Endogenous, tissue-specific short-interfering RNAs silence the chalcone synthase gene family in Glycine max seed coats

We present results from deep sequencing of small RNA populations from several genotypes of soybean and demonstrate that the CHS siRNAs accumulated only in the seed coats of the yellow varieties having either the dominant I or i-i alleles and not in the pigmented seed coats with homozygous recessive i genotypes. However, the diagnostic CHS siRNAs did not accumulate in the cotyledons of genotypes with the dominant I or i-i alleles thus demonstrating the novelty of an endogenous inverted repeat region of CHS genes driving RNA silencing in trans of non-linked CHS family members in a tissue-specific manner. The phenomenon results in inhibition of a metabolic pathway by siRNAs in one tissue allowing expression of the flavonoid pathway and synthesis of secondary metabolites in other organs as the chalcone synthase small RNAs are found in the seed coats of yellow seeded soybean varieties but not in the cotyledons of the same genotype. Overall design: In order to compare the population of chalcone synthase related small RNAs, we sequenced 3 to 6 million small RNAs using the Illumina Genome Analyzer from the following four soybean cultivars and tissues with specific genotypes at the I locus: Richland immature seed coats (homozygous for the dominant I allele that specifies yellow seed coat); Williams immature seed coats (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum) Williams (i-i/i-i yellow) immature cotyledons (homozygous for the dominant i-i allele that specifies yellow seed coat with pigmented hilum); Williams 55 immature seed coats (a Williams isogenic line homozygous for the recessive i allele that specifics pigmented seed coats. All seed coats and cotyledons were dissected from green stage immature seeds within the fresh weight range of 50-75 mg.

本研究报道了多个基因型大豆的小RNA（small RNA）群体的深度测序结果，证实查尔酮合酶小干扰RNA（CHS siRNAs）仅在携带显性I或i-i等位基因的黄色种皮大豆品种中积累，而在纯合隐性i基因型的有色种皮大豆样本中无积累。然而，携带显性I或i-i等位基因的基因型的子叶（cotyledon）中并未检测到特征性CHS小干扰RNA的积累，这表明查尔酮合酶基因的内源反向重复区域（inverted repeat region）可通过组织特异性方式，介导非连锁查尔酮合酶家族成员的反式RNA沉默，该现象具有新颖性。该现象体现为：某一组织中的小干扰RNA可抑制一条代谢通路，而在其他器官中则允许类黄酮代谢通路（flavonoid pathway）的表达与次级代谢产物（secondary metabolites）的合成——这是因为查尔酮合酶小RNA仅存在于黄色种皮大豆品种的种皮中，而非该基因型的子叶中。整体实验设计：为比较查尔酮合酶相关小RNA的群体差异，本研究利用Illumina基因组分析仪（Illumina Genome Analyzer）对以下4个大豆品种、不同组织及I位点具有特定基因型的样本进行了3~6百万条小RNA测序：1. Richland未成熟种皮（携带显性I等位基因的纯合基因型，对应黄色种皮）；2. Williams未成熟种皮（携带显性i-i等位基因的纯合基因型，对应带种脐着色的黄色种皮）；3. Williams (i-i/i-i黄色)未成熟子叶（携带显性i-i等位基因的纯合基因型，对应带种脐着色的黄色种皮）；4. Williams 55未成熟种皮（为Williams的近等基因系（isogenic line），携带隐性i等位基因的纯合基因型，对应有色种皮）。所有种皮与子叶均取自鲜重50~75 mg的绿期未成熟种子。