Rice Science ›› 2020, Vol. 27 ›› Issue (2): 81-85.DOI: 10.1016/j.rsci.2019.04.006
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Long Zhang1,2, Ning Li1,2, Huimin Fang3, Linglong Zhao1,2, Ke Guo1,2, Jing Zhang1,2, Qiaoquan Liu1,2, Ling Jiang4, Cunxu Wei1,2(
)
Received:2019-01-29
Accepted:2019-04-26
Online:2020-03-28
Published:2019-11-28
Long Zhang, Ning Li, Huimin Fang, Linglong Zhao, Ke Guo, Jing Zhang, Qiaoquan Liu, Ling Jiang, Cunxu Wei. Morphological Characteristics and Seed Physiochemical Properties of Two Giant Embryo Mutants in Rice[J]. Rice Science, 2020, 27(2): 81-85.
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| Trait | WT | M12 | M13 |
|---|---|---|---|
| No. of days to heading (d) | 52.7 ± 0.9 a | 53.5 ± 1.4 a | 52.5 ± 1.0 a |
| No. of tillers per plant | 22.7 ± 2.2 a | 22.9 ± 2.0 a | 22.5 ± 1.8 a |
| Plant height (cm) | 61.3 ± 1.9 a | 60.3 ± 1.0 a | 60.2 ± 0.9 a |
| Panicle length (cm) | 11.5 ± 0.3 a | 11.4 ± 0.4 a | 11.6 ± 0.3 a |
| NPBP | 5.4 ± 0.8 a | 5.5 ± 0.8 a | 5.7 ± 0.9 a |
| NSBP | 4.2 ± 0.6 a | 3.9 ± 0.7 a | 4.0 ± 0.8 a |
| Seed-setting rate (%) | 88.3 ± 4.2 a | 86.6 ± 8.5 a | 70.1 ± 7.9 b |
Supplemental Table 1. Comparison of agronomic traits among wild type (WT), mutants M12 and M13 plants.
| Trait | WT | M12 | M13 |
|---|---|---|---|
| No. of days to heading (d) | 52.7 ± 0.9 a | 53.5 ± 1.4 a | 52.5 ± 1.0 a |
| No. of tillers per plant | 22.7 ± 2.2 a | 22.9 ± 2.0 a | 22.5 ± 1.8 a |
| Plant height (cm) | 61.3 ± 1.9 a | 60.3 ± 1.0 a | 60.2 ± 0.9 a |
| Panicle length (cm) | 11.5 ± 0.3 a | 11.4 ± 0.4 a | 11.6 ± 0.3 a |
| NPBP | 5.4 ± 0.8 a | 5.5 ± 0.8 a | 5.7 ± 0.9 a |
| NSBP | 4.2 ± 0.6 a | 3.9 ± 0.7 a | 4.0 ± 0.8 a |
| Seed-setting rate (%) | 88.3 ± 4.2 a | 86.6 ± 8.5 a | 70.1 ± 7.9 b |
Fig. 1. Phenotypic analyses of giant embryo mutants M12 and M13.A, Morphologies of grains. Bar = 1 mm. B, Morphologies of brown rice. Bar = 1 mm. C, Quantification of brown rice length (BRL) (n = 20). D, Quantification of brown rice width (BRW) (n = 20). E, Quantification of brown rice thickness (BRT) (n = 20). F, 1000-brown rice weight (TBRW) (n = 3). G, The ratio of embryo size and seed size (n = 30). H, Weight of 100-embryo (n = 3). I, Longitudinal sections of the embryo of wild type (WT) and two mutants. CP, Coleoptile; RD, Radicle; ST, Scutellum. Bar = 0.2 mm. J, Embryo area of WT and two mutant seeds (n = 3). Data represent Mean ± SD. Different letters indicate a significant difference (P < 0.05).
Supplemental Fig. 1. The allelic test of M12 and M13.The F2 seed morphologies of reciprocal crosses from M12 and M13. Bars, 1 mm.
