水稻剑叶角度qFla-8-2位点的精细定位

doi:10.16819/j.1001-7216.2016.5101

摘要/Abstract

摘要：

为更好地理解水稻大剑叶角的分子遗传机制,对已初定位的控制剑叶角度的qFla-8进行精细定位和候选基因预测。利用以863B为受体亲本、A7444为供体亲本衍生的BC₃F₃群体中仅在目标基因区段杂合的172个单株自交构建的次级分离群体,对qFla-8所在的RM6215-RM8265区间9个SSR标记的基因型进行鉴定,结合表型数据进一步缩短qFla-8所在染色体区段。结果发现qFla-8位点所在染色体区段存在两个紧密连锁的位点qFla-8-1和qFla-8-2。其中,qFla-8-1位于RM6215和RM3153之间,可解释22.33%的表型变异;qFla-8-2位于RM1309和RM3491之间,可解释23.81%的表型变异。进一步对加性效应较大的qFla-8-2精细定位,通过开发插入缺失(InDel)分子标记,将qFla-8-2位点限定于InDel标记Z7和SSR标记RM23071之间,该区间的物理距离为67 kb。该区段包含3个预测基因Os08g0408200,Os08g0408300和Os08g0408500,分别编码功能类似GAMYB的WD40结构域蛋白、假定蛋白以及类APETALA2蛋白。

关键词: 水稻, 剑叶角度, QTL, 精细定位

Abstract:

To better understand the genetic mechanism regulating large rice flag leaf angle, qFla-8 detected previously was further fine-mapped and the candidate genes were predicted. Using a secondary segregating population, composed of 172 selfing individual plants with heterozygous region of target gene merely in BC₃F₃ population derived from the backcross between 863B (recipient parent) and A7444 (donor parent), genotypes of nine SSR markers in the interval of RM6215-RM8265 containing qFla-8 were identified. Combining the phenotypic data, chromosome segment containing qFla-8 was further narrowed and there are two closely linked loci, qFla-8-1 and qFla-8-2.qFla-8-1 was localized between RM6215 and RM3153, explaining 22.33% of phenotypic variation,and qFla-8-2 was localized between RM1309 and RM3491, explaining 23.81% of phenotypic variation. By developing insertion-deletion (InDel) markers, qFla-8-2 with higher additive effect was finally limited to 67 kb region between InDel marker Z7 and SSR marker RM23071,which contains three predicted genes, Os08g0408200 encoding WD40 domain containing protein similar to GAMYB-binding protein, Os08g0408300 encoding hypothetical protein and Os08g0408500 encoding APETALA2-like protein.

Key words: rice, flag leaf angle, QTL, fine mapping

中图分类号:

朱长丰, 梁利君, 曾思远, 李天伟, 董冠杉, 洪德林. 水稻剑叶角度qFla-8-2位点的精细定位[J]. 中国水稻科学, 2016, 30(1): 27-34.

Chang-feng ZHU, Li-jun LIANG, Si-yuan ZENG, Tian-wei LI, Guan-shan DONG, De-lin HONG. Fine Mapping of qFla-8-2 for Flag Leaf Angle in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(1): 27-34.

图/表 7

图1 863B和A7444不同生育期的剑叶角度 A-863B(左)和A7444(右)孕穗期的剑叶角度,bar=5 cm; B-863B(左)和A7444(右)灌浆期的剑叶角度,bar=10 cm; C-863B(左)、CF14-3(中,863B近等基因系)和A7444(右)成熟期的剑叶角度,bar=10 cm。白色箭头指的是剑叶角度。

Fig. 1. Flag leaf angle of 863B and A7444 at different growth stages. A, Flag leaf angle of 863B (left) and A7444 (right) at the booting stage, bar=5 cm; B, Flag leaf angle of 863B (left) and A7444 (right) at the filling stage, bar=10 cm; C, Flag leaf angle of 863B (left), CF14-3 (middle, NIL of 863B) and A7444 (right) at the mature stage, bar=10 cm. Flag leaf angle was showed by the white arrows.

