‘芙蓉李’转录组SSR信息分析与分子标记开发

Analysis on SSR information in‘Furongli'plum transcriptome and development of molecular markers in Prunus salicina Lindl.

【目的】明确‘芙蓉李’转录组SSR总体特征,开发SSR引物,为李种质资源多样性分析和品种鉴定等奠定基础。【方法】采用MISA分析‘芙蓉李’转录组SSR位点信息,利用Primer3设计引物,并随机选择40对引物对8个李品种进行多态性分析。【结果】在‘芙蓉李’转录组中,共检测到7 601个SSR位点,分布于5 434条Unigene,SSR位点出现频率为54.51%(SSR位点数7 601与大于1 kb的Unigene数13 944的比值)。其中,单核苷酸重复是主要类型,占总SSR的42.19%。其次是二、三核苷酸重复,分别占总SSR的37.72%和18.48%。A/T和AG/CT是单核苷酸和二核苷酸的优势重复基元。对7 601个SSR位点设计出4 235对SSR引物。随机选择40对引物进行PCR扩增,其中17对在8个李品种中表现出多态性。【结论】李转录组数据是开发SSR标记的有效来源,开发的SSR标记可为李种质资源遗传多样性分析等提供了丰富的候选标记。

【Objective】The objective of this study was to characterize plum SSRs derived from transcriptome of‘Furongli＇plum and to develop SSR markers for the studies of plum genetic diversity and cultivar identification. SSR markers have many advantages and have been widely applied in fruit research. A shortage of plum SSR markers limits their application. A rapid and cost-effective approach to develop SSR markers for plum is required. SSRs can be divided into genomic SSRs and EST-SSRs. Genomic SSRs are costly to isolate and have uncertain linkage to the transcribed regions of the genome. EST-SSRs specifically target the transcribed regions of the genome and have increased potential for linkage to loci that contribute to agronomic phenotypes.【Methods】MISA software was employed to analyze the distribution frequency and the basic characteristics of repeat motifs of SSR loci from the transcriptome of‘Furongli＇plum fruit at different stages during ripening. Only unigenes longer than 1kb were subjected to SSR loci searching. SSR primers were designed using Primer 3. The major parameters for primer pair design were primer length of 18-27 bases, PCR product size of 100-280 bp, GC content of 40%-60%, and annealing temperatures of 57 ℃-63 ℃. Furthermore, 40 pairs of primers were synthesized for germplasm polymorphism detection in plum. Eight plum accessions, including‘Younai＇,‘Huanai＇,‘Heibaoshi＇,‘Wickson＇,‘Qiujili＇‘Zuili＇,‘Furongli＇and‘Sanhuali＇, were selected for polymorphism investigation with the SSRs. The PCR reaction cycling profile was 94 ℃ for 3 min followed by 35 cycles at 94 ℃ for 30 s, 55 ℃ for 30 s,72 ℃ for 30 s, and a final extension at 72 ℃ for 10 min. PCR amplifications were conducted in a final volume of 20 μL containing 30 ng template DNA, Dream taq green PCR master mix（Thermo scientific） and4 μmol · L- 1of each primer. The separation of alleles was performed on a 2% agarose gel. Gels were stained with ethidium bromide.【Results】A total of 7 601 SSR loci were found in the transcriptome of‘Furongli＇plum, 7 distributed in 5 434 unigenes with a frequency of 54.51%（the ratio of the total number of SSR loci to the number of unigenes longer than 1 kb）. 1 584 of the unigenes contained more than one SSR. The most abundant type of repeat motif was single nucleotide（42.19%）, followed by dinucleotide（37.72%）, trinucleotide（18.48%）, tetranucleotide（1.22%）, pentanucleotide（0.28%） and hexanucleotide（0.11%） repeat units. SSRs with 10-20 tandem repeats（30.35%） were most common, followed by nine tandem repeats（23.55%）, six tandem repeats（15.25%）, five tandem repeats（12.49%）, seven tandem repeats（8.46%）, eight tandem repeats（6.39%）, and 20 tandem repeats（3.51%）. The dominant repeat motif in SSRs was A/T（41.47%）, followed by AG/CT（31.55%）, AAG/CTT（5.67%）, AT/TA（3.95%）, and AGC/CTG（2.76%）. AG/CT（83.64%） was the most common dinucleotide repeat motif, followed by AT/AT（10.46%） and AC/GT（5.79%）. The dominant repeat motif in trinucleotide repeat SSR was AAG/CTT（30.68%）, followed by AGC/CTG（14.95%）, AGG/CCT（13.88%）, ATC/ATG（13.17%） and ACC/GGT（10.25%）. The dominant repeat motif in tetranucleotide repeat SSR was AAAG/CTTT（20.43%）, followed by AAAT/ATTT（18.28%） and AAAC/GTTT（10.75%）. 4 235 pairs of SSR primers were designed for7 601 SSR loci using Primer 3 and 40 pairs of primers were randomly selected to evaluate their application and the polymorphism across eight plum accessions. All of the primer pairs successfully amplified fragments. 17 of the 40 microsatellite loci showed allelic polymorphism.【Conclusion】Based on the unigene sequences generated from transcriptome sequencing, 7 061 SSR locus were identified and 4 235 microsatellite markers were developed. 40 primer pairs were randomly selected to detect polymorphism among eight plum accessions, and 17 of these primer pairs showed polymorphim. This study demonstrates that unigene sequences generated from transcriptome sequencing in‘Furongli＇plum is an effective source to develop SSR markers. The SSR markers identified and developed from transcriptome of‘Furongli＇plum will provide more candidate markers for marker-assisted selection and genetic polymorphism analysis for Prunus salicina Lindl..