抽穗期是决定作物品种生育期及地区适应性的重要性状，受自身遗传因子和外界环境因素两方面决定，是开花基因时空顺序表达的结果(Yano et al., 2001)。研究表明，拟南芥至少有4条调控开花时间的信号途径，即光周期途径(Photoperiod pathways)，春化途径(Vernalization pathway)，自主途径(Autonomous pathway)和赤霉素途径(Gibberellin pathway)等(Boss et al., 2004)。这些途径中的许多相关基因已经被克隆，这些基因在开花植物中是比较保守，已克隆水稻抽穗期基因大多与拟南芥相关基因同源，转拟南芥成花基因的水稻表现出相同的效应(Hayama et a1, 2003)。比较大麦、水稻、拟南芥间抽穗期基因保守结构域，发现长日拟南芥与短日水稻在诱导开花的基因上具有一定的保守性(Griffiths et a1, 2003)，如水稻中的OsGI、Hd1和Hd3a基因与拟南芥中的同源基因GI、CO和FT相对应(Izawa et al., 2002)。

粳稻、籼稻的全基因组序列测定已先后完成(Goff et al., 2002; Yu et al., 2002)，重测序项目发展迅速。序列数据库为侯选同源基因搜索提供了方便(Izawa et al., 2003)。SNP/InDel是一种在基因组中广泛存在的多态性。研究表明，测序粳稻日本晴和籼稻9311基因组中每268 bp就存在1个SNP，每953 bp就有1个InDel (Shen et al., 2004)。这些多态性位点是开发基因图位克隆分子标记及分子标记辅助育种的基础，也是揭示抽穗期基因多样性及其生态适应性分子机制的基础(岳兵和邢永忠, 2005)。

本文以哀牢山哈尼梯田的传统代表品种(月亮谷)全基因组重测序为材料，借助目前在水稻花期基因中的研究结果，通过序列的搜索比对分析其SNP/InDel的数量与分布，为揭示当地传统品种持续种植上百年，经久耐用，避病、避寒提供分子证据。

1结果分析
1.1抽穗期调控基因SNP/InDel多态性
对62 个水稻抽穗期候选基因的分析结果表明，供试候选基因中SNP/InDel多态性丰富，日本晴/9311/月亮谷中SNP总数为1 356个，InDel的总数为242个；传统水稻品种月亮谷中特有的SNP总数为165个，占总SNP 12.66%；特有InDel的总数为3个，占0.83%；多态性位点数目分布很不均匀(图1)。

图1 候选基因的SNP/InDel密度分布 Figure 1 The density of SNP/InDel distribution among the candidate gene

SNP/InDel基因间差异较大。PHYA、OsFT-L4、OsFT-L6和OsLUX没有SNP/InDel，而OsMADS51有414 个SNP/InDel位点。40.32%的SNP/InDel位点数量小于l0个，l0个到20个之间的基因为20.97%，大于20的为38.71%。 SNP/InDel多态性大都分布在基因组的非编码区，分布在CDS中SNP为318，占所有SNP位点总数的23.45％，而分布在CDS中的 InDel为24个，占9.92％，其中月亮谷中特有的SNP/InDel分布在CDS中SNP为50个，占月亮谷所有特异SNP位点总数的 30.03％，分布在CDS中的InDel为0个。在CDS中的分布同样也是不均匀的，RCN2、OsLKP2、OsCRY2、Hd3a和OsELF3分别有106、14、13、12和10个SNP；OsFT-L6、OsFT-L10、OsFT-L12、Ghd7、OsAP1、OsFLK、Hd6、RCN1、RCN3、OsFy、OspRR1和RFL中无SNP，其余的基因SNP小于10个。RCN2、OsFT-L10、Ehd1和OsGAI分别有8、2、2和2个InDel，OsPRR95、OsPRR73、OsLKP2、ZTL、RGL1、RCN1、OsFCA、Hd3a和OsFT-L9各有1个InDel，其余的基因无InDel。

通过合并多态性的方法对SNP/InDel含量较高的基因深入分析发现，大部分基因中存在SNP/InDel热点区域(SNP/lnDel密度较高的片段) (图2)。将100 bp或500 bp内的所有SNP/InDel合并，视为一个多态性位点做图，结果显示多数基因的多态性位点数目变化明显，多态性的位点降低超过50%。例如OsMADS51的SNP/InDel位点有414个，经100 bp合并后降为147个，经500 bp合并后降为44个。但是，也有基因变化不明显，例如OsGI其热点区域不明显，经100 bp、500 bp合并后，其SNP/InDel位点降低不超过50%。

图2 候选基因侧翼序列的SNP/InDel密度分布 Figure 2 The density of SNP/InDel distribution among the candidate gene flanking sequences

1.2抽穗期基因上游2Kbp调控区域SNP/InDel多态性
基因的侧翼序列中可能存在比较重要的调控序列。对62个水稻抽穗期候选基因的上游调控序列分析结果表明，水稻抽穗期调控候选基因侧翼中同样存在着丰富的 SNP/InDel多态性，日本晴/9311/月亮谷中SNP总数为1 372个，InDel的总数为263个，月亮谷中特有的SNP总数为251个，占总SNP的16.90%，InDel的总数为50个占19.01%，多态性位点数目也分布很不均匀(图3)。

