水稻白条纹叶及白穗突变体wlp6的鉴定与基因定位

doi:10.16819/j.1001-7216.2018.7144

摘要/Abstract

摘要：

目的叶色突变体是研究水稻光合作用,叶绿素生物合成和遗传发育调控机理的重要材料。发掘水稻叶色突变体,是水稻功能基因组学研究的重要遗传基础。方法在昌恢121中发现了一份白条纹叶及抽穗期白穗突变体,经过连续多代自交能稳定遗传,暂命名为wlp6(white striped leaf and white panicle 6)。在南昌分早、中和晚3季播种wlp6与野生型种子,考查了中稻与晚稻的部分农艺性状;测定3叶期、分蘖期、抽穗期叶片及颖壳的叶绿素含量;通过电镜观察抽穗期叶肉细胞发育情况。在光照培养箱中进行温光敏感实验;将wlp6与昌恢121及02428正反交,观察F₁植株表型,对F₂分离群体进行卡方测验,分析突变体遗传规律;以wlp6/02428衍生的F₂群体为材料,利用BSA法进行基因定位。结果 wlp6自第1片叶到成熟,叶片均呈白条纹,抽穗期颖壳及枝梗失绿,高温天气穗转绿。突变体株高、有效穗数和每穗粒数在早稻季和中稻季均显著低于野生型,晚稻季wlp6的结实率和千粒重也显著低降低。叶绿素含量测定表明,wlp6叶片叶绿素含量在不同生育期及不同季均显著低于野生型,早稻和晚稻季种植的wlp6颖壳叶绿素含量也比野生型低。电镜观察抽穗期的叶肉细胞发现,wlp6叶绿体数目减少,体积变小,没有分化出明显的片层结构。温光敏感实验表明,突变体对光照强弱钝感,叶色受温度和日照长短影响,随着温度升高和日照时间变长突变体叶绿素含量有上升趋势。遗传分析表明,该性状受隐性核基因控制,利用wlp6/02428得到的616个F₂单株将WLP6定位于第6染色体短臂InDel标记R-7与R-8间,物理距离137 kb,此区间预测了21个候选基因。经候选基因分析及测序发现,其中LOC_Os06g14620编码一个核糖核酸还原酶小亚基,编码区第142和158位碱基由T替换为C,第288位插入了碱基A,碱基的插入导致翻译提前终止,因此推测LOC_Os06g14620是WLP6的候选基因。结论 LOC_Os06g14620是已经克隆的白条纹叶基因St1的候选基因,推测WLP6与St1等位,但突变位点不同,且表型也有差异。

关键词: 水稻, 白条纹叶及白穗, 叶绿素, 温光敏感, 基因定位

Abstract:

【Objective】Leaf color mutants are important materials for studying the regulation of photosynthesis, chlorophyll biosynthesis and genetic development in rice. The discovery of leaf color mutants is an important genetic basis for functional genomics research in rice. 【Method】 A mutant of white stripe leaf and white panicle was found in Changhui 121, which could be inherited stably to successive generations, and temporarily termed wlp6 (white striped leaf and panicle 6). wlp6 and wild-type seeds were seeded in the early, middle and late seasons in Nanchang, Jiangxi Province and the agronomic traits of the middle and late rice were investigated. Chlorophyll contents in leaf and glume at the 3-leaf stage, tillering stage and heading stage were tested. The development of mesophyll cells at the heading stage was observed with a electron microscopy. Temperature and light sensitive experiments were carried out in an illumination incubator. The phenotype of reciprocal F₁between wlp6 and Changhui 121, 02428 was observed and chi-square test was conducted on F₂ segregating population to analyze the genetic pattern of the mutant. The F₂ population derived from wlp6/02428 was used as material, and the mapping was carried out by the BSA method. 【Result】 From 1-leaf stage to mature stage, all leaves of wlp6 showed white stripes. At the heading stage, glumes and branches were albino, and the panicle turned green under high temperature. The plant height, number of effective panicles and number of grains per panicle of the mutant were significantly lower than those of its wild type in the two seasons, and the seed-setting rate and 1000-grain weight of wlp6 in late season were also significantly lower. The leaf chlorophyll contents of wlp6 were significantly lower than those of the wild type at all growth stages. The husk chlorophyll contents of wlp6 were significantly lower than those of the wild type in early and late season. In wlp6, the number of chloroplasts decreased, the volume became smaller, and there was no obvious lamellar structure. Leaf chlorophyll contents increased with the temperature and sunshine duration increase. Genetic analysis showed that the trait was controlled by recessive nuclear genes. WLP6 was located in the interval InDel markers R-7-R-8(physical distance 137 kb), on the short arm of chromosome 6. In this interval, 21 candidate genes were predicted. Through the analysis of candidate genes and sequencing, we found that LOC_Os06g14620 encodes a small chain of RNA reductase, 142nd and 158th base of the coding region carried a substitution from T to C, and the base A was inserted at the 288th position. The insertion of the base leads to termination of the gene translation. Therefore, it is speculated that LOC_Os06g14620 is the candidate gene for WLP6.【Conclusion】LOC_Os06g14620 is the candidate gene of the cloned white stripe gene St1. WLP6 is the allele of St1, with different mutation sites and phenotypes.

