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BMC Plant Biology
A large deletion conferring pale green leaves
ofmaize
Guoqi Yao1,2,3,4†, Hua Zhang1,2,3,4†, Bingying Leng1,2,3,4, Bing Cao1,2,3,4, Juan Shan1,2,3,4, Zhenwei Yan1,2,3,4,
Haiying Guan1,2,3,4, Wen Cheng1,2,3,4, Xia Liu1,2,3,4,5* and Chunhua Mu1,2,3,4*
Abstract
Background The structural basis of chloroplast and the regulation of chloroplast biogenesis remain largely unknown
in maize. Gene mutations in these pathways have been linked to the abnormal leaf color phenotype observed
in some mutants. Large scale structure variants (SVs) are crucial for genome evolution, but few validated SVs have
been reported in maize and little is known about their functions though they are abundant in maize genomes.
Results In this research, a spontaneous maize mutant, pale green leaf-shandong (pgl-sd), was studied. Genetic analysis
showed that the phenotype of pale green leaf was controlled by a recessive Mendel factor mapped to a 156.8-kb
interval on the chromosome 1 delineated by molecular markers gy546 and gy548. There were 7 annotated genes
in this interval. Reverse transcription quantitative PCR analysis, SV prediction, and de novo assembly of pgl-sd
genome revealed that a 137.8-kb deletion, which was verified by Sanger sequencing, might cause the pgl-sd phe-
notype. This deletion contained 5 annotated genes, three of which, including Zm00001eb031870, Zm00001eb031890
and Zm00001eb031900, were possibly related to the chloroplast development. Zm00001eb031870, encoding a Degra-
dation of Periplasmic Proteins (Deg) homolog, and Zm00001eb031900, putatively encoding a plastid pyruvate dehy-
drogenase complex E1 component subunit beta (ptPDC-E1-β), might be the major causative genes for the pgl-sd
mutant phenotype. Plastid Degs play roles in protecting the vital photosynthetic machinery and ptPDCs provide
acetyl-CoA and NADH for fatty acid biosynthesis in plastids, which were different from functions of other isolated
maize leaf color associated genes. The other two genes in the deletion were possibly associated with DNA repair
and disease resistance, respectively. The pgl-sd mutation decreased contents of chlorophyll a, chlorophyll b, carot-
enoids by 37.2%, 22.1%, and 59.8%, respectively, and led to abnormal chloroplast. RNA-seq revealed that the transcrip-
tion of several other genes involved in the structure and function of chloroplast was affected in the mutant.
Conclusions It was identified that a 137.8-kb deletion causes the pgl-sd phenotype. Three genes in this deletion
were possibly related to the chloroplast development, which may play roles different from that of other isolated maize
leaf color associated genes.
Keywords Maize, Pgl, Map based cloning, Large deletion
†Guoqi Yao and Hua Zhang contributed equally to this work.
*Correspondence:
Xia Liu
snakepy@126.com
Chunhua Mu
maizesd@163.com
Full list of author information is available at the end of the article
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Yaoetal. BMC Plant Biology (2023) 23:360
Background
Mutants with the phenotype of abnormal leaf color have
been reported frequently, most of which can be grouped
as albina, xantha, alboviridis, viridis, and girina [15]. In
general, these phenotypes were related to mutation of
genes in the pathway of chlorophyll synthesis and deg-
radation or genes directly involved in the chloroplast
biogenesis. For example, green genes identified in Arabi-
dopsis [1], and rice genes Ygl1 [51], Ygl2 [8], Ygl3 [46],
Ygl7 [10] and Ygl80 [45] are responsible for chlorophyll
metabolism, while Arabidopsis genes Apg1 ( [37], Cao
[23], Egy1 [7] and Var3 [36], and rice genes Ygl138(T)
[55] and Vyl [11] encode proteins for the development
of chloroplast. In maize, more than 200 genes/Quantita-
tive Trait Loci (QTL) associated with leaf color have been
recorded in maizeGDB database [54], including 8 isolated
genes, Elm1, Elm2, Chr.1-ClpP5, Oy1, Oy2, Vyl, Ygl-1,
and Zb7. Elm1 encodes a phytochromobilin synthase.
elm1, a mutant of single base transition of Elm1, was defi-
cient in phytochrome response and had a lower content
of chlorophyll than wild plants under white light condi-
tion [39, 40]. Elm2, encoding a heme oxygenase, was
also involved in phytochrome biosynthesis. elm2 with a
21-bp deletion in Elm2 showed yellow green leaves [42].
Vyl and Chr.1-ClpP5 were a pair of ClpP5 homologs. a
141-bp insertion in Vyl led to virescent yellow-like leaves
[52]. Oy1 encodes the subunit I of magnesium chelatase
in the chlorophyll biosynthesis pathway. Semi-dominant
oy1 was a chlorophyll deficient mutant [41]. Oy2 possi-
bly encodes chelatase subunit D and a point mutation of
this gene likely conferred the yellow leaves of maize [54].
Ygl-1 possibly encodes a cpSRP43 protein required to
target light-harvesting chlorophyll protein to thylakoid
membrane. ygl-1, a mutant of single nucleotide deletion
of Ygl-1, showed yellow-green leaves [14]. Zb7 is an IPP
and DMAPP synthase involving in isoprenoid synthesis.
