【作者】 张刚；

【导师】 朱林；

【作者基本信息】 宁夏大学 ， 生态学， 2025， 硕士

【摘要】 稗属牧草（Echinochloa）是禾本科（Poaceae）具有重要饲用价值的一年生或多年生草本植物,以其突出的抗旱性、耐盐碱性、耐刈割性和营养丰富等特点,成为盐碱地改良利用的优质草种资源。作为植物从营养生长向生殖生长转变的关键发育阶段,抽穗期直接影响牧草产量、饲用品质和生态适应性,是品种选育的核心性状指标。然而,目前关于稗属抽穗期分子调控机制的研究仍较为缺乏,严重制约了优良品种的选育进程。本研究以湖南稷子（Echinochloa frumentacea）和宁夏无芒稗（Echinochloa crusgalli）构建的杂交后代F_2:3和F_2:4群体为研究对象,通过统计学、BSA-Seq、RNA-Seq等方法与技术系统开展了稗属牧草表型多样性分析、抽穗期关键调控基因挖掘及其遗传调控机制解析等研究。研究结果如下:（1）杂交子代群体的抽穗期和全生育期表现为负向中亲,且同时具有较高的遗传多样性（H’>1.70）和较大的变异幅度（CV>17.00%）,表明这两个性状可作为生育期遗传改良的重要选择指标。杂交提高了稗草的表型多样性,如株高性状表现出明显的超亲优势（OPH=2.70～6.68）,具有中等变异程度（CV=6.28%～11.88%）。叶片数性状呈现显著中亲优势（MPH=2.86～4.76）,变异程度较大（CV=24.15%～25.83%）。生育期（包括抽穗期和全生育期）与多个农艺性状呈显著正相关关系,其中与株高、茎粗、叶宽和叶片数等性状的相关性达到极显著水平（P<0.01）。回归分析进一步确定穗色、穗重、株高和叶宽是影响生育期的关键因子。基于28的遗传距离阈值进行聚类分析,将供试材料划分为5个生育期特征明显的类群。（2）通过全基因组测序（WGS）技术,共鉴定到11,347个SNP和1,992个Indel有效变异位点。基于BSA方法的Index算法定位到三个重要区间:qHD-16-1:193.14kb（2,296,912～2,490,050 bp）,包含17个基因;qHD-6-1:11.05 kb（16,802,203～16,813,256 bp）,包含2个基因;qHD-6-2:9.9 Mb（6,914,689～16,813,256bp）,包含944个基因。（3）基于RNA-Seq技术分析,以|log2FC|≥1且P_adjust<0.05为阈值,共鉴定到5,985个差异表达基因（DEGs）。KEGG富集分析显示这些DEGs显著富集于多个植物开花相关通路,包括脂肪酸代谢和类黄酮生物合成等。通过WGCNA和GSEA分析,进一步鉴定了43条核心代谢通路和33个关键枢纽基因。根据功能特征,这些通路可分为以下六大类:能量代谢、碳水化合物代谢、氨基酸代谢、核酸与蛋白质代谢、次级代谢物生物合成和信号转导与应答。（4）通过Venn分析整合BSA-Seq和RNA-Seq数据,筛选获得63个共有基因,其中包括qHD-16-1区间的Ec RCS3基因。进一步用随机森林（RF）和LASSO回归对qHD-6-2区间的62个基因进行机器学习筛选,两种方法共同鉴定出5个重叠基因（Ec IPI1、Ec LOC120683629、Ec ARF、Ec PIF6和Ec At4g33760）。经ANN和q RT-PCR验证后,最终确定这6个基因（Ec RCS3、Ec IPI1、Ec LOC120683629、Ec ARF、Ec PIF6和Ec At4g33760）为调控稗属牧草抽穗期的关键基因。基于Plant TFDB数据库对关键基因和枢纽基因进行靶基因预测,鉴定出425个共有转录因子。结合转录因子-靶基因互作关系及已有研究证据,聚焦到调控稗草抽穗期的3条分子途径,分别为光周期途径、赤霉素途径、逆境诱导途径。本研究初步鉴定了调控稗属牧草抽穗期关键基因6个,为创制不同生育期的稗草新种质提供了基因资源及分子靶点。聚焦到稗草抽穗期分子调控途径3条,深化了对调控稗属植物抽穗期分子机制的认识。研究结果对盐碱地生态修复的品种选育具有重要指导价值,为阐明调控稗草抽穗期分子机制提供了新见解。更多还原