Key words: rice, white stripe and white panicle, chlorophyll, temperature and light sensitive, gene mapping

中图分类号:

李玲锋, 熊玉毅, 欧阳林娟, 彭小松, 陈小荣, 贺晓鹏, 傅军如, 边建民, 胡丽芳, 徐杰, 贺浩华, 孙晓棠, 朱昌兰. 水稻白条纹叶及白穗突变体wlp6的鉴定与基因定位[J]. 中国水稻科学, 2018, 32(6): 538-548.

Lingfeng LI, Yuyi XIONG, Linjuan OUYANG, Xiaosong PENG, Xiaorong CHEN, Xiaopeng HE, Junru FU, Jianmin BIAN, Lifang HU, Jie XU, Haohua HE, Xiaotang SUN, Changlan ZHU. Identification and Gene Mapping of White Stripe Leaf and White Panicle Mutant wlp6 in Rice[J]. Chinese Journal OF Rice Science, 2018, 32(6): 538-548.

图/表 11

表1 wlp6与野生型部分农艺性状

Table 1 Part agronomic traits of wlp6 and its wild type(WT).

农艺性状 Agronomic trait	中季 Middle season			晚季Late season
农艺性状 Agronomic trait	突变体Mutant	野生型WT		突变型Mutant	野生型WT
生育期 Growth duration / d	116.0±3.7	121.0	±1.6	135.0±2.3	132.0	±3.6
株高 Plant height / cm	137.4±6.6	149.2	±5.2^*	118.0±4.7	134.6	±3.0^**
剑叶长 Length of flag leaf / cm	28.2±1.4	36.6	±3.3^**	31.1±3.9	34.8	±2.2
剑叶宽 Width of flag leaf / cm	1.8±0.1	2.0	±0.0	1.8±0.2	1.9	±0.1
有效穗数 No.of Effective panicle per plant	7.6±1.1	10.2	±2.4^*	7.4±0.9	10.8	±1.6^**
穗长 Panicle length / cm	26.2±1.8	27.7	±1.0	24.2±0.5	27.2	±1.2^**
穗粒数 Grain number per panicle	147.1±19.9	190.9	±13.7^*	142.8±5.9	204.4	±20.8^**
结实率 Seed-setting rate / %	89.0±4.7	89.3	±2.8	60.7±1.1	74.1	±5.9^**
千粒重 1000-grain weight / g	24.3±0.3	23.6	±0.3	22.1±1.6	24.5	±1.1^**

图1 wlp6与野生型表型 ^ A—3叶期;B—分蘖期;C—抽穗期;D—穗的颜色对比;E—抽穗期剑叶对比;F—成熟时籽粒。

Fig. 1. Phenotype of wlp6 and its wild type. ^ A, 3-leaf stage; B, Tillering stage; C, Heading stage; D, Panicle color; E, Flag leaf at heading; F, Mature grains.

