Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (5): 825-836.doi: 10.3864/j.issn.0578-1752.2022.05.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Mapping of QTLs for Chlorophyll Content in Flag Leaves of Rice on High-Density Bin Map

ZHAO Ling(),ZHANG Yong,WEI XiaoDong,LIANG WenHua,ZHAO ChunFang,ZHOU LiHui,YAO Shu,WANG CaiLin,ZHANG YaDong()   

  1. Institute of Food Crops, Jiangsu Academy of Agricultural Sciences/Jiangsu High Quality Rice R&D Center /Nanjing Branch of China National Center for Rice Improvement, Nanjing 210014
  • Received:2021-10-20 Accepted:2021-11-25 Online:2022-03-01 Published:2022-03-08
  • Contact: YaDong ZHANG E-mail:zhaoling@jaas.ac.cn;zhangyd@jaas.ac.cn

Abstract:

【Objective】Finding new loci and genes related to rice chlorophyll content, and providing new theoretical basis for the research on the genetic mechanism of rice chlorophyll content. 【Method】 A recombinant inbred line (RIL) population containing 186 lines was constructed by crossing the japonica rice TD70 and the indica rice Kasalath with obvious difference in the chlorophyll content of the flag leaf. The two parents and RIL population were re-sequenced to construct a high-density genetic linkage map with 12 328 recombination Bin markers. The RILs and two parents were planted in fields at the Jiangsu Academy of Agricultural Sciences, in Nanjing in 2011 and 2020. The contents of chlorophyll of flag leaves were directly measured using the chlorophyll meter SPAD-502 on the 3rd day after heading. QTLs that control the chlorophyll content of the flag leaf at the heading stage of rice were detected by IciMappingv3.4 software with inclusive compound interval mapping method. The photosynthesis parameters of 20 SPAD extreme strains in the RIL population were measured with a portable photosynthesis system. 【Result】19 QTLs controlling chlorophyll content of flag leaves were detected on 9 chromosomes except Chr.8, Chr.9 and Chr.10 in two years. The phenotype variation explained (PVE) of single QTL ranged from 3.09% to 13.13%, LOD value ranged from 2.74 to 14.08. After comparing the physical positions, 10 QTLs were found to locate in the same interval or adjacent to previously QTLs. qCHL2-1 and qCHL5-1 were detected every year showing their genetic stability. qCHL2-1 was mapped between the 7.63-7.71 Mb on chromosome 2, and the two-year LOD values are 14.08 and 7.93 with the PVE 13.13% and 7.94%, respectively. qCHL5-1 was mapped between the 23.44-23.49 Mb on chromosome 5, and the two-year LOD values are 4.31 and 3.76, respectively. After the annotation and sequences analysis of genes located in the region of qCHL2-1and qCHL5-1, two genes, Os02g0236000 and Os05g0476700, were found to be associated with chlorophyll content of flag leaves in the rice. There are differences in sequences of the two genes between TD70 and Kasalath. Os02g0236000 is the AAT1 gene encoding the Aspartate Aminotransferase, which is an important enzyme in nitrogen metabolism and related to protein and amino acid content of rice. Os05g0476700 encodes protein relating to spotted leaf, which might associate with leaf color. Based on the mutation of AAT1 at CDS+273 bp, the haplotypes of ATT1 were classified in RIL population. Among the 20 extreme SPAD RIL lines, there were significant differences between different haplotype of ATT1 in SPAD value, chlorophyll content, water use efficiency, transpiration rate, stomatal conductance and net photosynthetic rate of flag leaf. 【Conclusion】19 QTLs associated with chlorophyll content in flag leaf at heading stage of rice were detected and two stable QTL loci, qCHL2-1and qCHL5-1 were identified. Two candidate genes were obtained after annotation and sequence comparison. One of them, ATT1, was considered as the most possible candidate gene after effort analysis of different haplotypes in photosynthetic efficiency. The QTLs and gene we obtained could be used for subsequent functional studies of flag leaf chlorophyll regulation and molecular marker breeding.

Key words: rice (Oryza sativa L.), recombinant inbred lines, high-density bin map, chlorophyll content, QTL

Fig. 1

Flag leaves of RIL population and two parents at the heading time"

Table 1

Chlorophyll content of flag leaves among the RIL population and two parents at the heading time"

年份
Years
亲本Parents 重组自交系RIL population
TD70 Kasalath 平均值Average 变异范围 Range 变异系数 CV (%) 峰度 Kurtosis 偏度 Skewness
2011 48.20±0.70 34.50±0.78 42.08 30.51—49.00 9.29 -0.42 -0.42
2020 46.20±1.37 38.10±0.72 40.58 27.53—50.83 9.36 0.13 -0.11