A single nucleotide alteration of Zb7 decreased the bio-
synthesis of chlorophyll and carotenoid leading to trans-
verse yellow-green leaf phenotype [32].
ough most of identified genomic variants were sin-
gle nucleotide polymorphisms (SNPs) or Indel poly-
morphisms (IDPs), large scale structure variants (SVs),
classified as genome rearrangements typically larger than
100bp [18], were also abundant in the crop genome as
revealed in maize by genome sequence projects [28, 43].
SVs contributed to the adaptation of crops to environ-
ments and the variation source for breeding. For exam-
ple, a 38.3-kb deletion in rice harbored grain number,
plant height and heading date7 (Ghd7) [53], and a 254-
kb deletion in soybean was associated with a low level of
palmitic acid of seeds [13]. However, few SVs in maize
have been verified with experiments and little is known
about their biological functions. A 147-kb deletion
identified in maize containing maize wall-associated
kinase (ZmWAK) resulted in susceptibility to the fungal
disease head smut [58]. Han et al. [16] reported that a
5.16-Mb deletion led to a set of phenotypic abnormities,
including reduced plant height, increased stomatal den-
sity, and rapid water losing in maize.
In this research, a natural maize mutant with pale
green leaf, pale green leaf-shandong (pgl-sd), was studied.
Map-based cloning revealed that a 137.8-kb deletion con-
taining 5 genes in chromosome 1 of pgl-sd results in its
abnormal leaf color.
Results
Inheritance ofleaf color ofpgl‑sd
e mutant pgl-sd had leaves with nearly white color at
the base and pale green at the tip at seedling stages, but it
exhibited pale green leaves at later growth stages (Fig.1),
a phenotype similar to that of B73vyl [52]. e mutant
was shorter and had later anthesis and silking date rela-
tive to the three wild inbred lines, B73, Zheng58 and
Qi319, but could grow to maturity and produce seeds.
e F1 progeny of the crosses between pgl-sd with B73,
Zheng58 or Qi319 showed green leaves and grew nor-
mally as the wild parents. Among a BC1 population of the
cross between pgl-sd and B73, 41 of 98 individuals exhib-
ited pgl-sd mutant phenotype. e ratio of the mutant
to the wild-type was in agreement with a 1:1 segrega-
tion ratio (X2(1:1) = 2.61, P = 0.11). In a Zheng58/pgl-sd F2
population (Population 1), 26 mutant plants and 69 wild
were observed, respectively (X2(1:3) = 0.28, and P = 0.59).
ese data suggested that the pgl-sd mutant phenotype
was probably determined by a single recessive gene.
ese two populations were used for the initial mapping
of pgl-sd.
At the fine mapping stage, we further investigated
leaf color of 3635 individuals from a Zheng58/pgl-sd F2
population and 2521 plants from a Qi319/pgl-sd F2 pop-
ulation, respectively. A total of 598 plants in the former
population and 405 in the latter expressed the mutant
phenotype, respectively. e mutant frequencies of both
populations were approximately 16%, which were sig-
nificantly lower than expected 25%. In the BC1 popula-
tion mentioned above, the mutant frequency was not
significantly different from 50%, but it was closer to 40%
(P = 0.71), which was consistent with the segregation
ratio of 16 mutant to 84 wild type in the Zheng58/pgl-sd
F2 population (X2(16:84) = 0.55, P = 0.45) and Qi319/pgl-
sd F2 populations (X2(16:84) = 0.008, P = 0.92). We exam-
ined ears of Zheng58/pgl-sd F1 and that of Qi319/pgl-sd
F1, but no obvious abortion of kernels were observed.
In addition, no abnormality of F1 seeds germination or
of F2 seedling establishment was found for these two
crosses. us, we speculated that the mutation of pgl-sd
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Page 3 of 14
Yaoetal. BMC Plant Biology (2023) 23:360
could lead to some degree of abortion of gamete, which
resulted in the observed segregation distortion.
Initial mapping ofpgl‑sd
Initially, 5 mutant and 5 wild plants from the B73/*2pgl-
sd population were genotyped with polymorphic mark-
ers equally distributed across maize chromosomes along
with the parent lines. With these markers, pgl-sd was
located into a region around bin 1.05 and 1.06 of chro-
mosome 1. Because of low diversity observed between
pgl-sd and B73, we then used the Population 1 to further
construct a linkage map encompassing the pgl-sd muta-
tion with polymorphic simple sequence repeat (SSR)
markers located in the two bins or nearby. pgl-sd was
finally mapped into a 5.5-cM interval delimited by the
closest markers umc1906 and umc1396 (Fig. 2a). With
molecular markers developed at the fine mapping stage,
this interval was further resolved by gy496 and gy489 in
the long arm direction (Fig.2a).
