水稻抽穗期的遗传基础复杂,属于数量性状。目前发掘的水稻抽穗期相关的QTL已超过700个,位于第3号染色体的QTL数目最多,但目前仅有29个QTL被克隆[5]。Kojima等[6]将水稻抽穗期相关QTL精细定位到20kb的范围内,并在该区间克隆到1个与水稻抽穗期相关的基因Hd3a。Zong等[7]研究发现,在长日照或短日照下,敲除Ghd7及DTH8会促使水稻抽穗,在长日照条件下,Hd1会促使Ghd7表达,最终导致水稻抽穗期推迟甚至不抽穗。Li等[8]发现,HGW突变体植株的抽穗期延迟,这一基因编码泛素相关的结构蛋白。尽管目前定位到的QTL数量很多,但大多数QTL的区间很大,且水稻抽穗期属于复杂的数量性状,因此,需要进一步挖掘新的QTL。
本研究利用空育131和小白粳子衍生的重组自交系群体（RIL）,通过靶向测序基因型检测技术（genotyping by target sequencing,GBTS）进行基因型分析,构建了包含527个bin标记的高密度遗传图谱,发掘新的抽穗期相关主效QTL,为水稻基因克隆及分子标记辅助选择育种提供参考。
1 材料与方法
1.1 试验材料
以粳稻品种空育131为母本、陆稻品种小白粳子为父本配制杂交组合,以通过“单粒传”法得到的包含195个株系的F9群体为试验材料。
1.2 试验方法
所有材料种植于东北农业大学阿城水稻试验基地,每份材料种植8行,每行20株,行距30cm、株距13.3cm,均为单株插秧。按株系单独编号,调查并记录抽穗日期。当该株系的第1个稻穗尖露出剑叶叶鞘至少1cm时,记为该株系的抽穗日期,每隔1d调查1次,将株系从播种到抽穗的天数作为该株系的抽穗期表型值,方法参照文献[11],略有改动。田间管理依照常规大田生产。
1.3 数据统计分析
使用Excel 2016和SPSS Statistics 19.0进行相关性状表型数据统计分析。
1.4 遗传连锁图谱的构建及QTL分析
利用10K SNP芯片（石家庄博瑞迪生物技术有限公司）的靶向测序基因型检测技术进行RIL群体的基因型分析,经由双亲多态性分析及IciMapping-Bin模块的去冗余分析,将RIL群体最小重组区间内的多个SNP标记合并为1个“bin”,最终获得527个高质量多态性bin标记。通过JoinMap 4.0进行遗传连锁图谱的构建,该图谱共覆盖水稻基因组1874.85cM,标记间的平均遗传距离为3.56cM。采用QTL IciMapping 4.2软件的完备区间作图法（ICIM）进行QTL定位（
2 结果与分析
2.1 水稻抽穗期的表型分析
分别对亲本及RIL群体的抽穗期进行基本统计分析（表1）。亲本的抽穗期处于RIL群体范围内,抽穗期在双亲之间表现出极显著差异,小白粳子的抽穗期明显长于空育131。RIL群体的抽穗期具有超亲分离现象,平均为97.74d,变异范围为76.00~111.00d,变异系数为7.68%。通过数据正态分布的适合性检验后发现,抽穗期的偏度和峰度绝对值均小于1,说明性状的表型符合正态分布,具有典型的数量性状遗传特征。
表1 水稻抽穗期的基本统计分析
Table 1
| 项目 Item | 亲本Parents | 重组自交系群体RIL population | |||||||
|---|---|---|---|---|---|---|---|---|---|
| 空育131 Kongyu 131 (d) | 小白粳子 Xiaobaijingzi (d) | 平均值 Mean (d) | 标准差 Standard deviation | 变异范围 Range (d) | 变异系数 Coefficient of variation (%) | 峰度 Kurtosis | 偏度 Skewness | ||
| 抽穗期Heading date | 91.00 | 104.00** | 97.74 | 7.51 | 76.00~111.00 | 7.68 | -0.42 | -0.23 | |
“**”表示在0.01水平上极显著差异
“**”indicates extremely significant difference at 0.01 level
2.2 水稻抽穗期QTL分析
