中国水稻科学 ›› 2015, Vol. 29 ›› Issue (6): 601-609.DOI: 10.3969/j.issn.1001G7216.2015.06.006
李智强1,2,3, 朱丹1,2,3, 王志龙1,2, 丁波3, 王国梁1,2,3,*()
收稿日期:
2015-06-18
修回日期:
2015-07-09
出版日期:
2015-10-25
发布日期:
2015-11-10
通讯作者:
王国梁
作者简介:
*通讯录作者:E-mail:wang.620@osu.edu
基金资助:
Zhi-qiang LI1,2,3, Dan ZHU1,2,3, Zhi-long WANG1,2, Bo DING3, Guo-liang WANG1,2,3,*
Received:
2015-06-18
Revised:
2015-07-09
Online:
2015-10-25
Published:
2015-11-10
Contact:
Guo-liang WANG
About author:
*Corresponding author:E-mail:wang.620@osu.edu
摘要:
在粳稻品种Dongjin大田种植过程中,发现一个黄绿叶自然突变体,命名为djyg。该突变体在苗期表现明显的黄绿叶表型,抽穗以后,叶色逐渐恢复正常。叶绿素含量测定结果表明,在苗期、分蘖盛期及抽穗期叶绿素b的含量分别下降53%、62%、36%。电镜结果表明,分蘖期突变体中基粒、类囊体垛堆凌乱、排列疏松,类囊体基质较为稀薄。qRT-PCR结果证实,PORA、Cab1R、PsbA的表达量在突变体中均较野生型明显下调。遗传分析结果表明,黄绿叶突变体djyg由一对隐性主效核基因控制,图位克隆确定该候选基因为编码叶绿素合成酶基因YGL1的一个新等位基因。该突变体未影响植株的主要农艺性状,可作为一个理想的表型标记应用于杂交稻育种工作中。
李智强, 朱丹, 王志龙, 丁波, 王国梁. 水稻黄绿叶突变体djyg的遗传分析与基因定位[J]. 中国水稻科学, 2015, 29(6): 601-609.
Zhi-qiang LI, Dan ZHU, Zhi-long WANG, Bo DING, Guo-liang WANG. Genetic Analysis and Gene Mapping of a Yellow-Green Leaf Mutant djyg in Rice[J]. Chinese Journal OF Rice Science, 2015, 29(6): 601-609.
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') |
---|---|---|
HEMA1 | CGCTATTTCTGATGCTATGGGT | GAGCACAGCAAAATCCTAGACG |
PORA | TGTACTGGAGCTGGAACAACAA | GAGCACAGCAAAATCCTAGACG |
V1(NUS1) | TGGAGGTCGGGACAGAGGA | CGAGGAGCACCACCATCAC |
Cab1R | AGATGGGTTTAGTGCGACGAG | TTTGGGATCGAGGGAGTATTT |
CAO1 | GATCCATACCCGATCGACAT | CGAGAGACATCCGGTAGAGC |
RbcL | CTTGGCAGCATTCCGAGTAA | ACAACGGGCTCGATGTGATA |
OsPPR1 | CTAAGACCGAATGACAAATGC | GCACTGCCAACAAGAATACC |
V2 | CGACAAGCAGAGCGAAGCG | AGGTTGCTGCTCCTTGAATGT |
Cab2R | TGTTCTCCATGTTCGGCTTCT | GCTACGGTCCCCACTTCACT |
PsaA | GCGAGCAAATAAAACACCTTTC | GTACCAGCTTAACGTGGGGAG |
PsbA | CCCTCATTAGCAGATTCGTTTT | ATGATTGTATTCCAGGCAGAGC |
SPP | CGGAGAGGAAACATAATGAC | ATAGGCATTTGTCTTTGTCTC |
表1 荧光定量PCR所需引物
Table 1 Primer for quantitative real-time PCR.
