青藏高原一年生野生大麦耐酸(铝)胁迫生理与分子机理的研究

Tibetan wild annual barley is rich in genetic diversity and provides a treasure trove of useful genes for crop improvement. Novel acid/Al toxicity tolerance wild barley germplasms were identified in our previous study. In this study a series of experiments will be conducted with the main objectives as follows: (1) to elucidate physiochemical mechanism of acid/Al tolerance in Tibetan wild barleys XZ16, XZ166 (high acid/Al tolerant) using XZ61 (acid/Al sensitive) and a Al-resistant-cultivar Dayton as control. (2) to map the novel acid/Al tolerance gene(s) using SSR, SNP and DArT markers in the DH population derived by F1 hybrids of genotypes differing in Al tolerance (XZ16, Al-tolerant wild genotype; Buloke, Al-sensitive cultivar). Meanwhile, the molecualr markers, and the WGS polymorphisms obtained via conduction of the whole genome shotgun (WGS) sequencing of the two parental varieties will be aligned to the barley MTP-BAC sequence in the target genome region for acid/Al tolerance. (3) to identify stress-specific proteins and candidate genes for acid/Al tolerance in the wild barley using two-dimensional gel electrophoresis coupled with mass spectrometry and cDNA microarrays by comparative analyzing the proteomics and transcriptional response in barley genotypes differing in acid/Al tolerance. (4) to identify and clone the functional gene(s) controlling acid/Al tolerance in the wild barley. Furthermore, near-isogenic lines with different Al tolerance are selected for whole genome transcriptomics analysis. Differentiated expression genes will be identified and mapped back to the target gene region for identification of the candidate gene. Meanwhile, the transcriptomic data will be used to establish the gene network for acid/Al tolerance. We expect to develop diagnostic molecular markers for marker-assisted breeding and germplasm characterization, and to confirm the candidate genes through combination of BAC, RNA and protein profiles, microarray, and BSMV-VIGS and transformation results. These results will be valuable for theoretical and technological guidance to molecular breeding for acid/Al-tolerant barley variety.

青藏高原一年生野生大麦蕴藏着多种抗逆特异基因。本研究拟以已获得耐酸(铝)野生大麦种质为材料，探讨野生大麦耐酸(铝)生理机理；以耐酸(铝)野生大麦基因型与酸(铝)敏感栽培品种杂交后代构建的DH群体为材料，构建SNP、SSR、DArT高密度图谱和定位耐酸(铝)相关基因(分子标记)，两亲本进行全基因组鸟枪法（WGS）测序，耐酸(铝)相关分子标记和WGS 多态性将锚定于大麦MTP-BACs的目标基因区域；利用比较蛋白/基因组学等技术,筛选耐酸(铝)特异蛋白和基因；应用生物信息学手段，通过耐酸(铝)差异显著的近等基因系(NIL)的全基因组表达谱分析,筛选最佳候选基因, 开发基因特异性分子标记用于进一步精细作图；综合分析生理、BAC、RNA谱、蛋白鉴定、基因芯片等结果，筛选分离克隆最佳候选基因，转基因功能验证；探明野生大麦耐酸(铝)生理与分子机制,促进我国大麦耐酸(铝)育种与生产的发展。

青藏高原一年生野生大麦为我国独有的珍贵野生资源，蕴藏着多种抗逆特异基因。本研究以酸(铝)野生大麦特异种质XZ16为材料，以酸(铝)胁迫敏感野生大麦XZ61和耐酸(铝)栽培大麦Dayton为对照，利用MIFE技术检测了三个基因型酸和铝胁迫下根尖伸长区和成熟区根际pH以及H+、K+、Ca2+离子流的变化，分析相关生理生化性状，揭示了耐/抗酸(铝)遗传差异的生理机制。利用XZ16×Buloke杂交后代构建的DH 群体，建立遗传连锁图谱，QTL分析检测到若干耐酸/铝相关QTLs； 利用基因芯片、全基因组重测序、高通量测序等技术，分析比较不同耐酸(铝)大麦基因型在正常环境和酸(铝)胁迫下基因、miRNA等的表达差异，筛选到耐酸(铝)候选基因；分离克隆耐酸铝相关候选基因HvHOX9、HvABCB25、HvAIR12和HvEXPA1，利用BSMV-VIGS、RNAi和同源过表达等技术，探讨了上述相关基因调控野生大麦耐铝性的机制。研究结果加深了青藏高原一年生野生大麦耐酸(铝)分子机制的认识，并为培育耐酸(铝)大麦品种提供了新的候选基因。