National Engineering Research Center for Sugarcane, Fujian Agriculture and Forestry University, Fuzhou 350002, China.
Guangdong Sugarcane Genetic Improvement Engineering Center, Institute of Nanfan and Seed Industry, Guangdong Academy of Sciences, Guangzhou 510316, China.
Int J Mol Sci. 2022 Aug 21;23(16):9435. doi: 10.3390/ijms23169435.
Erianthus arundinaceus is a valuable gene reservoir for sugarcane improvement. However, insufficient molecular markers for high-accuracy identification and tracking of the introgression status of E. arundinaceus chromatin impede sugarcane breeding. Fortunately, suppression subtractive hybridization (SSH) technology provides an excellent opportunity for the development of high-throughput E. arundinaceus-specific molecular markers at a reasonable cost. In this study, we constructed a SSH library of E. arundinaceus. In total, 288 clones of E. arundinaceus-specific repetitive sequences were screened out and their distribution patterns on chromosomes were characterized by fluorescence in situ hybridization (FISH). A subtelomeric repetitive sequence Ea086 and a diffusive repetitive sequence Ea009, plus 45S rDNA-bearing E. arundinaceus chromosome repetitive sequence EaITS were developed as E. arundinaceus-specific molecular markers, namely, Ea086-128, Ea009-257, and EaITS-278, covering all the E. arundinaceus chromosomes for high-accuracy identification of putative progeny. Both Ea086-128 and Ea009-257 were successfully applied to identify the authenticity of F1, BC1, BC2, BC3, and BC4 progeny between sugarcane and E. arundinaceus. In addition, EaITS-278 was a 45S rDNA-bearing E. arundinaceus chromosome-specific molecular marker for rapid tracking of the inherited status of this chromosome in a sugarcane background. Three BC3 progeny had apparently lost the 45S rDNA-bearing E. arundinaceus chromosome. We reported herein a highly effective and reliable SSH-based technology for discovery of high-throughput E. arundinaceus-specific sequences bearing high potential as molecular markers. Given its reliability and savings in time and efforts, the method is also suitable for development of species-specific molecular markers for other important wild relatives to accelerate introgression of wild relatives into sugarcane.
菰是甘蔗改良的宝贵基因库。然而，由于缺乏用于准确鉴定和跟踪菰染色质渗入状态的分子标记，甘蔗育种受到阻碍。幸运的是，抑制性差减杂交（SSH）技术为以合理的成本开发高通量菰特异性分子标记提供了极好的机会。在本研究中，我们构建了菰 SSH 文库。总共筛选出 288 个菰特异性重复序列克隆，并通过荧光原位杂交（FISH）对其在染色体上的分布模式进行了表征。一个端粒重复序列 Ea086 和一个弥散重复序列 Ea009，以及一个携带 45S rDNA 的菰重复序列 EaITS 被开发为菰特异性分子标记，即 Ea086-128、Ea009-257 和 EaITS-278，可用于对可能的后代进行高精度鉴定。Ea086-128 和 Ea009-257 均成功应用于鉴定甘蔗与菰之间的 F1、BC1、BC2、BC3 和 BC4 后代的真实性。此外，EaITS-278 是一个携带 45S rDNA 的菰染色体特异性分子标记，可用于快速跟踪该染色体在甘蔗背景中的遗传状态。三个 BC3 后代显然失去了携带 45S rDNA 的菰染色体。本研究报道了一种基于 SSH 的高效可靠技术，可用于发现具有高通量和高潜力的菰特异性序列，这些序列可作为分子标记。鉴于其可靠性和节省时间和精力，该方法也适用于开发其他重要野生近缘种的种特异性分子标记，以加速野生近缘种向甘蔗的渗入。
