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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (9): 1301-1310.doi: 10.3724/SP.J.1006.2018.01301

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Phenotypic Analysis and Gene Mapping of the Rice Narrow-leaf Mutant nal20

Hai-Xin LONG(),Hai-Yang QIU,UZAIR Muhammad,Jing-Jing FANG,Jin-Feng ZHAO,Xue-Yong LI()   

  1. National Key Facility for Crop Gene Resource and Genetic Improvement, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2018-02-07 Accepted:2018-06-12 Online:2018-09-10 Published:2018-07-02
  • Contact: Xue-Yong LI E-mail:lhxbling@126.com;lixueyong@caas.cn
  • Supported by:
    This study was supported by the National Major Project for Developing New GM Crops (2016ZX08009-003)

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Abstract:

Leaf is the major organ for photosynthesis in plant, and its area influences the light energy utilization efficiency and grain yield. To study the molecular mechanism of rice leaf morphology, we mutagenized the japonica cultivar Chunjiang 06 through 60Co-γ radiation. A stable narrow leaf mutant named as narrow leaf20 (nal20) was obtained in the M2 population. The nal20 mutant showed narrow leaf, reduced plant height, increased tiller number, shortened internodes, and earlier heading date. Here we mainly focused on the morphology of the flag leaf, the second top leaf and the third top leaf. We found that the blade width of the mutant reduced by 50%, compared with the wild type. Meanwhile, the variation on the blade length was relatively small. Cytological observation of leaf epidermal cells indicated that the reduced leaf width was mainly due to the decreased cell number, but not the cell size. Genetic analysis indicated that the narrow leaf phenotype was controlled by a recessive gene. Using the mutant plants from the F2 mapping population derived from a cross between nal20 and Dular, the candidate mutation locus was mapped to 1.9 Mb within the centromere of chromosome 7 by using polymorphic InDel markers. The next-generation sequencing result showed that a deletion of 455 kb occurred in the predicted region. Our study lays a good foundation for the cloning and functional study of the NAL20 gene and provides gene resources and breeding materials for the improvement of rice plant architecture.

Key words: rice, leaf, narrow-leaf mutant, heading date, gene mapping

Fig. 1

Leaf width of wild type (WT) and the nal20 mutantA: flag leaf size of wild type and the nal20 mutant at heading stage (bar = 10 mm); B: leaf width comparison of flag, top second, and top third leaves between WT and nal20. P-values were analyzed using Student’s t-tests. ** significantly different at P < 0.01 (n = 20)."

Fig. 2

Leaf length of wild type (WT) and the nal20 mutantA: top leaf size of wild type and the nal20 mutant at heading stage (bar = 5 cm); B: length comparison of flag, second, and top third leaves between WT and nal20. P-values were analyzed using Student’s t-tests. * P < 0.05, ** P < 0.01 (n = 20)."

Table 1

Comparison of leaf width and heading date under different daylength conditions"

性状
Trait		 日照条件
Daylength condition		 野生型春江06
Wild-type Chunjiang 06		 nal20
Mutant nal20
剑叶宽度 长日照(北京昌平) Long-day (Changping, Beijing) 15.10±0.67 8.60±0.49**
Flag leaf width (mm) 短日照(海南三亚) Short-day (Sanya, Hainan) 15.62±0.85 8.27±0.46**
抽穗天数 长日照(北京昌平) Long-day (Changping, Beijing) 131.65±2.04 94.92±1.57**
Heading date (d) 短日照(海南三亚) Short-day (Sanya, Hainan) 89.96±1.82 92.31±1.79

Fig. 3

Leaf vein number of wild type (WT) and the nal20 mutantA, C, E: leaf vein shape of the flag leaf, top second leaf and top third leaf in the wild type (bars = 1 mm); B, D, F: leaf vein of the flag leaf, top second leaf and top third leaf in the mutant (bars = 1 mm); G: comparison of large vein number in the wild type and mutant; H: comparison of small vein number in the wild type and mutant. P-values were analyzed using Student’s t-tests. ** P < 0.01 (n = 20)."

Fig. 4

Epidermal cell width and cell number of wild type (WT) and the nal20 mutant A, B: Epidermal cell shape of flag leaf in the wild-type (A) and mutant (B) (bars = 50 μm); C, D: comparison of epidermal cell width and cell number along leaf-width axis. P-values were analyzed from Student’s t-tests. ** P < 0.01 (n = 20)."