Fig. 2. Map-based cloning of the M12 and M13 mutation. A, Map-based cloning and identification of the mutation locus. The mutation locus was mapped to a 84.3 kb region between markers Z7-13 and Z7-18 on chromosome 7. A single nucleotide substitution of guanine to adenine occurred in the exon 1 and exon 2 in M12 and M13, respectively. B, Sequence chromatogram of M12. C, Sequence chromatogram of M13 mutation. The mutation site is noted with red box. D, Amino acid sequence of GE gene in M12 and M13. The two point mutations create Ala-113 replacement by Thr and a premature stop codon of Trp-340 in M12 and M13, respectively.
| Number | Locus name | Gene product name |
|---|---|---|
| 1 | LOC_Os07g41230 | esterase, putative, expressed |
| 2 | LOC_Os07g41240 | cytochrome P450, putative, expressed |
| 3 | LOC_Os07g41250 | peptide transporter PTR2, putative |
| 4 | LOC_Os07g41260 | PPR repeat domain containing protein, putative |
| 5 | LOC_Os07g41270 | retrotransposon protein, putative, unclassified |
| 6 | LOC_Os07g41280 | 6-phosphogluconolactonase, putative |
| 7 | LOC_Os07g41290 | DEFL13-Defensin and Defensin-like DEFL family |
| 8 | LOC_Os07g41300 | respiratory-chain NADH dehydrogenase |
| 9 | LOC_Os07g41310 | COBRA, putative, expressed |
| 10 | LOC_Os07g41320 | COBRA-like protein precursor, putative, expressed |
| 11 | LOC_Os07g41330 | mitochondrial import inner membrane translocase subunit Tim17 |
| 12 | LOC_Os07g41340 | B12D protein, putative, expressed |
| 13 | LOC_Os07g41350 | B12D protein, putative, expressed |
| 14 | LOC_Os07g41360 | alpha-1,4-glucan-protein synthase, putative, expressed |
Supplemental Table 2. Fine mapping region contained 14 predicted genes.
| Number | Locus name | Gene product name |
|---|---|---|
| 1 | LOC_Os07g41230 | esterase, putative, expressed |
| 2 | LOC_Os07g41240 | cytochrome P450, putative, expressed |
| 3 | LOC_Os07g41250 | peptide transporter PTR2, putative |
| 4 | LOC_Os07g41260 | PPR repeat domain containing protein, putative |
| 5 | LOC_Os07g41270 | retrotransposon protein, putative, unclassified |
| 6 | LOC_Os07g41280 | 6-phosphogluconolactonase, putative |
| 7 | LOC_Os07g41290 | DEFL13-Defensin and Defensin-like DEFL family |
| 8 | LOC_Os07g41300 | respiratory-chain NADH dehydrogenase |
| 9 | LOC_Os07g41310 | COBRA, putative, expressed |
| 10 | LOC_Os07g41320 | COBRA-like protein precursor, putative, expressed |
| 11 | LOC_Os07g41330 | mitochondrial import inner membrane translocase subunit Tim17 |
| 12 | LOC_Os07g41340 | B12D protein, putative, expressed |
| 13 | LOC_Os07g41350 | B12D protein, putative, expressed |
| 14 | LOC_Os07g41360 | alpha-1,4-glucan-protein synthase, putative, expressed |
Fig. 3. Contents of γ-aminobutyric acid (GABA) (A), protein (B), starch (C) and amylose (D) in brown rice flour of wild type (WT) and two mutants (M12 and M13). Different letters indicate a significant difference (n = 3, P < 0.05).