Fine mapping ofpgl‑sd
To fine mapping of pgl-sd, molecular makers umc2230
and umc1396 were used to identify recombinants
between markers and pgl-sd (Fig. S1). Besides the 24
mutants from the Population1, other 591 mutants with
no confused phenotype from the Zheng58/pgl-sd F2
were genotyped with the two markers. A total of 116
recombinants were obtained with one or both of the two
markers loci carrying the allele(s) from the wild parent
Zheng58. ese recombinants were further genotyped
with markers between umc2230 and umc1396 to reveal
their detailed structure in this region. To increase reso-
lution of the molecular map, we searched MaizeGDB
database (https:// www. maize gdb. org) and literatures
for SSR or IDP markers possibly located in the target
region. SSRs identified in the sequence of these region
were also used to design molecular markers. ese mark-
ers were screened for polymorphism between pgl-sd and
B73, Zheng58, or Qi319. pgl-sd was further delimited to
a ~ 6.4-Mb region enclosed by gy524 (at the position of
173.7Mb on chromosome 1 of Zm-B73-REFERENCE-
GRAMENE-4.0 (RefGen_v4)) and gy541 (180.0 Mb),
with 7 recombinants between gy524 and pgl-sd and 2
between gy541 and pgl-sd being identified, respectively
(Fig. S1). Recombination events were also identified by
another SSR marker gy533 originated from the MISA
Fig. 1 The phenotype of pgl-sd. a seedlings of B73 (column 1), pgl-sd (column 2), Zheng58 (column 3), Qi319 (column 4). b seedlings of a mutant
(left) and a wild plant (right) from B73/*2pgl-sd BC1 population. c adult plants of pgl-sd (left) and Qi319 (right) growing in the field
Fig. 2 Mapping of pgl-sd. a linkage mapping of pgl-sd with Zheng58/pgl-sd F2 population (units = cM). b physical coordinates of markers
on the chromosome 1 for fine mapping of pgl-sd with Qi319/pgl-sd F2 population based on RefGen_v5 (units = bp). The number of recombinant
mutants between each marker and pgl-sd was showed in bracket following the marker. c genotypes of representative recombinants
from the Qi319/pgl-sd F2 population. Black color indicates the pgl-sd genotype and white indicates heterozygous genotype. Codes of these plants
from top to below are b3.08.01, b3.11.11, b3.67.09, b3.16.07, b3.31.07, b3.33.09, b2.11.09, b2.26.02, b1.67.10, b3.12.03, b1.13.09, b2.57.07, b3.01.18,
and b1.66.10 respectively. d annotated genes in pgl-sd interval delimited by gy546 and gy548. e the deletion containing five genes in the pgl-sd
(See figure on next page.)
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Yaoetal. BMC Plant Biology (2023) 23:360
Fig. 2 (See legend on previous page.)
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Page 5 of 14
Yaoetal. BMC Plant Biology (2023) 23:360
search, which was located at 176.5Mb on chromosome
1 of the RefGen_v4. But gy533 was not placed on the
physical map because of the difficulty to distinguish the
polymorphism.
en, 387 mutants from Qi319/pgl-sd F2 were
screened for recombinants with umc1076 and gy502
selected according to the genetic map constructed with
Zheng58/pgl-sd F2. A total of 92 recombinants were
obtained. pgl-sd was delimited to a region with 6.7-Mb
physical distance delineated by the closest markers gy521
(171.7Mb) and gy527 (177.8Mb) originating from a liter-
ature (Jie etal. 2013) (Fig.1). However, this interval could
not be further resolved due to lack of more polymorphic
markers.
To overcome the shortage of polymorphic markers,
pgl-sd genome was sequenced on Illumina platform and
the resulted reads were aligned to RefGen_v4 along with
downloaded sequence reads of Zheng58 and Qi319 to
identify variants. IDPs with length difference less than
4bp between pgl-sd and Zheng58 or Qi319 in the inter-
val delimited by gy524 (173.7Mb) and gy533 (176.5Mb)
were screened for mapping pgl-sd. It was interesting
that only 3 variants between Zheng58 and pgl-sd were
obtained in this region, while 21 were gotten between
Qi319 and pgl-sd under this threshold. We designed
primers for 7 IDPs, 6 of which successfully detected
the expected polymorphisms between Qi319 and pgl-
sd, but none did between Zheng58 and pgl-sd as antici-
pated. With these markers, pgl-sd was further mapped
to a 155.8-kb region bracketed by gy546 and gy548, with
gy547 cosegregating with it (Fig. 1b). 3 recombinants
between gy546 and pgl-sd as well as 2 between gy548
and pgl-sd were observed, respectively. Coordinates for
gy546, gy547 and gy548 were 176.20 Mb, 176.34 Mb,
176.35Mb on chromosome 1 of the RefGen_v4. ere
were only 2 annotated genes in this region including
Zm00001d031078 and Zm00001d031079. However, care-
ful inspection of this region revealed a large gap exist-
ing in the interval of the assembly of RefGen_v4, while
no gap was present in the corresponding regions brack-
eted by gy546 and gy548 of both Zm-B73-REFERENCE-
NAM-5.0 (RefGen_v5) and B73 RefGen_v3 (RefGen_v3).