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') |
---|---|---|
HEMA1 | CGCTATTTCTGATGCTATGGGT | GAGCACAGCAAAATCCTAGACG |
PORA | TGTACTGGAGCTGGAACAACAA | GAGCACAGCAAAATCCTAGACG |
V1(NUS1) | TGGAGGTCGGGACAGAGGA | CGAGGAGCACCACCATCAC |
Cab1R | AGATGGGTTTAGTGCGACGAG | TTTGGGATCGAGGGAGTATTT |
CAO1 | GATCCATACCCGATCGACAT | CGAGAGACATCCGGTAGAGC |
RbcL | CTTGGCAGCATTCCGAGTAA | ACAACGGGCTCGATGTGATA |
OsPPR1 | CTAAGACCGAATGACAAATGC | GCACTGCCAACAAGAATACC |
V2 | CGACAAGCAGAGCGAAGCG | AGGTTGCTGCTCCTTGAATGT |
Cab2R | TGTTCTCCATGTTCGGCTTCT | GCTACGGTCCCCACTTCACT |
PsaA | GCGAGCAAATAAAACACCTTTC | GTACCAGCTTAACGTGGGGAG |
PsbA | CCCTCATTAGCAGATTCGTTTT | ATGATTGTATTCCAGGCAGAGC |
SPP | CGGAGAGGAAACATAATGAC | ATAGGCATTTGTCTTTGTCTC |
引物名称 Primer name | 引物类型 Primer type | 正向引物(5'-3') Forward primer (5'-3') | 反向引物(5'-3') Reverse primer (5'-3') | 产物大小(bp) Product size(bp) | |
---|---|---|---|---|---|
HM-1 | InDel | TTAGGCACACTTGGCAGACA | AGAGATGACACGGTTTGGGG | 200 | |
HM-2 | InDel | GGCATAAGAGGTACTTCTGGGA | TGAAGGCACAATTTAGGTGAACG | 183 | |
HM-3 | InDel | CCCTGCAATGCAAACGAGAG | TCCTCATTCGATTAGTCGGCG | 168 | |
HM-4 | InDel | AATTGGGCCATCTGTACGCA | ATTTTTGCCCTTCAACCGCC | 176 | |
HM-5 | InDel | CCTGGGTTAAGGCAGGTTCG | AGAGTGAGTACCACCACCGTA | 204 | |
HM-6 | InDel | GGGGTCGCGTGGAAAATTAC | TCCTACCGTGTGGTTACGTG | 249 | |
HM-7 | InDel | AGTACGCTTCGTGTGGATGG | GGAAGTGCCAGCTCATGATT | 237 | |
HM-8 | InDel | TCGACATATACAGCGCGGAC | TAGGGAGTACGCTCCCATGT | 235 | |
HM-9 | InDel | TGCCTATGCTTCCTTTCGTGA | CACATGTGCAAACGTGTGAT | 165 | |
HM-10 | InDel | TCCCGCCATGGTTATGCTTT | TACCGAGTGCAGGGTAGTCA | 225 | |
HM-11 | InDel | CATCCGGTCAAACGTCCGAT | GCCGGCCTGCTATGTTTTTA | 115 | |
HM-12 | InDel | TCCATTTCGGCTCTAGTTGTG | TATAAGGGCTATGGCTGGTTC | 123 | |
HM-13 | InDel | GCCTCGTACCATCGGATACTCT | ATTTCCATACGCGGTTGCTC | 112 | |
HM-14 | InDel | ACTACGAATCTGGACATACATA | AGAGTGAGAAGCACATATTGTA | 156 | |
HM-15 | InDel | CTTCGTGCTTACGGTGGCG | GGCATGACAGGCAAACTGC | 178 | |
HM-16 | InDel | CCTACCCTCCAACCCATAC | ACAAAGCCGACGCTAATCT | 168 | |
HM-17 | InDel | AGGGTCGTCGGAACAAGG | TGCCGTAGGGCGTCATC | 250 | |
HM-18 | InDel | GCTGTCCTTCTTATAGGCG | CGGAGGCATAGCTTGC | 177 | |
HM-19 | InDel | ACTACTCCGTATGTCCGTGCTA | GTCTACGCATGGGCTTCC | 167 | |
HM-20 | InDel | TTGATTCAGGTGGTGATGT | CTTGGATTATTTGCGAGAC | 200 | |
HM-21 | InDel | TACTCCCTCCATTTCAGCATA | GTCATTAACTAGCACCTCTTTT | 183 | |
HM-22 | InDel | TACTGTTGCACATAGGGTTC | TACTTCAAATGTATGTCCGT | 153 | |
HMS1 | djyg测序 djyg sequencing | GGGTCAAAAACATCGCGTTA | AACCTAGCTCCACCAACAGT | 1309 |
表2 精细定位和测序所用分子标记
Table 2 Markers used for fine mapping and sequencing.
