橡胶树愈伤乳管与树皮乳管的形态学和转录组学比较研究及胶乳产量早期预测

Rubber tree breeding cycle is more than 40 years, and it usually takes 2 generations to breed one variety. Early yield prediction may shorten breeding cycle significantly. Laticifer cells are the only place where natural rubber is synthesized and stored, and are the key factor that determine latex yield.The once adopted early prediction methods for rubber yield were based on the number of primary laticifer cells in juvenile trees. However, because the primary laticifer cell number in juvenile trees is not positively correlated with secondary laticifer cell number in mature trees, the once adopted methods were not accurate and never succeeded in breeding any varieties and were given up. We recently found laticifer cells in rubber tree callus cultures, and laticifer cell number in callus was positively correlated with latex yield of rubber trees, and we presumed that callus may be served as a model to predict the yield of a genetic line. More over, all reported studies on laticifer differentiation have been performed using rubber tree bark as a model. However, the bark model has many disadvantages, including uncontrollable environmental factors, and long-waiting time for tree growth, difficult application of potential growth simulators, and potential damage to commercially viable trees. This program will use comparative cytology, transcriptomic method to study laticifer differentiation, aiming at developing an early yield prediction method and a novel research model for laticifer differentiation using callus culture as a study model.

橡胶树常规育种周期40年以上，通常要两代人才能将一个新品种培育成功。产量早期预测能显著缩短育种年限。乳管是天然橡胶合成和贮存的场所，成龄树次生乳管数量是决定橡胶产量的最重要因素之一。过去曾经采用过的早期测产主要基于幼树初生乳管产胶能力，由于次生乳管和初生乳管产胶能力没有明显相关关系，曾经采用过的方法都存在"不准"的缺点。我们发现橡胶树愈伤组织中存在乳管细胞，而且乳管细胞数量与母树胶乳产量正相关。本项目拟系统研究橡胶树愈伤乳管与树皮乳管的细胞学、转录组学相似性和差异，以及产量水平不同的品种之间、杂交后代群体株系之间愈伤乳管数与胶乳产量的相关关系，揭示愈伤乳管与树皮次生乳管和胶乳产量之间的内在联系，建立基于愈伤组织的准确、高效的早期产量预测方法，缩短橡胶树育种年限；同时建立以愈伤组织为基础的新型、高效乳管分化发育研究模型，克服目前以田间树皮为模型研究乳管分化存在的环境因素难以控制等诸多缺点。

橡胶树是多年生经济作物，育种周期长达30年。由于幼树中缺乏与成年树产量相关性状，导致早期选择效率低下。本项目通过成年橡胶树次生乳管、初生乳管以及幼嫩枝条愈伤组织乳管的比较解剖学研究，发现愈伤乳管在形态上和表达谱上与成年树次生乳管相似。用传统的石蜡切片观察，初生乳管与次生乳管的差异不大。我们研究出了将乳管细胞从组织中分离出来的方法，对分离的乳管进行显微观察。结果表明，次生乳管是具有光滑侧壁的有节乳管，初生乳管是不分枝的无节乳管，侧壁由于侵入性生长形成串珠状结构，发育成熟后外表具有瘤状凸起。愈伤乳管形态上与次生乳管一样，侧壁光滑有横壁。超微结构研究结果表明，愈伤乳管细胞壁的相对厚度与次生乳管相似，是初生乳管的1.5倍左右。比较转录组学研究结果表明，与细胞壁合成和修饰相关的基因在初生乳管中高效表达，揭示初生乳管瘤状突起产生的原因；与抗生物胁迫相关和橡胶合成相关的基因在初生乳管上调表达，而与抗非生物胁迫相关的基因在次生乳管中上调表达，说明次生乳管的主要生物功能是抗非生物胁迫，而初生乳管的主要功能是抗生物胁迫。这与幼嫩组织容易受病虫侵害有关。愈伤乳管的表达谱与次生乳管表达谱的相关系数达到0.99，而与初生乳管的相关系数仅0.9。进一步从分子水平验证了愈伤乳管与次生乳管更相似。理论上可以用于橡胶产量的早期预测。选择11个具有不同产量的橡胶树品种，从其幼嫩枝条诱导愈伤组织，分析愈伤中乳管细胞频率与树皮乳管及干胶产量的相关关系。结果表明，愈伤乳管频率以及树皮乳管的列数呈高度基因依赖，都与干胶产量极显著正相关，相关系数分别为0.846和0.899（P< 0.002)。因此认为愈伤组织乳管可以作为一种细胞标记用于橡胶树杂交后代产量的早期预测，缩短育种年限。2018年12月，在马来西亚的一个学术研讨会会上，项目负责人在国际上首次提出了细胞标记辅助育种的概念。