We aligned the sequence of target interval of RefGen_v5
with that of RefGen_v3 by using MUMer and found that
these two assemblies agreed well with each other in this
region (Fig. S2). According to the annotation of RefGen_
v5, an unmapped contig B73V4_ctg98 from RefGen_v4
was found sharing high identity with the sequence of the
target region of RefGen_v5. is was the reason why Ref-
Gen_v5 was used as reference to give physical positions
of markers in the fine mapping of pgl-sd finally (Fig.2 b).
ere were 8 annotated genes in the interval of RefGen_v5,
which included Zm00001eb031850, Zm00001eb031860,
Zm00001eb031870, Zm00001eb031880, Zm00001eb031890,
Zm00001eb031900, Zm00001eb031910 and Zm00001eb031920.
Among these 8 genes, Zm00001eb031920 was in a tandem array
with Zm00001eb031910, and both of them corresponded to the
same annotated gene Zm00001d031079 in RefGen_v4. ere-
fore, only Zm00001eb031910 was considered in further stud-
ies for these two gene models. Marker gy547 was located at the
interval between Zm00001eb31900 and Zm00001eb31910.
en, we reanalyzed the sequence reads of pgl-sd, Zheng58 and
Qi319 using RefGen_v5 as the reference genome in order to
find more variants for developing markers to saturate the target
region. Intriguingly, genotype information was missing for pgl-
sd at many variant positions in this region reported by GATK
[35], which was confirmed by the observation that there were
no mapped reads for pgl-sd in many parts of the region. ree
new markers were obtained, including gy550, gy553, gy554.
gy550 was designed from Zm00001eb031850, the gene nearest
to gy546 in the target interval and gy553 was from the sequence
between Zm00001eb31880 and Zm00001eb31881. gy554 was
developed from the sequence between Zm00001eb31900 and
Zm00001eb31910 like gy547, but it was a little closer to gy548
than gy547. However, all of the three markers cosegregated with
pgl-sd (Fig.2), with gy553 as a dominant marker having no spe-
cific amplification in mutant plants. ese results indicated that
it was likely to be difficult to further resolve the region around
pgl-sd mutation though recombinants could be found between
gy546 and gy548.
A large deletion inpgl‑sd leading tothephenotype
ofpgl‑sd mutation
Among the 7 annotated genes in the target region,
Zm00001eb031870/Zm00001d000230 had At4G18370
as the ortholog in Arabidopsis (http:// www. maize GDB.
org) which encodes Degradation of periplasmic pro-
teins 5 (Deg5), a protein located in chloroplast thy-
lakoid lumen [44]. It was the only gene which had its
high expression present in only leaves according to the
RNA-seq expression data in maizeGDB (https:// www.
maize gdb. org) (Fig. S3). Marker gy562, developed from
Zm00001eb031870, cosegregated with pgl-sd (Fig. 2b).
So we speculated that Zm00001eb031870 was the candi-
date gene responsible for pgl-sd. To test the hypothesis,
reverse transcription PCRs (RT-PCRs) / reverse tran-
scription quantitative PCRs (RT-qPCRs) were conducted
to examine the expression of these 7 genes in leaves of
pgl-sd and the wild line Qi319 (Table1). All primer pairs
designed from Zm00001eb031910/Zm00001d031079
failed to specifically amplify products of expected size.
e expression of Zm00001eb031850/Zm00001d031078
was too low to be detected in leaf, but it was high in
the root of B73 (data not shown), which was consistent
with RNA-Seq expression data in maizeGDB (https://
www. maize gdb. org). As expected, the transcription of
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Yaoetal. BMC Plant Biology (2023) 23:360
Zm00001eb031870 was significantly down-regulated
in pgl-sd relative to Qi319. However, the expression of
Zm00001eb031880/Zm00001d000229 was detected in
all lines (B73, Zheng58 and Qi319) except pgl-sd, and the
expression levels of all the other 3 genes were also signifi-
cantly lower in pgl-sd than that in Qi319 (Table1).
We then tried to amplify the full-length genome
sequence and the coding sequence (CDS) of
Zm00001eb031870 in pgl-sd and Qi319 with the primer
pair gy573. However, PCR products of expected size
were only obtained from Qi319 (Fig. 3a). Impressively,
same results were gotten with three additional primer
pairs designed from different parts of this gene (data
not shown), indicating that Zm00001eb031870 was pos-
sibly deleted in pgl-sd. Combining these results with
the gene expression analysis, gy553 being a dominant
marker, and many missing data for pgl-sd at variant posi-
tions in the target interval together, we suspected that
a large deletion might exist in the target genome region
of the mutant. en, we scrutinized regions beyond
Zm00001eb031870 in the whole interval by developing
additional primer pairs, including all the other 6 genes
and intergenic regions following Zm00001eb031850 and
preceding Zm00001eb031910. All these primer pairs
amplified products with expected size from Qi319 but
did not from pgl-sd, except gy580 derived from the region
between Zm00001eb031850 and Zm00001eb031860 and
gy586 from Zm00001eb031890, which generally agreed
with our hypothesis.
