1–PCR amplification polymorphism. We conclude that CR1–PCR provides an efficient and reliable mechanism for genome mapping in avians that can correlate linkage and physical mapping approaches.