基于集群基因组扫描发掘甘蔗抗褐条病性状关联标记与基因

Sugarcane brown streak is a strong epidemic fungal disease, which is threatening to sugarcane production safety. The resistance of sugarcane genotype is the key factor, which determines the outbreak and epidemic of the disease in suitable climate. The breeding population of sugarcane is huge due to the probability of seedlings becoming a released variety only about 1/100,000-300,000. Thus, The core to improve breeding efficiency is development of accuracy and efficient evaluation techniques. For sugarcane, unfortunately, it is very limited. In this project, two main cultivars ROC22 (resistance, R) and Yuetang93-159 (susceptible, S) are used for parents to make reciprocal cross in order to produce two segregated populations with different disease resistance. After finishing the evaluation of phenotypic disease resistance in two plant cane crops and two ratoon crops, one population among two will be selected for construction of mixed pools from extreme opposite R or S individuals according to their quality and amount. Two parents and two mixed pools will be deeply sequenced by SLAF-seq, for parents and pools are 20X and 50X to 75X respectively depended on the number of individuals in the pools. Then, the analysis on the target trait i.e. phenotype of disease resistance and markers will be conducted to obtain the resistance related markers and verification of these markers in extensive genotypes by progressively including the pools, two population and other sugarcane genotypes. The 30 individuals with extreme R or S from the same population are selected for artificial inoculation in greenhouse and sampling for bulked segregant transcriptome analysis together with two parents at the determinate time point based on the preliminary experiments. The different expressed genes obtained based on the analysis of the transcriptome data will be verified by RT-PCR, and then the related genes will be obtained. In addition, PCR markers can be developed based on the related genes and used for verification in pools, two populations and in more different sugarcane genotypes, which will result in obtaining the stable related markers. Besides, the resistance related genes shared by both the related markers (derived from SLAF-seq) and the related genes (derived from RNA-seq) by comparing of the two sets of data. This project is hopefully to provide the key technology for sugarcane breeding resistant to brown stripe disease and provides the new idea and new technology for related research of mining the related genes/markers, and for multiploid and highly heterozygous sugarcane which has a large genome and in the absent of a genome sequence. Thus, this project has important application prospects and theoretical significance.

褐条病为强流行性真菌病害，适宜气候下易大面积发生，爆发流行的关键是品种抗性。甘蔗育种群体巨大，育成品种概率极低（1/10-30万），提高育种效率的核心是开发准确高效的鉴定技术。以主栽品种ROC22（抗）和粤糖93-159（感）正反交，创制2个分离群体。进行两新两宿抗病性鉴定（已完成一新一宿），选择1个群体构极端抗、感个体混合池，确保个体质量与数量。对亲本和混合池进行深度SLAF-seq，再行目标性状与标记关联分析，筛选抗性关联标记并扩大验证。同一群体选极端个体各30个，温室人工接种，按预实验确定时间点取样构池，对亲本和混池行集群分离转录组分析，差异表达基因经验证后获抗性关联基因，开发标记扩大验证。把关联标记与关联基因比对，筛选共有关联基因。项目有望为甘蔗抗褐条病育种提供核心技术，也为无参、大基因组、多倍体、高度杂合甘蔗和相关研究提供新思路和技术方法。项目有重要应用前景和理论意义。

1、项目背景.项目针对甘蔗育种技术落后，育种目标性状鉴定依赖表型，选择准确性差，从而不得不依赖超大群体，我国年种植实生苗100万株，第一代要淘汰95-97%，收获物为蔗茎，属营养体，环境互作效应大，育种效率低，费用高，且抗病性难以保证，急需开发关联标记。.2、主要研究内容.①创制甘蔗标记分析群体；.②杂种鉴定引物筛选与应用；.③实施了5个作物季褐条病抗性鉴定与重要农艺性状调查；.④SLAF-seq、BSA-seq与SNP筛选与验证；.⑤基于转录组测序和割手密基因组挖掘抗病相关基因并进行功能分析。.⑥甘蔗SNP芯片研发与有效性测试。.3、重要结果、关键数据及其科学意义.①粤糖 93-159（感）✕新台糖22（抗）杂交，创制了适合用于标记分析的永恒群体。.②杂种鉴定引物的筛选与分离群体鉴定。筛选出引物SEP23，SEP59，SMC119，SMC286，SMC31CUQ和SMC569，可扩增出亲本特征带，获324个真杂种。.③标记群体对褐条病抗性鉴定在适宜发病的云南陇川实施，共5个作物季。经三次多项式拟合，结合散点图，判断该群体的抗病性呈正态分布，筛选出抗病综合性状好的株系6个。 .④SLAF和BSA-seq与关联SNP位点的筛选验证。对89个抗感个体SLAF-seq得到283个SNP，其中2个有苗头但未通过验证；对优化的抗池R1和感池S1（35个）和重构极端抗、感池R2和S2（均13个）并加深BSA-seq，对精选出的8个SNP进行CAPS/dCAPS验证，多数难以扩增出来，仅1个位点能得到勉强的验证。.⑤基于转录组测序和割手密基因组分析，挖掘抗病相关ScMED7、ScPR10、NLR和LRR家族基因并进行功能分析。.⑥甘蔗SNP芯片研发与有效性测试。合作研发了甘蔗100K SNP芯片，并证明了其有效性，将应用于褐条病抗病性关联标记筛选。.3个博士研究生和2个硕士生参与，已毕业1位博士和1位硕士。.基于申报时可供选择的技术和甘蔗基因组破解，设计了SLAF-seq与标记群体结合的技术路径，试图筛选与抗病性关联的标记，但实施结果证明该策略和技术路径对甘蔗不适用，具有一定的科学意义。研发了高质量的甘蔗100K SNP芯片，作为甘蔗基因型的分型工具，具有好的应用前景。