图2 早、中、晚3季wlp6与野生型在3叶期、分蘖期和抽穗期的叶片及颖壳的叶绿素含量比较^**表示突变体与野生型间差异达0.01显著水平(双尾t检验)。

Fig. 2. Comparison of chlorophyll contents of the leaves and glume between wlp6 and wild-type in early, middle and late seasons at the three-leaf stage, tillering stage and heading stage.^ **Difference between the mutant and WT was significant at 0.01 level, respectively(two-tailed t test).

图3 wlp6与野生型叶肉细胞超微结构 ^ A和B表示wlp6叶肉细胞; C和D表示野生型叶肉细胞。

Fig. 3. Ultrastructure of mesophyll cells in wlp6 and its wild type. ^ A and B represent mesophyll cells of wlp6; C and D represent wild-type mesophyll cells.

图4 不同温度下生长的wlp6(左)和野生型(右)^ A、B、C、D和E分别表示20℃、25℃、28℃、30℃和32℃下2叶期的幼苗。

Fig. 4. wlp6(left) and its wild type(right) at different temperatures. ^ A, B, C, D and E represent the 2-leaf stage seedlings at 20℃, 25℃, 28℃, 30℃ and 32℃, respectively.

图5 不同温度下生长的幼苗叶片叶绿素含量^**表示突变体与野生型间差异达0.01显著水平(双尾t检验)。

Fig. 5. Leaf chlorophyll contents at various temperatures.^ **Difference between the mutant and WT was significant at 0.01 level, respectively(two-tailed t test).

图6 不同光照时间下生长的幼苗叶绿素含量^**表示突变体与野生型间差异达0.01显著水平(双尾t检验)。

Fig. 6. Chlorophyll contents of seedlings grown under different photoperiods.^ **Difference between the mutant and WT was significant at 0.01 level, respectively(two-tailed t test).

图7 不同光照强度下wlp6与野生型幼苗的株高、可溶性糖含量、叶绿素含量及叶绿素a/b比值^**表示突变体与野生型间差异达0.01显著水平(双尾t检验)。

Fig. 7. Plant height, soluble sugar content, chlorophyll contents and chlorophyll a/b ratio of wlp6 and wild-type seedlings under different light intensities.^ **Difference between the mutant and WT was significant at 0.01 level, respectively(two-tailed t test).

表2 白条纹株与正常株在两个F2群体中的分离比

Table 2 ．Separation ratio of white stripe and normal plants in two F2 populations.

杂交组合 Cross-combination	白条纹 White striped plants	正常株 Normal plants	分离比 Ratio of segregation	卡方值 χ²
wlp6/昌恢121 wlp6/Changhui 121	838	2 546	1∶3.04	0.09
wlp6/02428	457	1 412	1∶3.09	0.27

表3 定位基因用到的重要引物

Table 3 Some important primers used for gene mapping.

标记 Marker	正向引物序列 Forward primer	反向引物序列 Reverse primer	物理距离 Physical distance / kb
RM19715	CCAATCTAAATTACGCCGCTAGG	GCCAGCTGTTGTTTGTAGTTTCG	7 869
RM19765	GCTCCACAGAAAAGCAAAGC	TGCAACAGTAGCTGTAGCCG	8 931
R-9	TTTTCATATGGCGACCAAGC	CATAAAATTTGGCTCAGTACAGC	8 363
R-8	CTCGGAGTAAGCCCAAATCA	CTGGTTGGCTTTTGCTGTG	8 325
R-7	GCTGATTTGTTTGGTCCGGG	TCTTTCCGGCATCCCATGTC	8 188
R-6	GTTTAGGCTGTCAGCAAGGC	TCAGTGACGACTCGCTCTTC	8 180

图8 WLP6的定位(A、B)及突变位点(C)^ A—WLP6的初定位;B—WLP6的精细定位;C—候选基因,粗箭头处表示发生突变的位置。

Fig. 8. Location of WLP6(A, B) and mutation site(C).^ A, The initial location of WLP6; B, Fine mapping of WLP6; C, Candidate gene, thick arrow indicating location of mutation.

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