Development and application of microsatellite markers within transcription factors in flare tree peony (Paeonia rockii) based on next-generation and single-molecule long-read RNA-seq

doi:10.1016/S2095-3119(20)63402-5

摘要/Abstract

摘要：

牡丹是起源于中国的具有观赏、药用和食用价值的珍贵作物，其中紫斑牡丹是芍药属中最具代表性的种质之一。EST-SSR标记的开发和应用对于遗传和育种应用具有重要的价值，但芍药属中的EST-SSR资源仍非常有限。在本研究中，我们首先报道了紫斑牡丹中基于下一代（NGS）和单分子长度长（SMLRS）转录组测序的转录因子内SSR标记的开发，从959个与产量相关的候选转录因子内鉴定了包含六种核苷酸重复类型的166个EST-SSR标记，平均每5.83个unigenes包含一个SSR，共有102（61.45 %）对引物在用于RNA-seq的两个品种中产生了扩增产物，其中来自于18个基因家族（AP2、bHLH、HSF等）的58（56.86 %）对引物在连锁作图群体的亲本和8个随机抽选的紫斑牡丹样本中均表现为多态性。通过在37个牡丹样本中的评估，58个 EST-SSR标记表现出了高水平的多态性，PIC值的范围为0.32 到0.91（平均值为0.70），转移性研究表明58对引物在芍药属的7个野生种中的扩增比率为89.66到100%。此外，在62个样本中开展了遗传关系的研究，聚类分析表明主要的聚类和已知的谱系树是基本一致的。综上所述，这些新开发的EST-SSR标记在牡丹种质的保护以及标记辅助选择育种中具有重要的潜在用途。

Abstract:

Tree peonies native to China are a precious crop with ornamental, medicinal and edible oil properties, of which flare tree peony (Paeonia rockii) is one of the most significant germplasms in Paeonia. The development and application of expressed sequence tag-simple sequence repeat (EST-SSR) markers are very valuable for genetic and breeding applications, but EST-SSR resources for the genus Paeonia are still limited. In this study, we first reported the development of SSRs within transcription factors (TFs) in P. rockii based on next-generation sequencing (NGS) and single-molecule long-read sequencing (SMLRS). A total of 166 EST-SSRs containing six nucleotide repeat types were identified from 959 candidate TFs associated with yield, with an average of one SSR per 5.83 unigenes. In total, 102 (61.45%) pairs of primers produced amplification products in the two RNA-seq cultivars. Among them, 58 (56.86%) pairs of primers from 18 gene families (AP2, bHLH, HSF, etc.) were identified to be polymorphic both in the parents of a linkage mapping population and in eight randomly selected accessions of P. rockii. Further, the 58 EST-SSRs indicated a high level of informativeness with PIC values ranging from 0.32 to 0.91 (mean 0.70) after assessment in 37 tree peony accessions. Transferability studies indicated that the amplification ratio of the 58 pairs of primers ranged from 89.66 to 100% across seven species of Paeonia. In addition, a genetic relationship study was performed in 62 accessions. Cluster analysis using the neighbour-joining (NJ) tree demonstrated that major clusters corresponded to the known pedigree trees. Taken together, these newly developed EST-SSRs have a potential use in the conservation of tree peony germplasm and marker-assisted selection (MAS) breeding.

Key words: flare tree peony (Paeonia rockii) , EST-SSR markers , polymorphism , genetic diversity , transferability , genetic relationships

. [J]. Journal of Integrative Agriculture, 2021, 20(7): 1832-1848.

LIU Na, CHENG Fang-yun, GUO Xin, ZHONG Yuan. Development and application of microsatellite markers within transcription factors in flare tree peony (Paeonia rockii) based on next-generation and single-molecule long-read RNA-seq[J]. Journal of Integrative Agriculture, 2021, 20(7): 1832-1848.

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