In plants, 22-nucleotide small RNAs trigger the production of secondary small interfering RNAs (siRNAs) and enhance silencing. DICER-LIKE2 (DCL2)-dependent 22-nucleotide siRNAs are rare in Arabidopsis (Arabidopsis thaliana) and are thought to function mainly during viral infection; by contrast, these siRNAs are abundant in many crops such as soybean (Glycine max) and maize (Zea mays). Here, we studied soybean 22-nucleotide siRNAs by applying CRISPR-Cas9 to simultaneously knock out the two copies of soybean DCL2, GmDCL2a and GmDCL2b, in the Tianlong1 cultivar. Small RNA sequencing revealed that most 22-nucleotide siRNAs are derived from long inverted repeats (LIRs) and disappeared in the Gmdcl2a/2b double mutant. De novo assembly of a Tianlong1 reference genome and transcriptome profiling identified an intronic LIR formed by the chalcone synthase (CHS) genes CHS1 and CHS3. This LIR is the source of primary 22-nucleotide siRNAs that target other CHS genes and trigger the production of secondary 21-nucleotide siRNAs. Disruption of this process in Gmdcl2a/2b mutants substantially increased CHS mRNA levels in the seed coat, thus changing the coat color from yellow to brown. Our results demonstrated that endogenous LIR-derived transcripts in soybean are predominantly processed by GmDCL2 into 22-nucleotide siRNAs and uncovered a role for DCL2 in regulating natural traits.

在植物中，长度为22个核苷酸（nt）的小RNA（sRNA）具有触发次级小干扰RNA（siRNA）产生并进一步增强沉默的独特能力。尽管依赖DCL2的22-nt siRNA在拟南芥中很少见，并且被认为除了病毒感染期间几乎没有其他功能，但它们在许多主要农作物（如大豆和玉米）中含量很高。在这里，我们通过应用CRISPR-Cas9基因组编辑技术同时在天龙1号品种中敲除大豆DCL2的两个副本GmDCL2a和GmDCL2b，研究了Glycine max中内源性22-nt siRNA。sRNA测序表明，大多数22-nt siRNA均来自长反向重复序列（LIR），并在Gmdcl2a / 2b双突变体中消失。通过从头组装Tianlong1参考基因组和转录组分析，我们发现了由查尔酮合酶（CHS）基因CHS1和CHS3形成的内含子定位的LIR。该LIR是靶向其他CHS家族基因并触发次级21-nt siRNA产生的主要22-nt siRNA的来源。在Gmdcl2a / 2b中破坏此过程会大大增加种皮中CHS mRNA的水平，并将颜色从黄色变为棕色。我们的结果表明，大豆中的内源性LIR转录本主要由GmDCL2加工成22 nt siRNA，并且发现了DCL2在调节自然性状中被忽略的作用。在Gmdcl2a / 2b中破坏此过程会大大增加种皮中CHS mRNA的水平，并将颜色从黄色变为棕色。我们的结果表明，大豆中的内源性LIR转录本主要由GmDCL2加工成22 nt siRNA，并且发现了DCL2在调节自然性状中被忽略的作用。在Gmdcl2a / 2b中破坏此过程会大大增加种皮中CHS mRNA的水平，并将颜色从黄色变为棕色。我们的结果表明，大豆中的内源性LIR转录本主要由GmDCL2加工成22 nt siRNA，并且发现了DCL2在调节自然性状中被忽略的作用。