目的 基于轉(zhuǎn)錄組測(cè)序數(shù)據(jù)鑒別和分析多花黃精EST-SSR位點(diǎn)，開(kāi)發(fā)可用于多花黃精種質(zhì)資源評(píng)價(jià)與利用的SSR標(biāo)記。方法 利用MISA工具從多花黃精轉(zhuǎn)錄組126 544條Unigenes中鑒定SSR位點(diǎn)并進(jìn)行分析；利用Primer 3.0設(shè)計(jì)SSR引物，隨機(jī)選擇50對(duì)SSR引物進(jìn)行有效性驗(yàn)證。結(jié)果 從9982條Unigenes中鑒定出12 317個(gè)包含2～6個(gè)核苷酸重復(fù)類型的SSR位點(diǎn)，SSR的分布頻率為9.73%，發(fā)生頻率為1/5.91 kb；SSR位點(diǎn)中的主導(dǎo)類型是二核苷酸重復(fù)，占總SSR的53.14%，其次是三核苷酸重復(fù)，占33.31%。SSR引物的有效性篩選顯示，50個(gè)SSR引物對(duì)中有29個(gè)（58%）可從多花黃精中擴(kuò)增出預(yù)期大小的SSR條帶。SSR熒光標(biāo)記的毛細(xì)管電泳檢測(cè)顯示在多花黃精的7個(gè)SSR位點(diǎn)共鑒定出了9種基因型，進(jìn)一步驗(yàn)證了SSR引物的有效性。結(jié)論 多花黃精轉(zhuǎn)錄組SSR位點(diǎn)出現(xiàn)頻率高，重復(fù)類型豐富，多態(tài)性較高，這將為多花黃精遺傳多樣性分析和遺傳圖譜構(gòu)建提供大量有價(jià)值的候選標(biāo)記，也可為黃精屬內(nèi)種間物種的分子鑒別、多花黃精優(yōu)良品種的分子輔助育種提供技術(shù)手段。

Objective EST-SSR loci were identified and analyzed based on the transcriptome sequencing data in Polygonatum cyrtonema, in order to develop SSR markers suitable for evaluation and application of germplasm resources on P. cyrtonema. Methods SSR loci were identified and analyzed in all of 126 544 Unigenes by using MISA tool. SSR primers were designed by using Primer 3.0 software and 50 pairs of SSR primers were randomly selected for validation test. Results A total of 12 317 SSR loci, including the types of 2-6 nucleotide repeats with occurring frequency of 1/5.91 kb, were identified from 9 982 Unigenes in P. cyrtonema transcriptome. The distribution frequency of SSRs was 9.73%. Dinucleotide repeat was the main type, accounting for as much as 53.14% of all SSRs, followed by trinucleotide repeat (33.31%). The validation test on 50 pairs of SSR primers showed that 29 of them (58%) generated fragments with expected molecular size from P. cyrtonema. The capillary electrophoresis using fluorescence-labeled SSR markers showed that nine genotypes were identified at seven SSR loci in P. cyrtonema, which further demonstrated the validity of these SSR primers. Conclusion There are numerous SSRs in P. cyrtonema transcriptome with high frequency, rich repeat types and relatively high polymorphic, which will provide abundant valuable candidate markers for genetic diversity analysis and genetic mapping construction in P. cyrtonema, also can be used as a technical tool for molecular identification among Polygonatum species and for molecular marker assistant breeding in superior cultivars of P. cyrtonema.

浙江省中央財(cái)政林業(yè)科技推廣項(xiàng)目（2017-TS04）；浙江省衢州市科技攻關(guān)項(xiàng)目（2018-K45）