图2 水稻剑叶角度测量方法(左)及863B和A7444灌浆期剑叶角度(右)

Fig. 2. Measuring method (left) of flag leaf angle and the flag leaf angle of 863B and A7444 at the filling stage (right).

表1 缩短qFla-8所在区间用到的SSR引物

Table 1 SSR primers used for further narrowing qFla-8 region.

引物名称 Primer name	正向引物(5'-3') Forward primer(5'-3')	反向引物(5'-3') Reverse primer(5'-3')	产物长度 Production length/bp
RM23021	CTCAATGATGTTCTCGGCTTCC	GCCACAACGGTCAAGTAAAGAGC	200
RM6215	TTCAGCAGAGAGATGACGCAAGG	GTACAAACCAGCCCTCCGAGACG	190
RM3153	GTGTGATGGTGACGGATTACATGTGC	CATGCTGCAGAATTTCCATGTTGG	402
RM3689	CGTCAGCCGAAACTACTATCTAAACC	GTTTCACTGCACTCTGGTTTGC	293
RM1309	GAGGACACTGACGACAGCTTGG	CGCGCAAATCATTAAGTTCAGG	186
RM3491	GTGTTCTGATGTTCCCTCTCTGC	CCAACAAGGACTCACATGTCTCG	120
RM8264	TTCTACGGAATTTCTCCCTCTGG	CTAATCAATCTCTCGCGTTCTTGG	162
RM8265	TCGGCTGATCTGACCGTACATCC	GTGCATGCAACCACCTCTTGG	185
RM5808	GGGAGTAGGAGGGAGGGAGAAAGAGG	CAGAACCAGCGAGGGAAACACC	112

图3 第8染色体上qFla-8的遗传解析黑色箭头表示qFla-8所在位置。

Fig. 3. Genetic analysis of qFla-8 on chromosome 8. The black arrow indicates qFla-8.

图4 单位点qFla-8-2 F2分离群体163个单株的剑叶角度分布

Fig. 4. Frequency distributions of the flag leaf angle of 163 plants in F2 segregating generation of qFla-8-2 locus.

表2 精细定位qFla-8-2所用SSR和InDel标记

Table 2 SSR and InDel markers used for fine mapping of qFla-8-2.

引物名称 Primer name	正向引物(5'-3') Forward primer(5'-3')	反向引物(5'-3') Reverse primer(5'-3')	产物长度 Production length/bp
RM1309	GAGGACACTGACGACAGCTTGG	CGCGCAAATCATTAAGTTCAGG	189
RM23065	CCACGAACTCTCCCTATATCTACTGC	CGTGCACACCTGAAGAGTATGG	135
RM23071	GTTCCGCCGTTGAGTGATGACC	TCCTCAGTCCTCCCTCTCCTTCC	287
Z5	GTCAAATCAGCTGGTTCAGTG	ATTTGACCTAACGATTGCGA	200
Z7	ATCCACGTCACCCACAACTC	CCCACGGAAAACCAAAACAT	102
RM3491	GTGTTCTGATGTTCCCTCTCTGC	CCAACAAGGACTCACATGTCTCG	266

图5 qFla-8-2的图位克隆 A-利用438个单株的作图群体对qFla-8-2基因进行的精细定位。黑色水平线下面的数字表示标记与基因之间的交换单株数。B-qFla-8-2基因最终被缩到67 kb的DNA区段上。粗黑色箭头代表的是预测基因。

Fig. 5. Map-based cloning of qFla-8-2. A, Fine mapping of qFla-8-2 genome region using mapping population with 438 plants. Numbers below the black horizontal line represent the numbers of recombinants between the marker and gene. B, qFla-8-2 was finally narrowed to a 67 kb DNA fragment. The thick black arrows mean predicted genes.

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