Fig. 5

Gross morphology of wild type (WT) and the nal20 mutantA: plant type of wild type and the nal20 mutant at heading stage (bar = 5 cm). Comparison of plant height (B) and tiller number (C) between WT and nal20. P-values were analyzed using Student’s tests. ** P < 0.01 (n = 20)."

Fig. 6

Panicle morphology and internode length of wild type (WT) and the nal20 mutantA: panicle shape of wild type and the nal20 mutant at heading stage; B: comparison of grain number per panicle. The P-values were analyzed from student’s t-tests. ** P < 0.01 (n = 20); C: internodes of wild type and the nal20 mutant at heading stage; D: comparison of internode length (from the top to bottom is panicle, panicle internode, the first internode, the second internode, the third internode, the fourth internode, the fifth internode, and the sixth internode)."

Table 2

InDel markers"

分子标记
Marker		 正向引物序列
Forward primer sequence (5°-3°)		 反向引物序列
Reverse primer sequence (5°-3°)
R7-3 GGCAAGTTAAAACCGAGCAG CCATGGAAGGCTGTAACCAT
R7-4 GATAGCTTGACAACGGTGGCAC CCATACATTGTTGCACTTGTGAC
R7-6 CCCCATGAGGCCTACACTT AGCAGCATAATCAGATGAGACG
R7-7 ATCGGTGCCGCTCCTAGAT CACTCCACAGACATGCAATTT
R7-8 TTCCAGGCTGCATCTTATTC GCAGGACCCATGCTGAAAAG
C7-1 ATCTAGCGGCTAGCACACTGG ATCACCTCATGTCTCCGGACG
C7-2 ACTGTGTGCTGCCTGACATAC AGACGAATGGTCAAACATGTG
C7-3 GATGGTAGGAGGCCGGACTGG GCCTCCTTTACTACCGACCGC
C7-4 GTGGTGACAATGTGGTACAAT CCACTTATACGTGCGTAACAC
C7-5 CGCCGTTCGCATAAAGTCCTG TTGGACTTGTTTGGGCATACG
C7-6 AAGGACTTGCCGTTTGATCTC CATGATCGGTACTAGCAATTC

Fig. 7

Gene mapping of the nal20 mutantA: primary mapping result; B: fining mapping result; C: target gene region. The dashed line represents the 455 kb deletion. In panels A and B, the InDel markers and number of recombinants are marked above and below the horizontal line, respectively."

Table 3

Genes annotated in the 455 kb deletion region"

基因
Gene		 功能注释
Function annotation
LOC_Os07g15640 CRR4, putative, expressed
LOC_Os07g15650 Expressed protein
LOC_Os07g15670 Peroxiredoxin, putative, expressed
LOC_Os07g15680 Phospholipase D, putative, expressed
LOC_Os07g15720 Hypothetical protein
LOC_Os07g15770 CCT motif family protein, expressed
LOC_Os07g15820 Expressed protein
LOC_Os07g15860 Expressed protein
LOC_Os07g15870 Expressed protein
LOC_Os07g15880 Mitochondrial prohibitin complex protein 2, putative, expressed
LOC_Os07g15910 Expressed protein
LOC_Os07g15920 Expressed protein
LOC_Os07g15930 Legume lectins beta domain containing protein, expressed
LOC_Os07g15940 Legume lectins beta domain containing protein, expressed
LOC_Os07g15950 Expressed protein
LOC_Os07g15959 Expressed protein
LOC_Os07g15970 Erythronate-4-phosphate dehydrogenase domain containing protein, expressed
LOC_Os07g15980 Expressed protein
LOC_Os07g16030 Expressed protein
LOC_Os07g16040 Erythronate-4-phosphate dehydrogenase domain containing protein, expressed
LOC_Os07g16054 Hypothetical protein
LOC_Os07g16130 Acetyltransferase, GNAT family, putative, expressed
LOC_Os07g16140 FAD binding protein, putative, expressed
LOC_Os07g16150 Expressed protein
LOC_Os07g16180 Hypothetical protein
LOC_Os07g16210 Expressed protein
LOC_Os07g16224 Piwi domain containing protein, putative, expressed
LOC_Os07g16260 Expressed protein
LOC_Os07g16270 Hypothetical protein
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