Genetic Analysis and Gene Mapping of a Yellow-Green Leaf Mutant djyg in Rice

doi:10.3969/j.issn.1001G7216.2015.06.006

Abstract

Abstract:

A spontaneous yellow-green mutant was obtained from the japonica rice cultivar Dongjin in the field, designated as djyg. The mutant showed obvious yellow-green leaf phenotype at the seedling stage but the chlorotic leaves returned to green after the heading stage. Consistent with the phenotype, the chlorophyll b content of the mutant was reduced by 53%, 62% and 36% at the seedling, tillering and heading stages, respectively, compared with that of the wild type (WT). At the tillering stage, we observed that the granas and thylakoid stacks displayed a mess and loose structure, as well as less density in the mutant djyg compared with the WT under a transmission electron microscopy. The transcriptional levels of PORA, Cab1R, PsbA were also decreased in the mutant djyg. Genetic analysis and map-based cloning demonstrated that the phenotype of the mutant was controlled by a major recessive nuclear gene, which is an allele of chlorophyll synthase YGL1. The mutant djyg can be used as a phenotypic marker in rice breeding program since the main agronomic traits of the mutant djyg are normal compared with those of the WT.

Key words: Oryza sativa L., yellow-green leaf, mutant, map-based cloning

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。

关键词: 水稻, 黄绿叶, 突变体, 图位克隆

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.

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.

Figures/Tables 9

References 28

[1]	Khush G S.Feeding five billion rice consumers-The role of rice breeding.Adv Rice Sci, 2004: 3-15.
[2]	Wang P R, Gao J X, Wan C M, et al.Divinyl chlorophyll (ide) a can be converted to monovinyl chlorophyll (ide) a by a divinyl reductase in rice.Plant Physiol, 2010, 153: 994-1003.
[3]	Kusumi K, Sakata C, Nakamura T, et al.A plastid protein NUS1 is essential for build-up of the genetic system for early chloroplast development under cold stress conditions.Plant J, 2011, 68(6): 1039-1050.
[4]	Su N, Hu M L, Wu D X, et al.Disruption of a rice pentatricopeptide repeat protein causes a seedling-specific albino phenotype and its utilization to enhance seed purity in hybrid rice production.Plant Physiol, 2012, 59(1): 227-238.
[5]	Shu Q Y, Liu G F, Xia Y W.Temperature-sensitve leaf color mutation in rice (Oryza sativa L.).Acta Agric Nucl Sini, 1995, 10: 6-10.
[6]	邓晓娟,张海清,王悦,等. 水稻叶色突变基因研究进展. 杂交水稻, 2012,27(5): 9-14.
[7]	Deng X J, Zhang H Q, Wang Y, et al.Mapped clone and functional analysis of leaf-color gene ygl7 in a rice hybrid (Oryza sativa L. ssp. indica).PloS One, 2014, 9(6): e99564.
[8]	Zhou Y, Gong Z Y, Yang Z F, et al.Mutation of the light induced yellow leaf 1 gene, which encodes a geranyl reductase, affects chlorophyll biosynthesis and light sensitivity in rice.PloS One, 2013(8): e75299.
[9]	Tian X Q, Ling Y H, Fang L K, et al.Gene cloning and functional analysis of yellow green leaf 3(ygl3) gene during the whole-plant growth stage in rice.Genes Genom, 2013, 35: 87-93.
[10]	Jiang H W, Li M R, Liang N T, et al.Molecular cloning and function analysis of the stay green gene in rice.Plant J, 2007, 52: 197-209.
[11]	董青,张迎信,张振华,等.水稻黄绿叶突变体W390的遗传分析和基因定位.中国水稻科学, 2015, 29(3): 241-249.
[12]	Huang X Q, Wang P R, Zhao H X, et al.Genetic analysis and molecular mapping of a novel chlorophyll-deficit mutant gene in rice.Rice Sci, 2008, 15: 7-12.
[13]	Wang P R, Ma X Z, Li C M, et al.Comparative analysis of three mutants of divinyl reductase gene in rice.Sci Agric Sin(Chinese Version), 2013, 46: 1305-1313.
[14]	Sakuraba Y, Rahman M L, Cho S H, et al.The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions.Plant J, 2013, 74: 122-133.
[15]	Wang P R, Wang B, Sun X Q, et al.Fine mapping and physiological characteristics of a green-revertible albino gene gra75 in rice.Sci Agric Sin (Chinese Version), 2013, 46: 225-232.
[16]	Wu Z, Zhang X, He B, et al.A chlorophyll-deficient rice mutant with impaired chlorophyllide esterification in chlorophyll biosynthesis.Plant Physiol, 2007, 145(1): 29-40.
[17]	Wang P, Li C, Wang Y, et al.Identification of a geranylgeranyl reductase gene for chlorophyll synthesis in rice.Springer Plus, 2014, 3(1): 201.
[18]	Wan C, Li C, Ma X, et al.GRY79 encoding a putative metallo-β-lactamase-trihelix chimera is involved in chloroplast development at early seedling stage of rice.Plant Cell Rep, 2015: 1-11.
[19]	Harmut K.Chlorophyls and carotenoids: Pigments of photosynthetic biomembranes.Methods Enzymol, 1987, 148(34): 350-382.
[20]	Nie H, Zhao C, Wu G, et al.SR1, a calmodulin-binding transcription factor, modulates plant defense and ethylene-induced senescence by directly regulating NDR1 and EIN3.Plant Physiol, 2012, 158: 1847-1859.
[21]	Pogers S O, Bendich A J.Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues.Plant Mol Biol, 1985, 5: 69-76.
[22]	Zhang H T, Li J J, Yoo J H, et al.Rice Chlorina-1 and Chlorina-9 encode ChlD and ChlI subunits of Mg-chelatase, a key enzyme for chlorophyll synthesis and chloroplast development.Plant Mol Biol, 2006, 62: 325-337.
[23]	Sun X Q, Wang B, Xiao Y H, et al.Genetic analysis and fine mapping of gene ygl98 for yellow-green leaf of rice.Acta Agrono Sinica(Chinese Version), 2011, 37: 991-997.
[24]	Rüdiger W.Biosynthesis of chlorophyll b and the chlorophyll cycle.Phot Res, 2002, 74:187-193.
[25]	Stenbaek A, Jensen P E.Redox regulation of chlorophyll biosynthesis.Phyto Chem, 2010, 71: 853-859.
[26]	Tang Y L, Huang J F, Wang R C.Change law of hyperspectral data in related with chlorophyll and carotenoid in rice at different developmental stages.Rice Sci, 2004, 11: 274-282.
[27]	Yasuhito S, Md L R, Cho S H, et al.The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions.Plant J, 2013, 74: 122-133.
[28]	Baumgartner B J, Rapp J C, Mullet J E, et al.Plastid genes encoding the transcription/translation apparatus are differentially transcribed early in barley (hordeum vulagare) chloroplast development (evidence for selective stabilization of psbA mRNA).Plant Physiol, 1993, 101: 781-791.

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	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		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

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