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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (2): 292-303.doi: 10.3724/SP.J.1006.2022.12013

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

Phenotypic and genetic analyses of a rice mutant eed1 with defected embryo and endosperm development

YANG Jin1,2(), BAI Ai-Ning3, BAI Xue3, CHEN Juan3, GUO Lin1, LIU Chun-Ming1,3,*()   

  1. 1Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    2Graduate School, Chinese Academy of Agricultural Sciences, Beijing 100081, China
    3Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
  • Received:2021-02-18 Accepted:2021-04-26 Online:2022-02-12 Published:2021-05-17
  • Contact: LIU Chun-Ming E-mail:jinyang201587@163.com;liuchunming@caas.cn
  • Supported by:
    This study was supported by the Beijing Scientific and Technological Research Program(Z181100002418010);the CAS-Commonwealth Scientific and Industrial Research Organization Bilateral Collaboration Project(151111KYSB20180049);the National Major Project for Developing New GM Crops(2019ZX08010-001)

Abstract:

A stably inherited embryo and endosperm defective mutant, named embryo and endosperm defective 1 (eed1), was obtained from the mutant population of rice Zhonghua 11, generated by ethylmethane sulfonate (EMS). The 1000-grain weight, grain length, grain width, grain thickness, germination rate, total starch, amylose, and storage protein contents in eed1 mature caryopses were significantly decreased compared with wild type. In eed1, the caryopses were shrunken and the endosperm was opaque. Scanning electron microscopy observation revealed that starch granules in eed1 endosperm cells were loosely packed, in single, disperse and spherical forms. The embryo of eed1 was abnormal, and some caryopses showed no sign of embryonic differentiation. Most genes involved in biosynthesis of starch and storage protein were down-regulated in eed1 endosperm by qRT-PCR. EED1 was mapped in an interval of 672 kb on chromosome 9 using a F2 population derived from a cross between eed1 and Nanjing 6. The region contained 114 open reading frames. This study lays a foundation for further studying EED1 gene in regulating development of rice embryo and endosperm.

Key words: rice, embryo and endosperm development, grain filling

Table 1

Primers for qRT-PCR used in this study"

基因名
Genes ID
正向引物序列
Forward sequences (5'-3')
反向引物序列
Reverse sequences (5'-3')
Ubiquitin CTGTCAACTGCCGCAAGAAG GGCGAGTGACGCTCTAGTTC
OsAGPL1 GGAAGACGGATGATCGAGAAAG CACATGAGATGCACCAACGA
OsAGPL2 AGTTCGATTCAAGACGGATAGC CGACTTCCACAGGCAGCTTATT
OsAGPL3 AAGCCAGCCATGACCATTTG CACACGGTAGATTCACGAGACAA
OsAGPS1 GTGCCACTTAAAGGCACCATT CCCACATTTCAGACACGGTTT
OsAGPS2a ACTCCAAGAGCTCGCAGACC GCCTGTAGTTGGCACCCAGA
OsAGPS2b AACAATCGAAGCGCGAGAAA GCCTGTAGTTGGCACCCAGA
OsSS I GGGCCTTCATGGATCAACC CCGCTTCAAGCATCCTCATC
OsS IIa GCTTCCGGTTTGTGTGTTCA CTTAATACTCCCTCAACTCCACCAT
OsSS IIc GACCGAAATGCCTTTTTCTCG GGGCTTGGAGCCTCTCCTTA
OsSS IIIa GCCTGCCCTGGACTACATTG GCAAACATATGTACACGGTTCTGG
OsSS IIIb ATTCCGCTCGCAAGAACTGA CAACCGCAGGATAACGGAAA
OsSS IVa GGGAGCGGCTCAAACATAAA CCGTGCACTGACTGCAAAAT
OsSS IVb ATGCAGGAAGCCGAGATGTT ACGACAATGGGTGCCAAGAT
OsGBSS I
OsGBSS II
AACGTGGCTGCTCCTTGAA
AGGCATCGAGGGTGAGGAG
TTGGCAATAAGCCACACACA
CCATCTGGCCCACATCTCTA
OsBE I
OsBE IIa
TGGCCATGGAAGAGTTGGC
GCCAATGCCAGGAAGATGA
CAGAAGCAACTGCTCCACC
GCGCAACATAGGATGGGTTT
OsBE IIb ATGCTAGAGTTTGACCGC AGTGTGATGGATCCTGCC
OsISA1 TGCTCAGCTACTCCTCCATCATC AGGACCGCACAACTTCAACATA
OsISA2 TAGAGGTCCTCTTGGAGG AATCAGCTTCTGAGTCACCG
OsISA3 ACAGCTTGAGACACTGGGTTGAG GCATCAAGAGGACAACCATCTG
OsPUL ACCTTTCTTCCATGCTGG CAAAGGTCTGAAAGATGGG
OsDPE2 CAAGTACACCACAAGACCAGCAA CGTCCAACAGCGAATCCAAT
OsPHOL TTGGCAGGAAGGTTTCGCT CGAAGCCTGAAGTGAACTTGCT
OsPHOH CACCAAGACGAAGCTCATCAAG TTCACTCGTTGCTGGGTTCTC
GluA1 CATTTGAGCCAATTCGGAGT GGCCTGATTGTTGGAACTGT
GluA2 GCAAGAGCAGGAACAAGGAC CCTCATGGTGCAAAAGGTCT
GluA3 TGAAAACCAACCCTGACTCC ACTCATCTCCCCTGTTGTGC
GluB1 GCCAAAGTCAGAGCCAAAAG GAACCAATGTGCAACACCAG
GluB4 GCGACCAGAAGGCTACAAAG TTGCTTGTTGATCGTTGCTC
Glutelinλ TGGCGACCATAGCTTTCTCT GGGTTGTGCCATGGATTTAC
Globulin1 ATCGAGAACGGCGAGAAGT GGACGGAGATGGTATGGAGA
Globulin2 CGACGAGGTGTTCTACGTCA GTGTTGGCGGAGTAGACGAT
11S-Globulin CACCAAACCCGATCTTCAGT CGGAACAGCTTCTCCATCTC
19kD-Globulin GCCAGTAATTGCAGGGGATA AGGTCACCACCAACGTAAGC
Prolamin TCTCCAACCAACAATAGCAATG TGCGTAGCTATCTGTGCCCGTC
10kD-Prolamin TGCAGTATTTCCCACCAACA ACATGAACATGGCTGTGGAG
13kD-Prolamin CACAGCGCAGTTTGATGTTT GCTTGCCGCAATGCTATACT
17kD-Prolamin TTTGATGCTTGCACCTATGG GCAGCTGCTCAGTTTTAGCC
RA16 AGGTAGTGATCTCGGCGTTG CCGATTCCTGGCTGACATAG
RA17 TTCTCGGTATTGCTCCTCGT CTTATTCCTGGCCGACATTG

