Genetic Analysis and Gene Mapping of a Pale Green Leaf Mutant HM133 in Rice

doi:10.16819/j.1001-7216.2016.6033

Abstract

Abstract:

The pale green leaf mutant HM133 was identified from an EMS-induced IR64 mutant bank. The contents of photosynthetic pigments including chlorophyll and carotenoid of HM133 were reduced significantly at 6 weeks and 15 weeks after sowing when compared with IR64.The net photosynthetic rate of HM133 was considerably lower than that of the wild-type IR64 at heading stage while the stomatal conductance was apparently increased. The agronomic traits including plant height, number of filled grain per panicle and seed-setting rate decreased significantly in the mutant compared with the wild-type. In addition, the mutant exhibited a less number of grana, irregular arrangement of thylakoid layer in the chloroplast at the tillering stage. Genetic and mapping analysis showed that the pale green phenotype was controlled by a single recessive gene located in the long arm of chromosome 3 between SSR markers RM143 and RM3684. The interval contains an ORF OsChlD encoding magnesium-chelatase D subunit. Sequence analysis revealed that the mutant allele carried a nucleotide substitution from G to A in the tenth exon of OsChlD, which led to the substitution of glutamic acid for arginine acid. Therefore, it is deduced that OsChlD is the candidate gene controlling the pale green leaf phenotype of HM133.

Key words: Oryza sativa, pale green leaf, gene mapping, magnesium-chelatase, photosynthesis

摘要：

EMS诱导籼稻品种IR64获得淡绿叶突变体HM133。与野生型IR64相比,HM133播种后的第6周和第15周的光合色素含量以及抽穗期的净光合速率显著降低,气孔导度则明显上升;此外,突变体株高、每穗实粒数和结实率等农艺性状也较野生型显著下降。叶绿体超微结构分析表明,分蘖期HM133类囊体基粒片层形状不规则,堆叠凌乱、排列疏松。遗传分析表明HM133淡绿叶性状受单隐性核基因控制。通过分子标记将该基因定位于第3染色体长臂RM143和RM3684之间。该区间内包含编码镁螯合酶D亚基的基因OsCHLD。序列分析表明HM133中该基因第10外显子上有一个从G突变为A的单碱基变异,导致编码的氨基酸由精氨酸变成谷氨酸,推测OsCHLD基因即为控制HM133淡绿叶表型的候选基因。

关键词: 水稻, 淡绿叶, 基因定位, 镁螯合酶, 光合作用

CLC Number:

Yong-feng SHI, Yan HE, Dan GUO, Xiang-guang LV, Qi-na HUANG, Jian-li WU. Genetic Analysis and Gene Mapping of a Pale Green Leaf Mutant HM133 in Rice[J]. Chinese Journal OF Rice Science, 2016, 30(6): 603-610.

施勇烽, 贺彦, 郭丹, 吕向光, 黄奇娜, 吴建利. 水稻淡绿叶突变体HM133的遗传分析与基因定位[J]. 中国水稻科学, 2016, 30(6): 603-610.

Figures/Tables 8

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基因 Gene	正向引物(5'-3') Forward primer(5'-3')	反向引物(5'-3') Reverse primer(5'-3')
OsChlD	GGAAAGAGAGGGCATTAG	CAATACGATCAAGTAAGTGTT
OsChlI	AGTAACCTTGGTGCTGTG	AATCCATCAACATTCAACTCTG
OsChlH	CTATACATTCGCCACACT	TATCACACAACTCCCAAG
HEMA1	CGCTATTTCTGATGCTATGGGT	TCTTGGGTGATGATTGTTTGG
PORA	TGTACTGGAGCTGGAACAACAA	GAGCACAGCAAAATCCTAGACG
CAO1	GATCCATACCCGATCGACAT	CGAGAGACATCCGGTAGAGC
Cab1R	AGATGGGTTTAGTGCGACGAG	TTTGGGATCGAGGGAGTATTT
Cab2R	TGTTCTCCATGTTCGGCTTCT	GCTACGGTCCCCACTTCACT
psaA	GCGAGCAAATAAAACACCTTTC	GTACCAGCTTAACGTGGGGAG
psbA	CCCTCATTAGCAGATTCGTTTT	ATGATTGTATTCCAGGCAGAGC
rbcL	CTTGGCAGCATTCCGAGTAA	ACAACGGGCTCGATGTGATA
rbcS	TCCGCTGAGTTTTGGCTATTT	GGACTTGAGCCCTGGAAGG
YGL1	CAGTCTCCAATGGCCACCT	TGCTTTCATCAGTGGCTGG
SPP	CGGAGAGGAAACATAATGAC	ATAGGCATTTGTCTTTGTCTC
PPR1	CTAAGACCGAATGACAAATGC	GCACTGCCAACAAGAATACC
DVR	CGAGCCCAGGTTCATCAAGGTGC	CCTCCCGATCTTGCCGAACTCC
Ubiquitin	GCTCCGTGGCGGTATCAT	CGGCAGTTGACAGCCCTAG

