红掌转录组SSR位点表征及分子标记开发

SSR loci characterization and molecular marker development based on Anthurium andraeanum L. transcriptomes

  • 摘要:目的】通过转录组测序挖掘红掌SSR位点,开发多态性EST-SSR分子标记,为红掌种质资源评价、遗传图谱构建及分子育种提供可靠的工具支撑。【方法】利用Illumina对5个不同花色的红掌品种佛焰苞进行转录组测序,再采用MISA对SSR位点进行筛选,并统计其类型、频率及分布特征。利用Primer 3.0设计EST-SSR引物,随机选取120对引物,经3个代表性品种初筛后,对22个红掌品种进行PCR扩增,并计算多态性信息含量(PIC)。最后,基于NTSYSpc 2.10e的非加权组平均法(UPGMA)进行聚类分析,验证EST-SSR标记的有效性。【结果】红掌转录组测序获得22381条Unigenes(总长34872210 bp),从中检测出11324个SSR位点,分布于7705条Unigenes中,出现频率为50.60%,平均分布距离为3.08 kb。SSR位点中优势重复基序为单核苷酸(24.55%)、二核苷酸(46.41%)和三核苷酸(27.69%),核苷酸优势重复类型为A/T(21.59%)、AG/CT(37.73%)、AC/GT(6.30%)。SSR位点长度主要集中在12~20 bp,重复次数以5~12次为主。对含有SSR位点的7705个Unigenes设计引物,共获得7456对EST-SSR引物。随机筛选120对EST-SSR引物进行多态性检测,其中107对成功扩增(成功率89.17%),其中75对引物具有多态性,进一步筛选条带清晰的20对引物,并以22个红掌品种DNA为模板对其进行有效验证,获得16对高多态性引物(PIC>0.5000),这些引物对22个红掌品种表现出良好的区分能力,可将红掌品种分为四大类群,与传统形态学分类结果一致。【结论】红掌转录组SSR位点丰度和多态性均较高,可作为高效分子标记开发来源。开发的EST-SSR分子标记具有多态性高、稳定性好等特点,为红掌种质资源评价、遗传图谱构建和分子标记辅助育种提供了有效工具,有助于后续红掌重要观赏性状的遗传解析和品种改良。

     

    Abstract:Objective】This study aimed to discover SSR loci of Anthurium andraeanum L. through transcriptome sequencing and develop polymorphic EST-SSR molecular markers, providing reliable tools for evaluating germplasm resources, constructing genetic maps, and molecular breeding in Anthurium andraeanum L.【Method】Transcriptome sequencing of spathes of Anthurium andraeanum L. cultivars of five colors using Illumina, and MISA was employed to screen SSR loci, analyze their types, frequency, and distribution characteristics. EST-SSR primers were designed using Primer 3.0. A total of 120 pairs of primers were randomly selected. After preliminary screening of three representative cultivars, PCR amplification was conducted for 22 Anthurium andraeanum L. cultivars, with calculation of polymorphism information content (PIC). Finally, the effectiveness of EST-SSR markers was validated by unweighted pair group method with arithmetic mean (UPGMA) using NTSYSpc 2.10e.【Result】Transcriptome sequencing of Anthurium andraeanum L. generated 22381 unigenes, with the total length of 34872210 bp, from which 11324 SSR loci were detected, distributed across 7705 unigenes, with an occurrence frequency of 50.60% and an average distribution distance of 3.08 kb. The predominant repeat motifs were mononucleotide (24.55%), dinucleotide (46.41%), and trinucleotide (27.69%), with the predominant nucleotide repeat type of A/T (21.59%), AG/CT (37.73%), and AC/GT (6.30%). SSR locus lengths were primarily between 12-20 bp, with repeat numbers predominantly ranging from 5 to 12. A total of 7705 primers containing SSR loci were designed for unigenes, 7456 pairs of EST-SSR primers were obtained. Polymorphic detection was performed for the 120 pairs of EST-SSR primers, in which 107 pairs successfully amplified (with the success rate of 89.17%), with 75 pairs of primers showing polymorphism. A total of 20 pairs of primers were further selected, and the DNA of 22 Anthurium andraeanum L. cultivars was taken as templates for validation, yielding 16 pairs of highly polymorphic primers (PIC>0.5000). These primers demonstrated excellent discriminatory capability for the 22 Anthurium andraeanum L. cultivars that were classified into four groups, consistent with traditional morphological classification.【Conclusion】SSR loci of Anthurium andraeanum L. transcriptomes exhibit high abundance and polymorphism, which can be used for efficient molecular marker development. The developed EST-SSR markers show characteristics such as high polymorphism and stability, providing effective tools for germplasm evaluation, genetic map construction, and breeding assisted by molecular markers, which can facilitate genetic elucidation and cultivar improvement of major ornamental traits of Anthurium andraeanum L.

     

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