Thanks to the massively parallel sequencing
of NGS, the obtaining of chloroplast genomes
from five grass species was provided as a
promising barcode marker. The number of
assembly gaps increased with evolutionary distance from the reference of Oryza sativa. A
number of 91 SNPs were identified between the
closely related species of O. meridionalis and O.
sativa japonica (Nock et al. 2011). The complete
chloroplast genomes of eight Fritillaria species
were found to contain multiple polymorphic
SSR, large repeat sequences and highly variable
regions, which was invaluable for the species
identification and the relation establishment in
Fritillaria (Bi et al. 2018).
10.4.2 Engineering the Chloroplast
Genomes
for Biotechnology
Applications
Genetic engineering is the biotechnology of
directly transferring foreign genes into target
plants in order to improve its desirable traits,
such as yield, nutrition enhancement, and resistance to pathogen. An efficient genetic transformation protocols were developed in Lemna gibba
and L. minor with a binary vector containing
b-glucuronidase and nptII expression cassettes
(Yamamoto et al. 2000). Transgenic duckweed
could be regenerated after three months of
agrobacterium-mediated transformation. The
addition of the poorly assimilated carbohydrates
of galactose or sorbitol yielded high levels of
callus (Li et al. 2004). The stable and transgenic
Spirodela oligorrhiza showed that the transgene
protein of GFP expression reached more than
25% of total soluble proteins (Vunsh et al. 2007).
An artificial microRNA gene silencing system
was generated in L. minor fronds that the
expression of CH42 was significantly inhibited,
resulting in the reduction of chlorophyll pigmentation (Canto-Pastor et al. 2015). However,
all these studies were nuclear-level modification,
and there was no reported study about chloroplast transgene in duckweeds yet.
The duckweeds as an alternative biofuel and
bioremediator have attracted extremely interests
in recent years. The complete chloroplast genomes of duckweeds provide the framework to
explore their potential values and to accelerate
the chloroplast genetic engineering (Fig. 10.2).
The chloroplast could be also engineered to
produce high-value agricultural and biomedical
products with highly expression by the fact of
abundant chloroplast genome copies in a plant
cell. As the transgene is inserted within the IRs
region, its copy number of transgenes will be
doubled theoretically. However, achieving the
homoplastic state of genetically modified
chloroplast is a non-trivial task. It requires two or
three rounds of selection to eliminate all
untransformed copies (>1000 per cell) of the
native chloroplast genome. A comprehensive
summary demonstrated the power of chloroplast
genetic engineering, including 114 transgenes,
the integration sites, and engineered traits
(Daniell et al. 2016). The principle of chloroplast
genome engineering has to incorporate the foreign genes into intergenic spacer regions without
disrupting the native chloroplast genes. The
cassette usually contains gene(s) of interest, a
selectable marker gene, and two chloroplast
genes used as flanking sequences. However,
most intergenic spacer region is not conserved
and the ideal sites are still absent.
Sequence Information
The duckweed chloroplast genomes are deposited into GenBank with the ID of DQ400350 for
L. minor. JN160603, JN160604 and JN160605.
References
Ardui S, Ameur A, Vermeesch JR, Hestand MS (2018)
Single molecule real-time (SMRT) sequencing comes
of age: applications and utilities for medical diagnostics. Nucleic Acids Res 46(5):2159–2168
Badouin H, Gouzy J, Grassa CJ, Murat F, Staton SE,
Cottret L, Lelandais-Briere C, Owens GL, Carrere S,
Mayjonade B, Legrand L, Gill N, Kane NC, Bowers JE, Hubner S, Bellec A, Berard A, Berges H,
Blanchet N, Boniface MC, Brunel D, Catrice O,
Chaidir N, Claudel C, Donnadieu C, Faraut T,
Fievet G, Helmstetter N, King M, Knapp SJ, Lai Z,
112
Y. Zhang and W. Wang
of NGS, the obtaining of chloroplast genomes
from five grass species was provided as a
promising barcode marker. The number of
assembly gaps increased with evolutionary distance from the reference of Oryza sativa. A
number of 91 SNPs were identified between the
closely related species of O. meridionalis and O.
sativa japonica (Nock et al. 2011). The complete
chloroplast genomes of eight Fritillaria species
were found to contain multiple polymorphic
SSR, large repeat sequences and highly variable
regions, which was invaluable for the species
identification and the relation establishment in
Fritillaria (Bi et al. 2018).
10.4.2 Engineering the Chloroplast
Genomes
for Biotechnology
Applications
Genetic engineering is the biotechnology of
directly transferring foreign genes into target
plants in order to improve its desirable traits,
such as yield, nutrition enhancement, and resistance to pathogen. An efficient genetic transformation protocols were developed in Lemna gibba
and L. minor with a binary vector containing
b-glucuronidase and nptII expression cassettes
(Yamamoto et al. 2000). Transgenic duckweed
could be regenerated after three months of
agrobacterium-mediated transformation. The
addition of the poorly assimilated carbohydrates
of galactose or sorbitol yielded high levels of
callus (Li et al. 2004). The stable and transgenic
Spirodela oligorrhiza showed that the transgene
protein of GFP expression reached more than
25% of total soluble proteins (Vunsh et al. 2007).
An artificial microRNA gene silencing system
was generated in L. minor fronds that the
expression of CH42 was significantly inhibited,
resulting in the reduction of chlorophyll pigmentation (Canto-Pastor et al. 2015). However,
all these studies were nuclear-level modification,
and there was no reported study about chloroplast transgene in duckweeds yet.
The duckweeds as an alternative biofuel and
bioremediator have attracted extremely interests
in recent years. The complete chloroplast genomes of duckweeds provide the framework to
explore their potential values and to accelerate
the chloroplast genetic engineering (Fig. 10.2).
The chloroplast could be also engineered to
produce high-value agricultural and biomedical
products with highly expression by the fact of
abundant chloroplast genome copies in a plant
cell. As the transgene is inserted within the IRs
region, its copy number of transgenes will be
doubled theoretically. However, achieving the
homoplastic state of genetically modified
chloroplast is a non-trivial task. It requires two or
three rounds of selection to eliminate all
untransformed copies (>1000 per cell) of the
native chloroplast genome. A comprehensive
summary demonstrated the power of chloroplast
genetic engineering, including 114 transgenes,
the integration sites, and engineered traits
(Daniell et al. 2016). The principle of chloroplast
genome engineering has to incorporate the foreign genes into intergenic spacer regions without
disrupting the native chloroplast genes. The
cassette usually contains gene(s) of interest, a
selectable marker gene, and two chloroplast
genes used as flanking sequences. However,
most intergenic spacer region is not conserved
and the ideal sites are still absent.
Sequence Information
The duckweed chloroplast genomes are deposited into GenBank with the ID of DQ400350 for
L. minor. JN160603, JN160604 and JN160605.
References
Ardui S, Ameur A, Vermeesch JR, Hestand MS (2018)
Single molecule real-time (SMRT) sequencing comes
of age: applications and utilities for medical diagnostics. Nucleic Acids Res 46(5):2159–2168
Badouin H, Gouzy J, Grassa CJ, Murat F, Staton SE,
Cottret L, Lelandais-Briere C, Owens GL, Carrere S,
Mayjonade B, Legrand L, Gill N, Kane NC, Bowers JE, Hubner S, Bellec A, Berard A, Berges H,
Blanchet N, Boniface MC, Brunel D, Catrice O,
Chaidir N, Claudel C, Donnadieu C, Faraut T,
Fievet G, Helmstetter N, King M, Knapp SJ, Lai Z,
112
Y. Zhang and W. Wang