基因 Gene	正向引物(5'-3') Forward primer(5'-3')	反向引物(5'-3') Reverse primer(5'-3')
OsChlD	GGAAAGAGAGGGCATTAG	CAATACGATCAAGTAAGTGTT
OsChlI	AGTAACCTTGGTGCTGTG	AATCCATCAACATTCAACTCTG
OsChlH	CTATACATTCGCCACACT	TATCACACAACTCCCAAG
HEMA1	CGCTATTTCTGATGCTATGGGT	TCTTGGGTGATGATTGTTTGG
PORA	TGTACTGGAGCTGGAACAACAA	GAGCACAGCAAAATCCTAGACG
CAO1	GATCCATACCCGATCGACAT	CGAGAGACATCCGGTAGAGC
Cab1R	AGATGGGTTTAGTGCGACGAG	TTTGGGATCGAGGGAGTATTT
Cab2R	TGTTCTCCATGTTCGGCTTCT	GCTACGGTCCCCACTTCACT
psaA	GCGAGCAAATAAAACACCTTTC	GTACCAGCTTAACGTGGGGAG
psbA	CCCTCATTAGCAGATTCGTTTT	ATGATTGTATTCCAGGCAGAGC
rbcL	CTTGGCAGCATTCCGAGTAA	ACAACGGGCTCGATGTGATA
rbcS	TCCGCTGAGTTTTGGCTATTT	GGACTTGAGCCCTGGAAGG
YGL1	CAGTCTCCAATGGCCACCT	TGCTTTCATCAGTGGCTGG
SPP	CGGAGAGGAAACATAATGAC	ATAGGCATTTGTCTTTGTCTC
PPR1	CTAAGACCGAATGACAAATGC	GCACTGCCAACAAGAATACC
DVR	CGAGCCCAGGTTCATCAAGGTGC	CCTCCCGATCTTGCCGAACTCC
Ubiquitin	GCTCCGTGGCGGTATCAT	CGGCAGTTGACAGCCCTAG

材料 Material	株高 Plant height /cm	有效穗数 No. of productive panicles	穗长 Panicle length /cm	每穗实粒数 Number of filled grains per panicle	结实率 Seed-setting rate/%	千粒重 1000-grain weight/g
IR64	113.0±1.7	14.0±1.0	25.0±1.4	81.2±7.1	74.6±1.6	27.52±0.38
HM133	108.7±0.6^*	12.7±2.1	24.9±0.3	62.9±7.3^*	68.8±2.1^*	28.35±0.35

材料 Material	株高 Plant height /cm	有效穗数 No. of productive panicles	穗长 Panicle length /cm	每穗实粒数 Number of filled grains per panicle	结实率 Seed-setting rate/%	千粒重 1000-grain weight/g
IR64	113.0±1.7	14.0±1.0	25.0±1.4	81.2±7.1	74.6±1.6	27.52±0.38
HM133	108.7±0.6^*	12.7±2.1	24.9±0.3	62.9±7.3^*	68.8±2.1^*	28.35±0.35

取样时间 Sampling time	材料 Material	叶绿素a Chlorophyll a content	叶绿素b Chlorophyll b content	总叶绿素 Total chlorophyll content	类胡萝卜素 Carotenoid content
播种后6周 6 weeks after sowing	IR64	3.76±0.57	1.00±0.14	4.78±0.67	0.85±0.19
	HM133	2.11±0.06^**	0.46±0.04^**	2.58±0.03^**	0.52±0.10^*
播种后15周 15 weeks after sowing	IR64	2.86±0.29	0.88±0.13	3.77±0.42	0.63±0.04
	HM133	1.54±0.08^**	0.42±0.03^**	1.97±0.12^**	0.33±0.01^**