甘蔗近缘属多倍体植物斑茅染色体的分选与测序研究

Genomic assembling is a tight bottleneck, after the whole genome sequencing has made in the allopolyploid plant, especially in sugarcane. The relevant researches are inevitably influenced by it. Erianthus arundinaceus, a close relative genus to Saccharum, belongs to a polyploid plant and had been used in the sugarcane breeding program. The information of its genome sequencing will has a profound significance for relevant researches of sugarcane. On the basis of our previous research, this study is intended to further perform the backcrossing between the progeny of Erianthus arundinaceus and sugarcane for creating and selecting the population with only one or two Erianthus arundinaceus chromosomes. Using specific repeat sequences of Erianthus arundinaceus, the chromosomal karyotype of Erianthus arundinaceus from the different clones could be identified. The metaphase cells are abundantly accumulated through the root tip cell cycle synchronization in candidate materials. The fluorescent chromosome suspension could be successfully prepared by using Erianthus arundinaceus genomic DNA as probe to conduct GISH in the chromosomes suspension. Using flow cytometry, Erianthus arundinaceus chromosome could be sorted and enriched. Then, the third generation sequencing technology is used for sequencing, assembling, bioinformatic analysis, and mining the stress-tolerance genes. Ultimately, a new system will be established by a combination of the Erianthus arundinaceus chromosome sorting and sequencing technology. The study not only will lay the foundation for precise sequencing, precise assembling and analyzing for the whole genome of Erianthus arundinaceus, but also is of high significance for sugarcane genomic research and devising strategies of sugarcane molecular breeding.

测序后组装是甘蔗等异源多倍体植物全基因组研究的瓶颈，影响相关研究开展。斑茅是最受关注的、得到杂交利用的甘蔗近缘属多倍体植物，其基因组测序对甘蔗相关研究具有重要意义。在本课题组前期研究基础上，本研究拟通过对甘蔗与斑茅杂交后代的高代材料进一步回交，创制、筛选出仅含1～2条斑茅染色体的甘蔗材料群体；再用斑茅特异重复序列探针对斑茅染色体进行FISH核型鉴定，确定各材料染色体核型；含1～2条斑茅染色体的甘蔗材料经根尖细胞周期同步化后，富集中期相细胞，制成染色体悬浮液；以斑茅基因组为探针，在染色体悬浮液中进行GISH，制成荧光染色体悬浮液，再利用流式细胞术实现斑茅染色体分选、富集；尔后利用三代基因组测序技术进行测序、组装以及生物信息学分析，挖掘抗逆基因，建立斑茅染色体分选和测序组装技术体系。本研究将为斑茅全基因组精细测序、准确组装和分析奠定基础，对甘蔗基因组学研究和分子育种策略制定具有重要意义。

测序后组装是甘蔗等异源多倍体植物全基因组研究的瓶颈，影响相关研究开展。斑茅是最受关注的、得到杂交利用的甘蔗近缘属多倍体植物，其基因组测序对甘蔗相关研究具有重要意义。本研究通过对甘蔗与斑茅杂交后代的高代材料进一步回交，创制、筛选出仅含1～2条斑茅染色体的甘蔗材料群体，然后对斑茅染色体进行FISH核型鉴定，确定各材料染色体核型；通过细胞周期同步化富集中期相细胞，制成染色体悬浮液；以斑茅基因组为探针，在染色体悬浮液中进行GISH，制成荧光染色体悬浮液，再利用流式细胞术实现斑茅染色体分选、富集；尔后利用三代基因组测序技术进行测序、组装以及生物信息学分析，建立斑茅染色体分选和测序组装技术体系。.通过四年的研究，课题组创制了14个单条斑茅染色体无性系并进行了FISH核型鉴定；建立了甘蔗根尖细胞周期同步技术体系和斑茅染色体分选技术体系；从两个单条斑茅染色体材料中分选出了斑茅染色体；这2条染色体经二代测序比对后都为斑茅1号染色体，并对1号斑茅染色体进行三代基因组测序、组装并进行生物信息学分析；为识别不同号染色体，进行了染色体识别研究，利用Oligo-FISH识别染色体获重要进展，开发了能区分甘蔗属复合体不同号染色体的Oligo标记，研究了甘蔗属复合体的染色体构成与进化，研究了甘蔗与斑茅杂交后代F1、BC1、BC4、BC5的染色体传递及机制，并发现了后代出现“单条染色体加倍”的现象；发表研究论文11篇，其中SCI收录论文9篇，其中TOP期刊1篇，2区期刊3篇；获国家授权发明专利2件，申报1件专利获实审；培养博士3名毕业，硕士毕业10名，在读博士1名。本研究将为斑茅全基因组精细测序、准确组装和分析奠定基础，为后续甘蔗基因组学研究，甘蔗重要性状分子育种策略制定具有重要意义。