采用IciMapping 4.2软件的完备区间作图法,对水稻抽穗期进行QTL分析（图1和表2）,在水稻第1、3和7号染色体上共检测到4个与水稻抽穗期相关的QTL。4个QTL的LOD值在3.0181~13.2288之间,表型贡献率介于4.0619%~21.7016%。其中,qHD-3-1和qHD-7-1的表型贡献率均大于10%,属于主效QTL。除qHD-7-1的增效等位基因来自于小白粳子外,其余3个QTL的增效等位基因均来自于空育131。通过与已克隆水稻抽穗期相关基因比较发现,qHD-1-1、qHD-3-2和qHD-7-1区间内分别含有已知基因OsGI、Hd6和Ghd7。而qHD-3-1区间内未发现已克隆基因,是控制水稻抽穗期的新位点。qHD-3-1贡献率为10.5098%,位于标记C3_799346和C3_1493308之间,物理距离为0.69Mb。
图1
图1
水稻抽穗期QTL在染色体上的分布
Fig.1
Distribution of QTLs on chromosomes at heading stage in rice
表2 水稻抽穗期QTL及其遗传效应
Table 2
| 数量性状位点 QTL | 染色体 Chromosome | 标记区间 Marker interval | LOD值 LOD peak | 贡献率 Contribution rate (%) | 加性效应 Additive effect | 已知基因 Known gene | |
|---|---|---|---|---|---|---|---|
| qHD-1-1 | 1 | C1_4330799 | C1_4645558 | 3.0181 | 4.0619 | 1.5969 | OsGI |
| qHD-3-1 | 3 | C3_799346 | C3_1493308 | 7.0671 | 10.5098 | 2.7637 | |
| qHD-3-2 | 3 | C3_29189855 | C3_33130038 | 3.2297 | 4.5338 | 1.6866 | Hd6 |
| qHD-7-1 | 7 | C7_8809828 | C7_13201476 | 13.2288 | 21.7016 | -3.8409 | Ghd7 |
3 讨论
水稻抽穗期与其产量、品质及适应性密切相关[14]。但传统的遗传标记（例如SSR、RFLP等）存在密度低、数量少及精度不足甚至双交换位点漏测等问题,影响了抽穗期相关QTL的发掘[15]。相比之下,通过bin标记构建的高密度遗传图谱省时省力、标记密度高且能够直接发掘区间内的候选基因,并且由于单个bin内部包含多个不重组的SNP,避免了双交换位点漏测的问题。本研究利用包含527个bin标记的高密度遗传图谱,平均遗传距离仅为3.56cM,能够快速高效地发掘抽穗期相关QTL,并进一步预测候选基因。通过对由空育131和小白粳子衍生的RIL群体进行抽穗期调查,发现水稻抽穗期在双亲中表现出极显著差异,RIL群体变异范围较广,有明显的超亲分离现象,表型符合正态分布,具有典型的数量性状遗传特征,适于进行QTL分析。
本研究所定位的4个与抽穗期相关QTL中,其中3个含有与抽穗期相关的基因。Izawa等[16]发现,Os-GI突变体在短日照下表现为晚花;Takahashi等[17]研究表明,来源于亲本Kasalath的Hd6等位基因在长日照及自然生长条件下均延迟抽穗,反之,在短日照下则不能延迟抽穗。Xue等[18]发现,长日照条件提高Ghd7的表达,能推迟抽穗期。因此,Os-GI、Hd6和Ghd7能够对提高全球水稻产量及水稻季节适应性起到重要作用。此外,只有位于3号染色体上的qHD-3-1中未包含报道过的已知基因,且其贡献率高达10.5098%,是新的抽穗期相关QTL,本结果能够为水稻抽穗期QTL的精细定位及克隆提供参考,同时为分子标记辅助育种提供了新的资源。
4 结论
基于GBTS技术对双亲空育131、小白粳子及其衍生的RIL群体进行基因型分析,构建了包含527个bin标记的高密度遗传图谱,通过QTL分析,鉴定到4个与水稻抽穗期相关的QTL。其中除qHD-3-1外,其余3个与抽穗期相关QTL区间内均包含已知的抽穗期基因,说明qHD-3-1是控制水稻抽穗期的新位点。
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