野生‘龙门香橙’的植物学特征观察及起源研究

【目的】广东省龙门县境内南昆山主峰天堂顶海拔1 000 m处有当地称之为‘龙门香橙’的野生柑橘，论文拟从形态学和DNA分子证据上探讨其与已知柑橘资源的异同，为合理利用该资源奠定基础。【方法】实地调查‘龙门香橙’的生长环境，观察记录叶、花、果实的形态特征。分析果实的可溶性固形物、糖、酸和抗坏血酸含量。在电子显微镜下观察花粉的显微特征。对比分析包括‘龙门香橙’在内的24个柑橘资源的AFLP图谱以及10个资源的8个SNP位点的基因型。克隆、测序分析‘龙门香橙’的β-胡萝卜素羟化酶（β-carotene hydroxylase，CHX）基因的2个等位基因，并与其他柑橘材料的CHX基因序列进行比对。【结果】生境调查结果表明‘龙门香橙’非人工种植，是真正的野生柑橘。其果实、种子的形状和单胚等特征与柚类相似，但果实与柚相比明显偏小、出汁率与可食率很低、味苦等性状提示其比较原始。叶片形状、线形翼叶、花丝联合等特征类似宽皮柑橘类，比较进化；但花丝和新叶染有紫红色等性状则比较原始。基于AFLP图谱的聚类分析显示其不与其他研究材料聚类，但介乎橘柚之间。8个SNP分子标记分析结果显示，‘龙门香橙’的3个SNP位点为宽皮柑橘纯合基因型，1个位点为橘柚杂合基因型，其余4个位点为柚子纯合基因型，这种基因型组合不同于其他任何所分析的材料。‘龙门香橙’的CHX基因的核苷酸序列显示其2个等位基因间相互差异较小，且含有其他已知柑橘CHX基因上未发现的SNP。CHX基因序列进化分析结果表明其与其他柑橘的遗传距离较远、差异似达种间水平。【结论】‘龙门香橙’是一个真正的野生柑橘资源。其形态性状兼具原始与进化特征。在DNA分子水平上，‘龙门香橙’与已知柑橘不同，甚为独特。

Morphological and Molecular Studies on a Wild Citrus ‘Longmen Xiangcheng’

【Objective】 Wild citrus plants are growing in the Nankun mountain, Longmen county, Guangdong Province, which is named ‘Longmeng Xiangcheng’ by the locals. This study aimed to reveal the genetic differences between ‘Longmeng Xiangcheng’ and other citrus species by using morphological and molecular analysis. 【Method】The habitat environment of ‘Longmen xiangcheng’ was investigated, and the morphology of leaf, flower and fruit was described. Fruit were analyzed for content of total soluble solid, sugar, titratable acid and ascorbic acid. Pollens were observed under scanning electronic microscope. The phylogenetic relationship between ‘Longmen xiangcheng’ and other citrus species was investigated using AFLP and SNP markers. The two alleles of β-carotene hydroxylase gene (CHX) were cloned, sequenced and compared with those of other citrus. 【Result】 The natural habitant environment of ‘Longmeng Xiangcheng’ indicates that it is truly a wild citrus. ‘Longmeng Xiangcheng’ resembles to pummelo in fruit and seed morphology and is also mono-embryonic. But it is smaller than pummelo in size, extremely low content of juice and edible portion, and bitter taste of the fruit indicates that it is more primitive. Its lanceolate leaves, linear leaf wings and united filaments show clearly similarities to those of mandarins. However, its young leaves, young flowers, and filaments are tinged with purple, indicating its primitiveness. The phylogenetic tree based on AFLP profiles show that it is not clustered with any of the other 23 analyzed citrus, though it is placed in between pummelos and mandarins. The genotyping results showed that its genotypes at 3, 1, 4 of the 8 SNP loci are mandarin homozygotes, mandarin-pummelo heterozygote, and pummelo homozygotes, respectively, and no other analyzed citrus accessions contained such a unique combination of genotypes. The CHX gene sequence data revealed that ‘Longmen Xiangcheng’ possesses two very similar alleles carring some very unique SNPs not found in any known citrus CHX gene sequences. Phylogenetic analyses based on CHX gene sequences showed that ‘Longmeng Xiangcheng’ is distant from other citrus, and ‘Longmen Xiangcheng’ can be placed as an independent clade in trees.【Conclusion】‘Longmen Xiangcheng’ is truly a wild citrus. Its morphology is characterized by possessing with both primitive and advanced traits. It is quite unique and different from any known citrus as reveled by molecular DNA data.