草莓印记基因及其调控sRNAs筛选与功能验证

The ploidy levels of genus Fragaria are diverse, interspecific and interploidy hybridization barriers occur generally in the genus. Genomic imprinting is an inheritance process independent of the classical Mendelian inheritance. Sexual reproductive isolation in flowering plants is closely related to the genomic imprinting mechanism. This research will aim to identify the imprinted genes and their regulatory small RNA (sRNA) molecules in strawberry for providing a basis of epigenetics in interspecific and interploidy hybridization barriers of Fragaria spp. First, RNA-seq and sRNA-seq high-throughput sequencing are used to screen the candidate imprinting genes and regulatory sRNA in endosperm of reciprocal cross of two octoploid Fragaria ×ananassa genotypes, based on differently expressed level as the 2:1 ratio of maternal to paternal genomes, then some imprinting genes and regulatory sRNAs will be validated by allele-specific PCR. Second, the methylation states of imprinting genes and their flanking regions are analyzed by targeted regions methylation sequencing to verify how sRNAs and DNA methylation regulate the expression of imprinting genes in endosperm together. Third, the function of imprinting genes and sRNAs in overcoming the interspecific/interploidy hybridization barrier will be analyzed using the diploid Fragaria viridis knock-out mutants, are created using CRISPR/Cas9 gene-editing technology, reciprocally hybriding with Fragaria ×ananassa, then interaction models of some imprinting genes and sRNAs in cross barrier will be constructed.

草莓属植物倍性水平多样，存在种间和倍性杂交障碍。植物的生殖隔离与基因组表观印记机制密切相关，鉴定草莓的印记基因及其调控sRNA分子，可为草莓种间杂交及倍性杂交障碍机制提供表观遗传学基础。以栽培草莓两个基因型为试材进行正反杂交，对胚乳RNA-seq与sRNA-seq进行高通量测序，按照明显偏离2M:1P的标准，筛选出候选印记基因及其调控sRNAs，利用等位基因特异PCR对印记基因与sRNAs进行验证，鉴定出印记基因及其调控的sRNAs，再利用目标区域捕获甲基化测序对印记基因及其侧翼区域的DNA甲基化状态进行分析，明确sRNAs、DNA甲基化共同调控胚乳印记基因表达的作用机制；利用CRISPR/Cas9构建2x绿色草莓部分印记基因及sRNAs突变体，通过与8x栽培草莓种间倍性杂交，确定部分印记基因和sRNAs在杂交障碍中的功能并构建其调控机制模型。

草莓胚和胚乳的发育过程通过瘦果直接影响着果实的大小和品质，对草莓胚和胚乳基因组印记以及相关的表观遗传学研究还未见报道。通过对栽培草莓（Fragaria ×ananassa Duch.）‘红颊’与‘甜查理’正反交授粉后12天的胚和胚乳进行转录组测序以及亲本的重测序，在胚中鉴定了296个印记基因和21个印记lncRNA，在胚乳中鉴定了620个印记基因和29个印记lncRNA；这些印记基因具有一定的保守性，大多数涉及与籽粒发育相关的叶酸碳池与碳代谢等信号途径；根据印记基因的保守性，从森林草莓（F. vesca）鉴定出5个印记基因，这些印记基因大部分在胚乳中高表达，仅有FvDRIP2在胚中表达量较高；对上述胚和胚乳组织进行了sRNA测序和全基因组DNA甲基化测序，表明胚乳中24-nt sRNA的丰度远低于胚，胚乳的DNA甲基化水平明显低于胚，进一步分析发现胚乳印记基因区域的CHH甲基化密度明显低于非印记基因区域，推断CHH甲基化在胚乳的印记表达机制中发挥着重要作用；对部分印记基因的功能验证表明，FaTRG-31参与了胚乳发育且在非生物胁迫中发挥作用，过表达FaAIRP2促进了种子的萌发；对胚和胚乳中的miRNA、PHAS loci和siRNA进行了全面分析，大部分的miRNA-PHAS loci对与生长素信号传导途径关联，说明在果实的早期发育中生长素的生物发生在胚和胚乳中发挥了重要作用，验证了fan-miR159n和fan-miR159r与FaARF23的靶向关系并研究了其在栽培草莓果实发育中的作用；另外，分别对二倍体森林草莓和八倍体栽培草莓sRNA路径AGO、DCL和RDR等3个基因家族进行了全基因组鉴定、基因特征、进化关系、组织表达模式以及植物激素响应分析，为进一步了解这些基因在草莓生长发育中发挥的功能以及siRNA介导的DNA甲基化（RdDM）调控印记基因表达机制提供了新线索。研究结果在理论上丰富了栽培草莓果实和种子（瘦果）发育的调控路径，为栽培草莓全基因组分子育种提供了基因资源和理论支撑。