基于SLAF-seq技术的石斛高密度遗传图谱构建及生物碱含量QTL定位

Dendrobium species，perennial herbs of the orchid family, are well-known orchids with highly ornamental and medicinal values. Alkaloid is one of mainly active ingredients in Dendrobium species, and it has the functions of promoting digestion, antioxidant and anticancer activities. The content of alkaloid in D. nobile is higher than other Dendrobium species, thus D. nobile is the mainly extracting materials for alkaloid, but these medicinal materials mainly depend on wild resources. Currently there is no information available concerning breeding for high alkaloid content. Moreover, Dendrobium species grow slowly，it needs a long period for new varieties by conventional cross breeding, thus it will be meaningful for molecular assisted breeding to study QTL mapping for alkaloid content based on genetic maps. In this study, we plan to construct the high-density genetic maps of D.nobile and D. wardianum using SNP markers developed by SLAF-seq (specific length amplified fragment sequencing), based on an F1 family produced by the controlled interspecific hybridization between one female D.nobile and one male D. wardianum. Then alkaloid content of each plant is evaluated for the stems at two year growth phase, which is used to do QTL analysis. This study can serve for finding molecular markers which link to alkaloid content, and provide scientific basis for germplasm improvement and molecular breeding with marker-assisted selection of Dendrobium species.

石斛为兰科的多年生草本植物，是一类重要的观赏兼药用花卉。生物碱是其主要药用活性成分之一，具有抗氧化、抗癌、健胃等作用。金钗石斛中生物碱含量远较其它石斛高，是生物碱的主要提取原料，但原料过度依赖于野生资源，以生物碱含量为育种目标的研究还未见报道。同时，石斛生长缓慢，传统杂交育种选育高生物碱含量品种的周期较长，因而基于遗传连锁图谱开展生物碱含量的QTL定位对其分子辅助育种具有重要意义。本课题拟以生物碱含量差异大的金钗石斛（母本）和大苞鞘石斛（父本）F1杂交群体为材料，利用SLAF-seq技术开发大量的石斛SNP标记，以此构建高密度的石斛遗传连锁图谱，并对石斛茎的生物碱含量进行QTL定位，从而为寻找与石斛生物碱含量紧密连锁的分子标记奠定基础，为石斛种质改良和分子辅助育种提供理论依据。

石斛为兰科的多年生草本植物，是一类重要的观赏兼药用花卉。同时，石斛生长缓慢，传统杂交育种选育高生物碱含量或高观赏价值品种的周期较长，因而基于遗传连锁图谱开展相关性状的QTL定位对其分子辅助育种具有重要意义。本研究以生物碱含量差异大的金钗石斛（母本）和大苞鞘石斛（父本）F1杂交群体为材料，利用RNA-seq测序技术对两亲本和100个子代进行RNA测序，共获得522.43Gb Clean Data，组装共得到177052条Transcript和66153条Unigene，Transcript与Unigene的N50 分别为1777和1271，组装完整性较高。共开发获得362,413多态性位点，包含331642个SNP和 30771 个InDel，最终构建了基于SNP标记的高密度遗传图谱，该图谱包含9645个SNP标记，19个连锁群，总图距为3612.12cM，相邻标记平均距离为0.41 cM。平均每个连锁群包含508个SNP标记，连锁群的长度则从110.77 cM (LG16) 到 285.61 cM (LG19)不等。. 对叶长、叶宽、茎长、茎粗等农艺性状进行了QTL分析。叶长定位在13号连锁群16.032cM到16.251cM的区间，该区间包含7个unigene和19个SNP标记。叶宽定位到13号连锁群16.251cM 到97.059cM的区间，该区间包含4个unigene和15个SNP标记。茎长定位到3号连锁群137.527cM的位置和19号连锁群49.37cM 的位置，这2个位置包含23个unigene和3个SNP标记。茎粗定位到13号连锁群96.951cM 到97.059cM的区间，该区间包含3个unigene和4个SNP标记。. 该遗传图谱是石斛属内的第一张高密度的遗传图谱，这为后期石斛分子辅助育种和相关研究奠定可基础。