| Amino acid | WT | M12 | M13 |
|---|---|---|---|
| Glutamic acid | 0.144 ± 0.002 a | 0.173 ± 0.000 b | 0.171 ± 0.002 b |
| Aspartic acid | 0.076 ± 0.001 a | 0.092 ± 0.001 b | 0.090 ± 0.001 b |
| Serine | 0.044 ± 0.001 a | 0.052 ± 0.001 b | 0.051 ± 0.001 b |
| Threonine | 0.031 ± 0.000 a | 0.037 ± 0.001 b | 0.036 ± 0.000 b |
| Glycine | 0.040 ± 0.001 a | 0.047 ± 0.001 b | 0.047 ± 0.001 b |
| Alanine | 0.048 ± 0.001 a | 0.057 ± 0.001 b | 0.058 ± 0.001 b |
| Cysteine | 0.016 ± 0.000 a | 0.018 ± 0.001 b | 0.017 ± 0.000 ab |
| Valine | 0.046 ± 0.000 a | 0.054 ± 0.000 b | 0.054 ± 0.001 b |
| Methionine | 0.015 ± 0.000 a | 0.017 ± 0.000 b | 0.017 ± 0.000 b |
| Isoleucine | 0.033 ± 0.000 a | 0.039 ± 0.000 b | 0.039 ± 0.001 b |
| Leucine | 0.068 ± 0.000 a | 0.080 ± 0.000 b | 0.079 ± 0.001 b |
| Tyrosine | 0.033 ± 0.001 a | 0.038 ± 0.000 b | 0.038 ± 0.000 b |
| Phenylalanin | 0.045 ± 0.000 a | 0.053 ± 0.000 b | 0.053 ± 0.001 b |
| Lysine | 0.034 ± 0.000 a | 0.041 ± 0.000 b | 0.041 ± 0.000 b |
| Histidine | 0.020 ± 0.000 a | 0.025 ± 0.000 b | 0.025 ± 0.000 b |
| Arginine | 0.068 ± 0.001 a | 0.081 ± 0.001 b | 0.083 ± 0.001 b |
| Proline | 0.033 ± 0.000 a | 0.038 ± 0.000 b | 0.039 ± 0.000 b |
| Total amino acid | 0.790 ± 0.007 a | 0.941 ± 0.006 b | 0.936 ± 0.012 b |
| Essential amino acid | 0.304 ± 0.002 a | 0.358 ± 0.001 b | 0.356 ± 0.004 b |
Table 1 Amino acid contents in brown rice. mg/g
| Amino acid | WT | M12 | M13 |
|---|---|---|---|
| Glutamic acid | 0.144 ± 0.002 a | 0.173 ± 0.000 b | 0.171 ± 0.002 b |
| Aspartic acid | 0.076 ± 0.001 a | 0.092 ± 0.001 b | 0.090 ± 0.001 b |
| Serine | 0.044 ± 0.001 a | 0.052 ± 0.001 b | 0.051 ± 0.001 b |
| Threonine | 0.031 ± 0.000 a | 0.037 ± 0.001 b | 0.036 ± 0.000 b |
| Glycine | 0.040 ± 0.001 a | 0.047 ± 0.001 b | 0.047 ± 0.001 b |
| Alanine | 0.048 ± 0.001 a | 0.057 ± 0.001 b | 0.058 ± 0.001 b |
| Cysteine | 0.016 ± 0.000 a | 0.018 ± 0.001 b | 0.017 ± 0.000 ab |
| Valine | 0.046 ± 0.000 a | 0.054 ± 0.000 b | 0.054 ± 0.001 b |
| Methionine | 0.015 ± 0.000 a | 0.017 ± 0.000 b | 0.017 ± 0.000 b |
| Isoleucine | 0.033 ± 0.000 a | 0.039 ± 0.000 b | 0.039 ± 0.001 b |
| Leucine | 0.068 ± 0.000 a | 0.080 ± 0.000 b | 0.079 ± 0.001 b |
| Tyrosine | 0.033 ± 0.001 a | 0.038 ± 0.000 b | 0.038 ± 0.000 b |
| Phenylalanin | 0.045 ± 0.000 a | 0.053 ± 0.000 b | 0.053 ± 0.001 b |
| Lysine | 0.034 ± 0.000 a | 0.041 ± 0.000 b | 0.041 ± 0.000 b |
| Histidine | 0.020 ± 0.000 a | 0.025 ± 0.000 b | 0.025 ± 0.000 b |
| Arginine | 0.068 ± 0.001 a | 0.081 ± 0.001 b | 0.083 ± 0.001 b |
| Proline | 0.033 ± 0.000 a | 0.038 ± 0.000 b | 0.039 ± 0.000 b |
| Total amino acid | 0.790 ± 0.007 a | 0.941 ± 0.006 b | 0.936 ± 0.012 b |
| Essential amino acid | 0.304 ± 0.002 a | 0.358 ± 0.001 b | 0.356 ± 0.004 b |
| Material | To (ºC) | Tp (ºC) | Tc (ºC) | ΔH (J/g) | PV (MPa/s) | HV (MPa/s) | BV (MPa/s) | FV (MPa/s) | SV (MPa/s) |
|---|---|---|---|---|---|---|---|---|---|
| WT | 61.9 ± 0.1 b | 70.0 ± 0.0 c | 77.2 ± 0.1 b | 4.8 ± 0.2 a | 1 314 ± 38 c | 955 ± 44 c | 359 ± 6 b | 2 002 ± 67 c | 1 047 ± 23 b |
| M12 | 60.3 ± 0.4 a | 68.4 ± 0.2 a | 75.7 ± 0.2 a | 4.7 ± 0.1 a | 1 104 ± 83 b | 709 ± 11 b | 395 ± 72 b | 1 529 ± 50 b | 820 ± 39 a |
| M13 | 60.8 ± 0.3 a | 69.0 ± 0.0 b | 76.2 ± 0.1 a | 4.7 ± 0.1 a | 684 ± 6 a | 461 ± 23 a | 223 ± 16 a | 1 239 ± 13 a | 778 ± 10 a |
Table 2 Thermal properties and pasting properties of brown rice flour.
