杉木与台湾杉EST-SSR标记的开发与应用

Development and Application of EST-SSR Markers for Cunninghamia lanceolata and Taiwania cryptomerioides

简单序列重复（Simple Sequence Repeats,SSR）标记是基于SSR中核苷酸单位串联重复次数不同而开发的一种分子标记技术,具有多态性丰富、呈共显性遗传、易于PCR检测,在基因组上分布均匀等特点（Powell et al.,1996）。直接利用现有的EST（Expressed Sequence Tag）序列开发SSR标记就是目前采用较多的一种策略。

Cunninghamia lanceolata is one of the most important fast-growing timber tree species in southern China. In order to develop an effective molecular maker,which is important for genetic diversity analysis and marker-assisted selection in C. lanceolata,all the EST sequences of C. lanceolata and Taiwania cryptomerioides were obtained from NCBI for the development of EST-SSR markers. As a result,311 non redundant EST sequences were assembled from 408 EST sequences of C. lanceolata. Among these sequences,a total of 28 SSRs were identified from 26 non redundant EST sequences,and the trinucleotide and tetranucleotide repeats were the dominant types with the frequency of 60. 71% and25%,respectively. A total of 384 non redundant EST sequences were found after assembly of 2 643 EST sequences of T.cryptomerioides and 32 SSRs were identified from 27 non redundant EST sequences. The trinucleotide and tetranucleotide repeats were also the dominant types with the frequency of 53. 13% and 25%,respectively. According to these sequences,50 pairs of EST-SSR primers were designed. Through PCR and sequencing,10 pairs of primers with polymorphism were isolated,among which,9 pairs of primers were from C. lanceolata and 1 pair of primers was from T. cryptomerioides. These polymorphic primers were further used to assess the genetic diversity of 30 different clones of C. lanceolata. The results showed that 30 clones were divided into 6 groups at coefficient rate 0. 80. All the results indicated that the developed SSR markers from EST sequences would have important application value in genetic analysis and molecular breeding of C.lanceolata.