引物名称 Primer name | 引物类型 Primer type | 正向引物(5'-3') Forward primer (5'-3') | 反向引物(5'-3') Reverse primer (5'-3') | 产物大小(bp) Product size(bp) | |
---|---|---|---|---|---|
HM-1 | InDel | TTAGGCACACTTGGCAGACA | AGAGATGACACGGTTTGGGG | 200 | |
HM-2 | InDel | GGCATAAGAGGTACTTCTGGGA | TGAAGGCACAATTTAGGTGAACG | 183 | |
HM-3 | InDel | CCCTGCAATGCAAACGAGAG | TCCTCATTCGATTAGTCGGCG | 168 | |
HM-4 | InDel | AATTGGGCCATCTGTACGCA | ATTTTTGCCCTTCAACCGCC | 176 | |
HM-5 | InDel | CCTGGGTTAAGGCAGGTTCG | AGAGTGAGTACCACCACCGTA | 204 | |
HM-6 | InDel | GGGGTCGCGTGGAAAATTAC | TCCTACCGTGTGGTTACGTG | 249 | |
HM-7 | InDel | AGTACGCTTCGTGTGGATGG | GGAAGTGCCAGCTCATGATT | 237 | |
HM-8 | InDel | TCGACATATACAGCGCGGAC | TAGGGAGTACGCTCCCATGT | 235 | |
HM-9 | InDel | TGCCTATGCTTCCTTTCGTGA | CACATGTGCAAACGTGTGAT | 165 | |
HM-10 | InDel | TCCCGCCATGGTTATGCTTT | TACCGAGTGCAGGGTAGTCA | 225 | |
HM-11 | InDel | CATCCGGTCAAACGTCCGAT | GCCGGCCTGCTATGTTTTTA | 115 | |
HM-12 | InDel | TCCATTTCGGCTCTAGTTGTG | TATAAGGGCTATGGCTGGTTC | 123 | |
HM-13 | InDel | GCCTCGTACCATCGGATACTCT | ATTTCCATACGCGGTTGCTC | 112 | |
HM-14 | InDel | ACTACGAATCTGGACATACATA | AGAGTGAGAAGCACATATTGTA | 156 | |
HM-15 | InDel | CTTCGTGCTTACGGTGGCG | GGCATGACAGGCAAACTGC | 178 | |
HM-16 | InDel | CCTACCCTCCAACCCATAC | ACAAAGCCGACGCTAATCT | 168 | |
HM-17 | InDel | AGGGTCGTCGGAACAAGG | TGCCGTAGGGCGTCATC | 250 | |
HM-18 | InDel | GCTGTCCTTCTTATAGGCG | CGGAGGCATAGCTTGC | 177 | |
HM-19 | InDel | ACTACTCCGTATGTCCGTGCTA | GTCTACGCATGGGCTTCC | 167 | |
HM-20 | InDel | TTGATTCAGGTGGTGATGT | CTTGGATTATTTGCGAGAC | 200 | |
HM-21 | InDel | TACTCCCTCCATTTCAGCATA | GTCATTAACTAGCACCTCTTTT | 183 | |
HM-22 | InDel | TACTGTTGCACATAGGGTTC | TACTTCAAATGTATGTCCGT | 153 | |
HMS1 | djyg测序 djyg sequencing | GGGTCAAAAACATCGCGTTA | AACCTAGCTCCACCAACAGT | 1309 |
材料 Material | 株高 Plant height /cm | 穗长 Panicle length /cm | 单株有效穗数 No. of panicles per plant | 每穗总粒数 No. of spikelets per panicle | 结实率 Seed-setting rate/% | 千粒重 1000-grain weight/g |
---|---|---|---|---|---|---|
Dongjin | 90.68±0.54 | 18.9±0.5 | 34±1 | 118±1 | 99.24±0.22 | 22.84±0.71 |
djyg | 90.55±0.45 | 18.1±0.9 | 33±1 | 116±1 | 98.82±0.21 | 22.38±0.28 |
表3 野生型Dongjin与黄绿叶突变体djyg主要农艺性状(平均数±标准差,t检验)
Table 3 Agronomic traits of wild-type Dongjin and mutant djyg(mean±SD, t-test).
材料 Material | 株高 Plant height /cm | 穗长 Panicle length /cm | 单株有效穗数 No. of panicles per plant | 每穗总粒数 No. of spikelets per panicle | 结实率 Seed-setting rate/% | 千粒重 1000-grain weight/g |
---|---|---|---|---|---|---|
Dongjin | 90.68±0.54 | 18.9±0.5 | 34±1 | 118±1 | 99.24±0.22 | 22.84±0.71 |
djyg | 90.55±0.45 | 18.1±0.9 | 33±1 | 116±1 | 98.82±0.21 | 22.38±0.28 |
图3 分蘖盛期野生型Dongjin与突变体djyg叶绿体亚显微结构(标尺=1 μm)
Fig. 3. Ultrastructure of chloroplasts of wild-type Dongjin and mutant djyg at the fully tillering stage(Bar=1 μm).
图4 qRT-PCR分析野生型Dongjin与突变体djyg中叶色相关基因的表达。
Fig. 4. Expression analysis of genes associated with leaf color by qRT-PCR in wild-type Dongjin and mutant djyg.
组合 Cross | 总株数 Total number of plants | 正常植株 No. of normal plants | 黄绿植株 No. of yellow green plants | χ2(3∶1) | |
---|---|---|---|---|---|
9311×djyg | 781 | 592 | 189 | 0.23 | 3.84 |
Dongjin×djyg | 822 | 629 | 193 | 0.93 | 3.84 |
表4 突变体djyg的遗传分析
Table 4 Genetic analysis of mutant djyg.
组合 Cross | 总株数 Total number of plants | 正常植株 No. of normal plants | 黄绿植株 No. of yellow green plants | χ2(3∶1) | |
---|---|---|---|---|---|
9311×djyg | 781 | 592 | 189 | 0.23 | 3.84 |
Dongjin×djyg | 822 | 629 | 193 | 0.93 | 3.84 |
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