图3 合并后候选基因的SNP/InDeL分布图 Figure 3 The distribution of SNP/InDel after united

按照基因上游2Kbp内SNP/InDel的分布数量，可以将供试62个候选基因分为以下2种类型：①保守型：经100 bp合并作图后，大多数基因的上游调控序列多态性位点降低一半以上，经500 bp合并后作图后多态性位点保持不变或差异较小的，如RCN4、RFT1、OsELF3、OsFCA和OsFPA等；②热点型：经500 bp合并后作图后多态性位点又减低一半以上的，如OsCDF1、OsELF6、OsFLD、TFL1和OsFT-L13等(图4)。

图4 候选基因侧翼的SNP/InDel分布图 Figure 4 The distribution of SNP/InDel of flanking sequences after united

2讨论
随着基因组研究的不断深入，积累了大量的在遗传学研究和分子育种中有重要的利用价值的数据库资源(闫双勇等, 2005)。重测序数据库资源的积累为研究同一物种内的遗传变异及适应机制创造了条件。本文以目前已经全基因组测序完成的2个栽培稻亚种：粳稻品种日本晴(Nipponbare)通过全基因组鸟枪法和逐步克隆法被测序；籼稻品种9311通过全基因组鸟枪法被测序，以及我课题组对元阳传统水稻品种月亮谷的全基因组重测序全基因组序列为材料，分析了62个水稻抽穗期候选基因的SNP/InDel多态性。结果表明，水稻抽穗期调控候选基因中存在着丰富的 SNP/InDel多态性。传统水稻品种月亮谷抽穗期相关基因中即有较多的SNP/InDel变异位点，同时分布在CDS的特异SNP位点数高于日本晴 /9311的SNP位点数。侧翼序列多态性的差别反映了调节序列或转座子的存在。多态性较低的侧翼序列可能存在调节序列，而非常保守的区域则可能存在比较重要的调节序列(闫双勇等, 2005)。推测传统水稻品种抽穗期基因所具有的较多SNP/InDel，可能与其相对于现代品种具有较强的适应性和稳定性有关。在水稻上包括花期相关基因Hd1、Edh1和Ghd7 (Yano et al., 2000; Doi et al., 2004; 薛为亚等, 2009)等与农艺性状自然变异有关的功能核苷酸多态性(functional nucleotide polymorphisms, FNPs)已经得到了鉴定。分析水稻品种间功能核苷酸多态性是揭示其分子生态适应性的重要途径(Takahashi et al., 2009)。

3材料与方法
3.1哈尼族传统水稻品种月亮谷全基因组测序
哈尼族当地传统水稻品种月亮谷全基因组序列采取重测序的方法，我课题组出资，由华大基因(深圳)完成，并分别以日本晴和9311为模板拼接成2套月亮谷全基因组序列。

3.2水稻花期调控途径的候选基因获取
本文所用候选基因分为两类，一类是文献中已经报道过的水稻抽穗期调控基因及水稻中拟南芥开花调控的直系同源基因，例如Hdl、OsMADS51等，直接在Gene Bank (http://www.ncbi.nlm.nih.gov/)中查询其登陆号及核酸序列；第二类是在TAIR (www.arabidopsis.ore)网站上获得拟南芥花期调控蛋白质氨基酸序(Swarbreck et al., 2007)，用该序列在Gramene (www.gramene.ore)网站上用BLASTP程序搜索水稻蛋白质数据库，获得显著相似的蛋白质序列，并获得相应的基因组序列(Ware et a1., 2002)，选E-value最小和对比序列长度比较接近的序列作为候选序列。从表1中可以看出侯选的序列都有比较小的 E-value值(E-value值最大的为6.5E-59)，序列大小也比较接近。

表1 水稻中抽穗期候选调控基因 Table 1 The candidate genes of heading date in rice

3.3月亮谷花期调控途径基因及上游2kbp调控序列的获取
利用BIOEDIT (Kostka et al., 2008)将测序水稻品种建立本地BLAST数据库。然后用侯选基因的序列作查询序列搜索BLAST数据库发现基因片段，同时截取基因及上游2kbp调控序列片段，序列比对用Clustal W软件进行，比对结果文件保存为.phy文件。

3.4 SNP/InDel位点提取和数目统计
SNP/InDel位点的提取，位点和数目统计用TASSEL软(http://sourceforge.net/projects/tassel)完成。

作者贡献
高东是本研究的实验设计和实验研究的执行人；高东、毛如志及杨香婷完成数据分析；毛如志，魏富刚论文初稿的写作；高东是论文的构思者及负责人，指导实验设计，数据分析，论文写作与修改。全体作者都阅读并同意最终的文本。

致谢
本研究国家重点基础研究发展计划(973项目)课题(2011CB100406)资助。

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