摘要
用从条斑紫菜EST数据库中筛选合成的微卫星引物对8个坛紫菜丝状体品系进行微卫星DNA指纹扩增。5个微卫星引物共扩增出32条带,其中3对引物所扩增出的5个条带(AU192094-127、AU187410-335、AU187410-190、AU194267-203和AU194267-328)被用来构建8个坛紫菜丝状体品系的DNA指纹。在这个图谱中,每个丝状体品系都有独一的指纹模式,彼此很容易被区分开。所获得的DNA指纹图谱,可用来进行孟德尔分离研究,及为坛紫菜纯系鉴定提供分子基础。
As a high polymorphic and codominant molecular marker, microsatellites, or simple sequence repeats (SSRs) have been used widely in genetic researches such as genetic maps construction and population polymorphism research. In Porphyra, no effort was devoted to the utilization of microsatellite marker. The biggest obstacle of microsatellite marker is the availability of interested microsatellite loci. One of the most convenient methods for obtaining microsatellite loci is to search the public database. In former work, online BLAST software was used to isolate all the di-nucleotide and tri-nucleotide microsatellite loci from the Genbank dbEST of Porphyra yezoensis. Total of 211 non-redundant microsatellite loci, amounted to 1.01% of the dbEST, were exploited from 20 979 EST sequences. Among these microsatellite sequences,35 dinucleotide micorsatellite repeats which core-unit repeats not less than 7 and 176 trinucleotide microsatellite which repeat numbers no less than 5 were isolated. Then some loci were selected out to design primers at the flanking regions according to the followed criteria: primer length within 20±2 bp, GC content between 50% and 75%, melting temperature between 50℃ and 65℃, expected product size between 100 and 300bp and without a secondary structure. 15 microsatellite primer pairs were synthesized and inter-species amplified on 8 filament lines of Porphyra haitanensis, the primer pairs which produced distinct amplified bands were selected out. In this research, total 32 bands were amplified by 5 microsatellite primer pairs in 8 filament lines of P. haitanensis. Among them, 5 bands (AU192094-127,AU187410-335,AU187410-190,AU194267-203,AU194267-328 note: the former was the name of primer, latter was the molecular weight of selected bands) were chosen to construct the DNA fingerprinting of 8 filament lines. For the rest two primer pairs of AV435669 and AV432452, multiple band pattern and false positive (microsatellite repeats were absent from the amplification bands) problems might exist in the inter-species amplification, so the amplified bands of these two primer pairs were excluded from the fingerprint construction.In the constructed DNA fingerprint, each filament line has its unique fingerprint pattern and can be easily distinguished from each other. According to this fingerprint, researchers can trace the genetic background of descendant blades which germinated from 8 filament lines.Besides, the molecular fingerprint can overcome the disability of morphological identification, which was susceptive to the environment affection. In addition, the co-dominant of microsatellite marker made it possible to study the Mendel segregation so as to identify the pure line filaments of Porphyra haitanensis. The fingerprint can also be used to confirm the stage of meiosis during the life history of P.haitanensis, for this purpose, the microsatellite primer should not have the problems of multiple bands and false positive problems, and the microsatellite locus of this primer is heterozygosis.In summary, this paper just built a molecular basis for the future genetic research of Porphyra haitanensis.
出处
《水产学报》
CAS
CSCD
北大核心
2005年第3期323-326,共4页
Journal of Fisheries of China
基金
国家自然科学基金资助项目(No.49976030)
关键词
DNA指纹
微卫星标记
坛紫菜
丝状体
DNA fingerprint
microsatellite marker
Porphyra haitanensis
filament