Table 2

Markers for mapping of EED1"

引物名
Primers ID
正向引物序列
Forward sequences (5'-3')
反向引物序列
Reverse sequences (5'-3')
OS907 CTTCTAACTTTTCCATCACATCG CCTCCAACTACCAGCCATAA
InDel950
InDel976
ACTTTCTAACTTCCTAATAAG
GTTAAATCTTCTCTCAATCT
GTTCTGGAATGAAATATTAGA
TCTCTCCGGTCTCTCCCTGC
RM107 AGATCGAAGCATCGCGCCCGAG ACTGCGTCCTCTGGGTTCCCGG
RM201 GTACTCTCGCCGTTCACAACTCC TTAGTGACCGGGATGACACAGC
RM245 ATGCCGCCAGTGAATAGC CTGAGAATCCAATTATCTGGGG
InDel908 TGGGAGAACTAAGGAGCAA CTGTTATCTGGCAATCTG

Fig. 1

Phenotypic analyses of mature caryopses of ZH11 and eed1 A: appearance comparison of the whole and mature caryopses between ZH11 and eed1, bar: 1 mm. B: cross-sections of ZH11 and eed1 mature caryopses, bar: 1 mm. C: comparison of mature caryopses size between ZH11 and eed1, n = 20. D: 1000-grain dry weight of ZH11 and eed1 mature grains, n = 3. Values are means ± SDs by Student’s t-test. **: P < 0.01. E-F: the scanning electron microscopic observation of mature caryopses of ZH11 (E) and eed1 (F), bar: 5 μm."

Fig. 2

Germination rate and cell viability of eed1 A: phenotypes of WT and eed1 mature caryopses at 7 days after imbibition, bar: 3 cm; B: germination rate of WT and eed1 mature caryopses at 7 days after imbibition; C-E: TTC staining in longitudinally sectioned mature caryopses of WT (C) and eed1 (D, E), bar: 1 mm; values are means ± SDs by Student’s t-test. **: P < 0.01."

Fig. 3

Caryopses phenotype analyses from regenerated plantlets of ZH11 and eed1 A-B: the eed1 callus can differentiate into adventitious buds and roots on RE2 medium (A), and eventually grow into plantlets (B), bar: 2 cm; C-D: changes in the fresh (C) and dry (D) weight of ZH11 and eed1 caryopses of different stages; E: phenotypes of fresh caryopses of ZH11 and eed1 at 0-30 days after pollination, bar: 1 mm; F: phenotypes of dry caryopses of ZH11 and eed1 at 24, 27, and 30 DAP (days after pollination), bar: 1 mm; values are means ± SDs by Student’s t-test. *: P < 0.05; **: P < 0.01."

Fig. 4

Starch contents and storage protein accumulations in eedl caryopses A-B: the determination of the contents of total starch (A), amylose (B) in ZH11, WT, and eed1 mature caryopses, n = 3. Values are means ± SDs by Student’s t-test; **: P < 0.01; C: SDS-PAGE profiles of total protein in ZH11, WT, and eed1 mature caryopses; αGT: glutelin acidic subunits; αGlb: α-globulin; βGT: glutelin basic subunits; Pro or Alb: prolamins or albumins."

Fig. 5

Relative expression profile of starch and storage protein biosynthesis genes in eed1 A: relative expression levels of genes involved in starch biosynthesis in endosperm of ZH11 and eed1; B: relative expression levels of genes involved in storage protein biosynthesis in endosperm of ZH11 and eed1."

Table 3

Genetic analysis of EED1 segregation using three heterozygous EED1/eed1 plants"

表型
Phenotype
杂合植株-1
Heterozygous plant-1
杂合植株-2
Heterozygous plant-2
杂合植株-3
Heterozygous plant-3
正常籽粒 Normal grains 157 152 133
胚胎且胚乳缺陷籽粒 eed1 grains 47 50 45
χ2(3:1) 0.418 < 3.84 (P > 0.05) 0.006 < 3.84 (P > 0.05) 0.007 < 3.84 (P > 0.05)

Fig. 6

Genetic mapping of EED1 A: EED1 was linked with makers OS907 and InDel908 on the long arm of chromosome 9; B: EED1 was mapped to a 672 kb region based on 61 segregated plants. The region contained 114 ORFs."

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