| Material | To (ºC) | Tp (ºC) | Tc (ºC) | ΔH (J/g) | PV (MPa/s) | HV (MPa/s) | BV (MPa/s) | FV (MPa/s) | SV (MPa/s) |
|---|---|---|---|---|---|---|---|---|---|
| WT | 61.9 ± 0.1 b | 70.0 ± 0.0 c | 77.2 ± 0.1 b | 4.8 ± 0.2 a | 1 314 ± 38 c | 955 ± 44 c | 359 ± 6 b | 2 002 ± 67 c | 1 047 ± 23 b |
| M12 | 60.3 ± 0.4 a | 68.4 ± 0.2 a | 75.7 ± 0.2 a | 4.7 ± 0.1 a | 1 104 ± 83 b | 709 ± 11 b | 395 ± 72 b | 1 529 ± 50 b | 820 ± 39 a |
| M13 | 60.8 ± 0.3 a | 69.0 ± 0.0 b | 76.2 ± 0.1 a | 4.7 ± 0.1 a | 684 ± 6 a | 461 ± 23 a | 223 ± 16 a | 1 239 ± 13 a | 778 ± 10 a |
| Primer name | Forward primer (5'-3') | Reverse primer (5'-3') |
|---|---|---|
| Z7-9 | TCTCCTCTTCCCCCGATC | ATAGCGGGCGAGGCTTAG |
| Z7-10 | ACAGTATCCAAGGCCCTGG | CACGTGAGACAAAGACGGAG |
| Z7-11 | GCCCACCTGTCATTGAGAGTA | GTTTTTGCGCTTTTGTTGCT |
| Z7-13 | GGAGTATTTTAGTAGGCTATTA | TAAAGTTCAAAGATACAAGAAAT |
| Z7-14 | TGCTTGCTTCGATCTGATC | GTTGTGACTTGTGAAGAAGG |
| Z7-15 | AAGAGCTTCTTGACGAGGTA | ATGGATGGATATGAACAGTGC |
| Z7-17 | CTCGGAGAAATTGCCGTTC | GTCACCTCACCACCTTCTCC |
| Z7-18 | AACACCTTGAATCTTTCCACGT | GAATTTGAACCATATTAGCTA |
Supplemental Table 3. Primers used in mapping GE gene
| Primer name | Forward primer (5'-3') | Reverse primer (5'-3') |
|---|---|---|
| Z7-9 | TCTCCTCTTCCCCCGATC | ATAGCGGGCGAGGCTTAG |
| Z7-10 | ACAGTATCCAAGGCCCTGG | CACGTGAGACAAAGACGGAG |
| Z7-11 | GCCCACCTGTCATTGAGAGTA | GTTTTTGCGCTTTTGTTGCT |
| Z7-13 | GGAGTATTTTAGTAGGCTATTA | TAAAGTTCAAAGATACAAGAAAT |
| Z7-14 | TGCTTGCTTCGATCTGATC | GTTGTGACTTGTGAAGAAGG |
| Z7-15 | AAGAGCTTCTTGACGAGGTA | ATGGATGGATATGAACAGTGC |
| Z7-17 | CTCGGAGAAATTGCCGTTC | GTCACCTCACCACCTTCTCC |
| Z7-18 | AACACCTTGAATCTTTCCACGT | GAATTTGAACCATATTAGCTA |
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