Fig. 2

Distribution of chlorophyll content of flag leaves among RIL population"

Table 2

Identification of QTL contributing to rice chlorophyll content of flag leaves in RIL population"

年份
Year
QTL 染色体
Chr.
标记区间
Marker interval
置信区间
Confidence interval (Mb)
LOD 贡献率
PVE (%)
加性效应
Additive effect
2011 qCHL1-1 1 RBN0894—RBN0895 25.69—25.72 6.46 6.47 -1.43
qCHL1-2 1 RBN1045—RBN1046 31.76—31.83 6.45 5.55 -1.18
qCHL2-1* 2 RBN1570—RBN1571 7.63—7.71 14.08 13.13 -1.81
qCHL2-2 2 RBN2335—RBN2336 35.12—35.15 2.74 3.76 -1.14
qCHL3-1 3 RBN3011—RBN3012 25.41—25.44 4.85 4.04 0.99
qCHL5-1* 5 RBN5254—RBN5255 23.44—23.49 4.31 3.57 0.91
qCH6-1 6 RBN6496—RBN6497 29.42—29.48 4.67 3.92 -1.11
qCH7 7 RBN7384—RBN7385 24.35—24.37 5.09 7.79 2.37
2020 qCHL1-3 1 RBN0126—RBN0127 3.16—3.22 8.22 10.95 1.42
qCHL1-4 1 RBN0145—RBN0146 3.93—3.95 9.19 9.64 1.32
qCHL2-1* 2 RBN1570—RBN1571 7.63—7.71 7.93 7.94 -1.24
qCHL2-3 2 RBN2330—RBN2331 35.03—35.10 3.27 8.01 -1.28
qCHL3-2 3 RBN2365—RBN2366 0.33—0.36 4.52 3.54 0.93
qCHL4 4 RBN4072—RBN4073 23.30—23.38 7.47 7.71 -1.17
qCHL5-2 5 RBN4489—RBN4490 0.19—0.26 4.72 4.68 1.11
qCHL5-1* 5 RBN5254—RBN5255 23.44—23.49 3.76 4.82 0.86
qCH6-2 6 RBN6322—RBN6323 24.40—24.50 5.63 5.68 -1.01
qCHL11 11 RBN10911—RBN10912 20.63—20.70 4.00 3.89 0.84
qCHL12-1 12 RBN11570—RBN11571 8.13—8.45 2.84 3.09 1.73
qCHL12-2 12 RBN12254—RBN12255 25.50—25.54 6.10 4.98 -1.03
qCHL12-3 12 RBN12262—RBN12263 25.70—25.74 3.84 5.31 -0.98

Table 3

Annotated genes in interval of qCHL2-1 and qCHL5-1"

QTL 染色体
Chr.
物理距离
Interval (Mb)
基因
Gene
基因功能注释
Annotation
qCHL2-1 2 7.63—7.71 Os02g0235000 肌动蛋白相关蛋白2/3复合亚基3类似蛋白
Similar to Actin-related protein 2/3 complex subunit 3
Os02g0235600 60S核糖体蛋白L11-2 (L16)类似蛋白
Similar to 60S ribosomal protein L11-2 (L16)
Os02g0235900 包含氧氧化还原酶共价FAD结合位点结构域的蛋白
Oxygen oxidoreductase covalent FAD-binding site domain containing protein
Os02g0236000 天冬氨酸氨基转移酶 Aspartate aminotransferase (EC 2.6.1.1)
qCHL5-1 5 23.44—23.49 Os05g0476466 CBL互作蛋白激酶28类似蛋白 Similar to CBL-interacting protein kinase 28
Os05g0476700 U-box E3泛素连接酶,斑点叶 U-box E3 ubiquitin ligase, spotted leaf
Os05g0477300 含有核糖体蛋白S26e结构域蛋白
Ribosomal protein S26e domain containing protein
Os05g0477500 含DUF3615结构域未知功能蛋白
Protein of unknown function DUF3615 domain containing protein
Os05g0477600 α-扩展蛋白OsEXPA4 Alpha-expansion OsEXPA4

Fig. 3

The effect of mutation of AAT1 at CDS+273 bp (T/C) on photosynthesis of 20 extreme SPAD RIL lines HapA: The base is T at CDS+273 bp of AAT1; HapB: The base is C at CDS+273 bp of AAT1. Different lowercase letters indicate significantly different (P<0.05)"

Table 4

Overlap of known QTLs contributed to leaves’ Chlorophyll content with QTLs detected in this study"