To further validate this hypothesis and determine the
precise position of the possible deletion, Manta was
used to detect signals of SVs in the targeted region with
the sequence read mapping result of pgl-sd. However,
no meaningful information was attained. en we tried
Breakdance. Fortunately, a 137,794-bp deletion start-
ing from 178,338,866bp and ending at 178,476,727bp
on the chromosome 1 of RefGen_v5 was identified with
4 supported reads. To catch more supported informa-
tion, we assembled sequence reads of pgl-sd by using
SOAPdenovo and found a 8204-bp contig C52375234
(data s1) matching the deletion-supporting reads from
the SV analysis using Breakdancer. Blastn search with
C52375234 against RefGen_v5 demonstrated that its
right part (from 1 to 6,010bp of its reverse complement)
matches the region from 178,332,925 to 178,338,934 of
B73 chromosome 1 completely and its left part (from
6,009 to 8,024 bp of its reverse complement) aligned
with the region from 178,476,727 to 178,478,922 of B73
chromosome 1 with 100% identity, repectively, suggest-
ing a deletion starting from 178,338,935bp and ending
at 178,476,729bp on the chromosome 1 in pgl-sd com-
pared with B73, which was consistent with the predic-
tion from Breakdancer but with a little difference at the
exact starting position. e primer pair gy598, flanking
the predicted deletion, did amplify products of expected
size from genome DNA of pgl-sd but did not from Qi319
due to too larger size of the segment flanked by gy598
(Fig.3b). Similar results were brought for the primer pair
gy599 which was internal to gy598 though it demon-
strated worse specificity than gy598 (Fig.3c). Sequencing
amplicons from pgl-sd with gy598 resulted an anticipated
6,119-bp sequence, which matched the C52375234 with
100% identity. us, these data confirmed our hypothesis
(Fig.3d, Data S1).
Mechanisms forthephenotype ofpgl‑sd
Because pgl-sd mutants exhibited pale green leaves, we
examined whether contents of chlorophyll a (Chla), chlo-
rophyll b (Chlb) and carotenoids (Car) were changed in
pgl-sd. It was detected that contents of all of three pig-
ments were significantly reduced in the mutant com-
pared with the wild-type at early seedling stages (P < 0.01)
Table 1 RT-qPCR analysis of candidate genes for pgl-sd
a average relative expression ± sd with FPGS used as reference gene, “u” and “low” indicated "undetermined" and CT > 34 in q-PCR analysis, respectively. bP value for
two tail T-test of expression dierence between pgl-sd and Qi319. cExpression dierences of Zm00001eb031880 and Zm00001eb031910 were not investigated due to
amplication failure of the former in leaf of pgl-sd and no appropriate primer pair were obtained for the latter
gene primer Relative expressionaPb
YLP Q319
Zm00001eb031850 gy555 u u -
Zm00001eb031860 gy556 0.25 ± 0.05 0.36 ± 0.05 0.05
Zm00001eb031870 gy557 u 1.56 ± 0.16 -
Zm00001eb031880cgy565 - - -
Zm00001eb031890 gy559 2.33 ± 0.77 7.14 ± 1.57 0.02
Zm00001eb031900 gy560 low 0.28 ± 0.05 -
Zm00001eb031910c- - -
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Page 7 of 14
Yaoetal. BMC Plant Biology (2023) 23:360
of the B73/2*pgl-sd population (Fig.4). e content of
Chla, Chlb and Car of the mutant was 62.8%, 77.9% and
40.2% compared to the wild-type, respectively. us, the
mutation of pgl-sd reduced the accumulation of these
major classes of photosynthetic pigments.
Further, scrutinization of seedling leaves with elec-
tron microscopy revealed that chloroplasts in mesophyll
cells of the mutant were larger in size than that of the
wild-type (Fig.5). Chloroplasts of mesophyll cells in the
mutant also displayed irregular shapes and had small
granal stacks compared with that in the wild-type. ese
results suggested that the pgl-sd mutation likely affected
the development of chloroplast directly, which then
resulted in the reduction of photosynthetic pigments
accumulation.
RNA-Seq analysis was also conducted to divulge
effects of the mutation of pgl-sd on gene expression in
the developing leaves. In the differentially expressed
gene (DEG) analysis, a total of 345 genes were identi-
fied under the threshold mentioned in the methods.
No over-represented Gene Ontology (GO) items were
observed with adjusted P-value < 0.05 in GO enrich-
ment analysis of DEGs, but 82 items were enriched under
threshold of P-value < 0.05. e top three significantly
enriched items classified as Biological Process (BP) were
GO:0009405 (pathogenesis), GO:0009750 (response
to fructose) and GO:0051341 (regulation of oxidore-
ductase activity), respectively. e top three items clas-
sified as Cell Component (CC) included GO:0032040
(small-subunit processome), GO:0015629 (actin cytoskel-
eton), GO:0000275 (mitochondrial proton-transporting
ATP synthase complex-catalytic core F(1)). e top three
items of Molcular Function (MF) comprised GO:0016831
(carboxy-lyase activity), GO:0004612 (phosphoenolpyru-
vate carboxykinase (ATP) activity) and GO:0008483
(transaminase activity). No information clearly related to
the trait pale green leaf was obtained from these data.