【Abstract】 Echinochloa,a genus of grasses in the Poaceae family,comprises annual or perennial herbaceous plants with significant forage value.Its outstanding characteristics,such as drought resistance,salt-alkali tolerance,cutting resistance,and rich nutrition,make it a valuable grass species resource for the improvement and utilization of saline-alkali lands.The heading date period is a critical developmental stage for plants as they transition from vegetative growth to reproductive growth,directly affecting forage yield,quality,and ecological adaptability.It is a core trait indicator in variety breeding.However,research on the molecular regulatory mechanisms of heading date in Echinochloa is still relatively lacking,severely restricting the breeding progress of superior varieties.This study systematically investigated the phenotypic diversity,key regulatory genes and genetic regulatory mechanisms controlling heading date in Echinochloa using statistical analysis,BSA-Seq,RNA-Seq and other techniques.The research was conducted on F_2:3and F_2:4hybrid populations derived from crosses between Echinochloa frumentacea and Echinochloa crusgalli.The results of the study are as follows:（1）The heading date and entire growth period of the hybrid progeny exhibited negative heterosis（mid-parent heterosis）and simultaneously displayed high genetic diversity（H’>1.70）and large variation（CV>17.00%）,suggesting that these two traits can serve as important selection indicators for the genetic improvement of growth duration.Hybridization increased the phenotypic diversity of Echinochloa.For example,The plant height trait showed significant over-parental heterosis（OPH=2.70～6.68）with moderate variation（CV=6.28%～11.88%）.The leaf number trait displayed significant mid-parent heterosis（MPH=2.86～4.76）with a large degree of variation（CV=24.15%～25.83%）.The growth duration（including heading time and Maturity stage）was significantly positively correlated with multiple agronomic traits,with correlations to plant height,stem thickness,leaf width,and leaf number reaching a highly significant level（P<0.01）.Regression analysis further confirmed that spike color,spike weight,plant height,and leaf width are the key factors affecting growth duration.Based on a genetic distance threshold of 28,clustering analysis divided the tested materials into five groups with distinct growth duration characteristics,providing important classification references for subsequent breeding work.（2）Using whole genome sequencing（WGS）technology,a total of 11,347 SNPs and1,992 valid Indel variations were identified.BSA analysis（Index algorithm）localized three important intervals:q HD-16-1:193.14 kb（2,296,912～2,490,050 bp）,containing 17 genes;q HD-6-1:11.05 kb（16,802,203～16,813,256 bp）,containing 2 genes;and q HD-6-2:9.9 Mb（6,914,689～16,813,256 bp）,containing 944 genes.（3）Based on RNA-Seq technology analysis,with|log2FC|≥1 and P_adjust<0.05 as thresholds,a total of 5,985 DEGs were identified.KEGG enrichment analysis indicated that these DEGs were significantly enriched in multiple flowering-related pathways,including fatty acid metabolism and flavonoid biosynthesis.Through WGCNA and GSEA analyses,43 core metabolic pathways and 33 key hub genes were further identified.According to functional characteristics,these pathways can be classified into the following six categories:energy metabolism,carbohydrate metabolism,amino acid metabolism,nucleic acid and protein metabolism,secondary metabolite biosynthesis,and signal transduction and response.（4）Through Venn analysis,we integrated BSA-Seq and RNA-Seq data to screen and obtain 63 shared genes,among which was the Ec RCS3 gene located in the q HD-16-1interval.We further employed Random Forest（RF）and LASSO regression for machine learning-based selection of the 62 genes in the q HD-6-2 interval,and both methods jointly identified 5 overlapping genes（Ec IPI1,Ec LOC120683629,Ec ARF,Ec PIF6,and Ec At4g33760）.Following validation by ANN and q RT-PCR,ultimately,these 6 genes were determined to be key genes regulating heading date in Echinochloa.Based on the Plant TFDB database,we conducted target gene predictions for the key genes and hub genes,identifying 425 shared transcription factors.By combining the interactions between transcription factors and target genes with existing research evidence,we focused on three molecular pathways regulating the early and late heading dates of Echinochloa:the photoperiod pathway,the gibberellin pathway,and the stress-induced pathway.This study identified 6 key genes regulating the heading date of Echinochloa,providing molecular targets and genetic resources for the development of new germplasm with varying growth periods.By focusing on the three molecular pathways regulating heading date,we deepened our understanding of the molecular mechanisms underlying heading date regulation in Echinochloa.The results have significant guiding value for the breeding of varieties for ecological restoration in saline-alkali lands and provide new insights into the molecular mechanisms regulating heading date in Echinochloa.更多还原