本研究This study 已发表的相关位点/基因 Known QTLs/Genes
QTL 染色体
Chr.
物理位置
Position (Mb)
性状/基因
Character/Gene
定位群体/基因登录号
Population /Acc. No.
物理位置
Position (Mb)
参考文献
Reference
qCHL1-1 1 25.69—25.72 抽穗后7 d叶绿素含量
Chlorophyll content at 7d after heading
日本晴/Kasalath//日本晴BILs
Nipponbare/Kasalath//Nipponbare BILs
25.13—26.19 [29]
qCHL1-2 1 31.76—31.83 干旱胁迫下的剑叶或倒2叶叶绿素含量
Chlorophyll content in drought stress
珍汕97B/IRAT109 RILs
Zhenshan 97B/IRAT109 RILs
30.1—33.86 [26]
分蘖期上部展开叶叶绿素含量
Chlorophyll content of up most fully expanded leaf at tillering period
ZYQ8/JX17 DHs 30.06—32.06 [27]
拔节期剑叶叶绿素含量
Chlorophyll content of flag leaf at jointing stage
沈农0530-9/北陆129 F2(F2:3
Shennong0530-9/Habataki F2 (F2:3)
30.17—34.1 [28]
qCHL2-1 2 7.63—7.71 成熟期剑叶叶绿素含量
Chlorophyll content of flag leaf at mature stage
沈农265/丽江新团黑谷RILs
Shennong 265/Lijiangxintuanheigu RILs
2.88—9.47 [11]
分蘖期剑叶叶绿素含量
Chlorophyll content of flag leaf at tillering stage
岗46B/A232 RILs
Gang 46B/A232 RILs
5.20—8.76 [30]
发育期剑叶叶绿素b含量
Leaf chlorophyll b content at developmental stage
ZS97/WY2 DHs 7.43—11.41 [31]
qCHL3-1 3 25.41—25.44 叶绿素b还原酶 Chlorophyll b reductase LOC_Os03g45194 25.52 [37]
qCHL4 4 23.30—23.38 分蘖期剑叶叶绿素含量
Chlorophyll content of flag leaf at tillering stage
岗46B/A232 RILs
Gang 46B/A232 RILs
23.17—31.27 [30]
苗期叶绿素含量
Chlorophyll content at seedling stage
珍汕97A/明恢63 RILs
Zhenshan 97A/Minghui 63 RILs
22.28—26.86 [36]
抽穗5 和25 d叶绿素含量的降低
Decreased chlorophyll content between leaves at 5 and 25 days after heading
Nipponbare/Kasalath BC1F1 22.27—26.38 [38]
qCHL5-1 5 23.44—23.49 抽穗7d剑叶叶绿素b含量
Chlorophyll b content of flag leaf at 7 days after heading
窄叶青8号/京系17 DH
Zhaiyeqing 8/Jingxi 17 DH
19.27—31.45 [32]
齐穗期剑叶叶绿素含量
Chlorophyll b content of flag leaf at full-heading stage
Dular/Lemont RILs 3.89—24.09 [33]
开花期剑叶叶绿素含量
Degree of greenness of flag leaf at heading stage
珍汕97B/IRAT109 RILs
Zhenshan 97/IRAT109 RILs
20.17—26.84 [34]
孕穗期剑叶叶绿素b含量
Chlorophyll b contents of flag leaf at booting stage
十和田/丽江新团黑谷RILs
Towada/Lijiangxintuanheigu RILs
0.46—23.95 [35]
谷氨酸-1-半醛转氨酶基因GSA
Glutamate -1-semialdehyde aminotransferase
LOC_Os05g39770 23.35 [11, 24]
qCHL5-2 5 0.19—0.26 干旱胁迫下的剑叶或倒2叶叶绿素含量
Chlorophyll content of leaves in drought stress
珍汕97B/IRAT109 RILs
Zhenshan 97B/IRAT109 RILs
0.1—0.18 [26]
qCHL6 6 24.40—24.50 干旱胁迫下的剑叶或倒2叶叶绿素含量
Chlorophyll content of leaves in drought stress
珍汕97B/IRAT109 RILs
Zhenshan 97B/IRAT109 RILs
24.03—28.13 [26]
抽穗后5d和25 d叶绿素含量的降低
Decreased chlorophy content between leaves at 5 and 25 days after heading
Nipponbare/Kasalath BC1F1 27.61—31.17 [38]
qCH7 7 24.35—24.37 羟甲基后胆色素原合酶
Hydroxymethylbilane synthase
LOC_Os07g40250 24.13 [11]
硝酸盐转运蛋白基因OsNPF7.1
Nitrate and di/tripeptide transporter OsNPF7.1
LOC_Os07g41250 24.72 [12]
qCHL11 11 20.63—20.70 成熟期剑叶叶绿素含量
Chlorophyll content of flag leaf at mature stage
沈农265/丽江新团黑谷RILs
Shennong 265/Lijiangxintuanheigu RILs
18.13—28.28 [11]
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