Among genes located into the deletion region, Zm000
01eb031870/Zm00001d000230, Zm00001eb031880/Zm0
0001d000229 and Zm00001eb031900/Zm00001d000227
were expressed in the wild-type but no expression
was detected for them in the mutant as expected
(Table 2). However, expression of Zm00001eb031860/
Zm00001d000231 was detected in the mutant at a low
level in comparison with the wild-type. Similarly, the
expression of Zm00001eb031890/Zm00001d000227 was
also observed in the mutant though it was significantly
lower than that of the wild-type (Table2). ese data
coincided with the results of RT-qPCR. It was possible
that the expression of highly homologous genes inter-
fered with the exact detection of Zm00001eb031860 and
Zm00001eb031890. In fact, Zm00001eb031890 shared
high identity to Zm00001eb159540 and the cDNA of
Zm00001eb159540 was amplified with a primer pairs
designed from Zm00001eb031890 (data not shown).
Zm00001eb031860/Zm00001d000231 could be assigned
to GO:0003678, classified as a MF item with the descrip-
tion of DNA helicase. Search against the InterPro protein
Fig. 3 The structure variation in pgl-sd. White triangles indicate expected PCR products. a amplification of Zm00001eb031870 with the primer
pair gy573. b, c, validation of the deletion in pgl-sd with gy598 and gy599, respectively. d sequence alignment of gy598 amplificon from pgl-sd
with the target region of RefGen_V5. M, molecular weight marker. For 1 and 2, genomic DNA of pgl-sd and Q319 were used, respectively. For 3
and 4, cDNA of pgl-sd and Q319 were used, respectively
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Page 8 of 14
Yaoetal. BMC Plant Biology (2023) 23:360
signature databases (https:// www. ebi. ac. uk/ inter pro)
showed that Zm00001eb031860 had a Pif1-like heli-
case domain. It was found that both GO:0031570 and
GO:0044774 in the BP, with the description of “DNA
integrity check point”, were enriched with P-value = 0.07,
indicating that deletion of Zm00001eb031860 might affect
the DNA repair system.
Zm00001eb031870/Zm00001d000230 could be assigned
to GO:0009535, a CC GO item with the description of
“the pigmented membrane of a chloroplast thylakoid”.
Zm00001d008209, a gene assigned to GO:0009535, was
significantly up-regulated at the transcriptional level in
the mutant compared with the wild-type. According to
the annotation of maizeGDB, Zm00001eb031870 might
be involved in photosystem II (PSII) repair. e GO item
GO:0009654 in CC, with description “photosystem II oxy-
gen evolving complex”, was enriched with P-value = 0.07.
2 DEGs were gouped to this GO item, among which the
expression of Zm00001d049390 was not detected in the
mutant. In addition, 2 genes assigned to GO:0009765 with
Fig. 4 Differences of leaf photosynthetic pigment contents between mutants and wild plants. Plants were sampled from B73/*2pgl-sd BC1
population. Data was showed as mean ± sd (n = 8 for mutants and 9 for the wild-type, respectively). ** indicates P < 0.01
Fig. 5 The structure change of chloroplasts in the mutant
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Yaoetal. BMC Plant Biology (2023) 23:360
the description of “photosynthesis, light harvesting” was
differentially expressed in mutants compared with the
wild-type. It was of note that both Zm00001eb031890/
Zm00001d000228 and Zm00001eb031900/Zm00001d00
0227 might participate in chloroplast development
too. Search against InterPro database revealed that
Zm00001eb031890/Zm00001d000228 had sequence
similarity to the pthr10566 which was described as “Pro-
tein Activity of BC1 Complex Kinase 8, Choloplastic”.
Zm00001eb031890 was grouped to GO:0009941 (“chloro-
plast envelope”) and GO:0046467 (“membrane lipid bio-
synthetic process”). Zm00001eb031900/Zm00001d000227
could also be assigned to GO:0009941 and it was homolo-
gous to AT1G30120 which encodes a part of plastid pyruvate
dehydrogenase complex.
Search with Conserved Domain Architecture Retrieval
Tool (CDART) (https:// www. ncbi. nlm. nih. gov/) dem-
onstrated that Zm00001eb031880/Zm00001d000229
had sequence similarity to a cotton fiber-like protein
(DUF761). In Arabidopsis, DUF761-containing proteins
likely had a role in plant development and disease resist-
ance [56]. It was interesting that GO:0009405, a BP item
with description of “pathogenesis”, was the most signifi-
cantly over-represented item (P-value = 0.0008). In addi-
tion, items GO:0010112 (“regulation of systemic acquired
resistance”) and GO:0009870 (“resistance gene-depend-
ent”) in the BP were also significantly enriched.
Taken together, the deletion in pgl-sd affected its chlo-
roplast development, which might lead to the decrease of
photosynthetic pigment contents in leaves. But not many
DEGs and no high enriched GO items were identified in
our research. e possible reason might be that the RNA
samples were prepared from plants of a B73/2*pgl-sd
population. e genetic background differences between
individuals resulted in high variations of gene expres-
sions within the mutant or wild pools, which reduced the
power for the DEG detection. But the deletion including
three genes possible related to the structure and function
of chloroplast affected the expression of several genes
involved in the structure and function of chloroplast.
Besides these, the mutation might also affect the DNA
repair system and the disease resistance as well.
Discussion
To date, 8 green-leaf-color genes have been isolated in
maize, which included Elm1, Elm2, Chr.1-ClpP5, Oy1,
Oy2, Vyl, Ygl-1, Zb7 [14, 32, 41, 42, 52, 54], none of which
was located in the deletion reported here, indicating
that pgl-sd involved in leaf color-related genes differ-
ent from those reported. ere were 5 annotated genes
located in this deletion according to the annotation of
B73 genome (https:// www. maize gdb. org), 3 of which
might be related to structure and function of chloro-
plast, including Zm00001eb031870/Zm00001d000230,
Zm00001eb031890/Zm00001d000228 and Zm00001eb03
1900/Zm00001d000227. In Arabidopsis protein data-
base (https:// www. arabi dopsis. org/), Zm00001eb031870
shared highest identity with At4g18370, known as Deg5.
In Arabidopsis, 4 Degs are located in chloroplast, three
of which, Deg1, Deg5 and Deg8, are present in thylakoid
lumen [44]. Aarabidosis mutant deg1 was small, and had
thin and pale green leaves compared with the wild-type
[4, 20], a phenotype sharing by pgl-sd. But deg1 flow-
ered earlier than the wild-type whereas pgl-sd flow-
ered later. But loss of function of Deg5, the ortholog of
Zm00001eb031870, in Aarabidosis, has no visible effects
on normal conditions [4, 44]. tcm5, a rice Deg mutant,
also exhibited albino phenotype and defective chloro-
plasts [57]. Zm00001eb031900/Zm00001d000227 was
orthologous to AT1G30120 which putatively encoded
a plastid pyruvate dehydrogenase complex E1 compo-
nent subunit beta (ptPDC-E1-β). e ptPDC provides
acetyl-CoA and NADH for fatty acid biosynthesis in
plastids [5, 21].e floury endosperm19 (flo19), a ptPDC-
E1-α1 mutant in rice, showed low plant height and slow
growth throughout the entire growth period rative to
the wild-type, a phenotype reminiscent of pgl-sd, in
addition to opaque of the interior endosperm [25]. It
was surprising to observed that Zm00001eb031870
and Zm00001eb031900 were the only two core genes
revealed by pan gene analysis of the deletion region with
data of MaizeGDB (https:// www. maize gdb. org) (data not
showed). ese data suggested that Zm00001eb031870
and Zm00001eb031900 might be the major causative
genes for the observed phenotype of pgl-sd. However
whether the deletion of Zm00001eb031890 contributes
to specificity of the phenotype of pgl-sd remains to be
elucidated.
Besides the aforementioned three genes, the pgl-
sd deletion region contained Zm00001eb031860
and Zm00001eb031880, which were possibly related
to DNA repair system and plant disease resistance,
respectively. ough limited information was obtained
Table 2 RNA-seq analysis of the expression of genes located in
the pgl-sd deletion
a data were presented with mean ± se. bP-value from analysis of DESeq2
Gene ID
(RefGen_v4) Gene ID
(RefGen_v5) Expression(FPKM)aPb
Mutant Wild
Zm00001d000231 Zm00001eb031860 0.04 ± 0.01 0.07 ± 0.02 0.71
Zm00001d000230 Zm00001eb031870 0.00 ± 0.00 19.78 ± 4.36 0.00
Zm00001d000229 Zm00001eb031880 0.00 ± 0.00 4.09 ± 0.97 0.00
Zm00001d000228 Zm00001eb031890 1.36 ± 0.35 6.68 ± 3.27 0.03
Zm00001d000227 Zm00001eb031900 0.00 ± 0.00 37.63 ± 5.03 0.00
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Yaoetal. BMC Plant Biology (2023) 23:360
from our RNA-seq data, it was observed that transcrip-
tion of some genes in related pathways were affected in
the mutant. But the traits we examined only included
plant height, leaf color, and plant growth. us, it is
necessary to find a appropriate set of traits to confirm
the possible effects of the deletion of these two genes.
SVs are crucial for genome evolution and abundant in
genomes, especially for species like maize, which, as an
ancient tetraploid, experienced many times of duplica-
tion events, but few validated SVs have been reported
in maize. e finding of the deletion of pgl-sd provided
valuable information for functions and mechanisms of
SVs in maize genome. Several mechanisms have been
proposed for occurrence of SVs, including non-allelic
homologous recombination (NAHR), microhomologous
recombination (MHR), non-homologous end joining
(NHEJ), microhomology-mediated end join (MHMEJ)
and microhomology-mediated break-induced replica-
tion (MMBIR), etc.[6, 17]. NAHR requires long stretches
of homologous sequences flanking the genomic region
of SVs. MHMEJ or MMBIR is characterized by short
homologous sequences (< 70bp) at the break-joint posi-
tion, whereas MMBIR was prone to result in complex SVs
[6, 17]. For pgl-sd, no long homologous segments were
observed flanking the deletion, but a 4-bp same nucleo-
tide, A, were present at the break-joining point (Fig.3),
indicating that MHMEJ might lead to the deletion.
Conclusions
In this study, we identified a 137.8-kb deletion through
map-based cloning of pgl-sd, in which no maize leaf color
associated genes were reported before. is deletion led
to abnormality of chloroplast development, reduced con-
tents of photosynthetic pigments in leaves, and affected the
expression of genes involved in the structure and function
of chloroplast. ree genes in this deletion were possibly
related to the plastid development with roles different from
that of other isolated maize leaf color associated genes.
As Zm00001eb031870, an ortholog of Arabidopsis Deg5,
and Zm00001eb031900, putatively coding ptPDC-E1-β,
were the only core genes in the identified deletion, the
mutation effects on maize phenotype suggested these two
genes may be necessary for normal maize development. e
reports on the function of Degs and ptPDCs in other plants
also point out the value of exploring the use of both genes
in breeding maize varieties with higher yield potential and
better stress tolerance to extreme environments, especially
characterized by high temperature and light, in the future.
Methods
Plant materials andphenotyping
e spontaneous mutant pgl-sd was isolated from
a breeding population. In consideration of possible
effects of genetic backgrounds on phenotypic expres-
sion of the mutation of pgl-sd, the mutant plants were
crossed with three elite wild inbred lines, including
B73, Zheng58 and Qi319. B73 and Zheng58 belong to
the Stiff Stalk group, and Qi319 is a line derived from
mixed origin. e BC1 population, B73/*2pgl-sd, was
developed by backcrossing B73/pgl-sd F1 individuals
with pgl-sd. F2 populations were created for the other
two crosses.
Phenotypes of the mutant pgl-sd, the three wild inbred
lines and their F1 progeny were investigated in green
house at seedling stages (from emergence stage, V0, to
vegetative phase 3 stage, V3), and in the field at all growth
stages. For segregation analysis, plants of BC1, F2 popula-
tions and their parents were grown in plastic trays in a
green house and leaf color was evaluated at stages from
V0 to V3.
Molecular markers development andgenotyping
SSR and IDP markers were retrieved from maizeGDB
database (https:// www. maize gdb. org) for mapping of pgl-
sd. At the fine mapping stage, IDP and SSR markers listed
in the two papers [19, 29] were used, but primer pairs
were redesigned in most cases. e nucleotide sequence
around the target region of RefGen_v4 were also used to
search for potential SSR with MISA [2] for fine mapping
of pgl-sd.
At the final stage of the mapping study, pgl-sd was rese-
quenced to develop more IDP markers. Leaf samples
of pgl-sd seedlings were used for genomic DNA extrac-
tion. A 350-bp insertion size library was constructed
and sequenced with Illumina NovaSeq6000 at Novogene
(Tianjin, China), yielding a total of 276,713,444 150-
bp paired-end reads (41.12G base). Sequence data of
Zheng58 (PRJEB30082) and Qi319 (PRJNA609577) were
download from EBI (https:// www. ebi. ac. uk). Reads with
poor quality was filtered with Trimmomatic [3] and eval-
uated with FastQC (https:// www. bioin forma tics. babra
ham. ac. uk/ proje cts/ fastqc/). Clean reads were mapped
to the RefGen_v4 or RefGen_v5 by using BWA mem [26]
with default parameters. e mapped reads were sorted
with SAMtools (Li et al. 2009) and marked duplication
with picard (https:// broad insti tute. github. io/ picard/),
then subjected to GATK (HaplotypeCallerfunction) [35])
for variant calling. Raw indels were filtered with expres-
sion "QD < 2.0 || FS > 200.0 || ReadPosRankSum < -20.0".
Primers were designed using Primer3 [38] and synthe-
sized at Sangon (Qingdao, China). Primer information
for markers used for genotyping and other analyses in
this study was listed in (Table3).
PCR amplifications were performed using TransGen
(Beijing, China) EasyTaq DNA Polymerase for page with
annealing temperature being set to 55 centigrade. PCR
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Page 11 of 14
Yaoetal. BMC Plant Biology (2023) 23:360
products were separated with electrophoresis on 8%
(W/V) polyacrylamide gel with a acrylamide to bisacryla-
mide ratio of 19:1 (W/W) or 39:1.
Linkage map construction
A linkage map was constructed with MAPMAKER 3.0b
[24] in the initial mapping. Linkage groups were deter-
mined with a minimum logarithm of odds (LOD) score
of 3.0 and max distance 50cM. Recombination frequency
was computed with Haldane’s mapping function. Linkage
maps were draw with MapChart [47].
Detection andvalidation ofSVs
Mapping data of genome sequencing reads for pgl-sd,
Zheng58 and Qi319 based on RefGen_v5 were used to
detect SVs in pgl-sd compared with B73 by using both
Manta [9] and Breakdancer [12], respectively. e genome
assembly of pgl-sd was performed by using SOAPde-
novo (SOAPdenovo-63, with K being set to 63) [33] with
cleaned reads subjected to duplication removing. e
final assembly was utilized to confirm possible structure
variants predicted by the software aforementioned when
necessary. PCRs for experimental SV validation were con-
ducted with Vazyme (Nanjing, China) Phanta Flash Mas-
ter Mix following the factory’s manual and PCR products
were separated with 1% (W/V) agarose gel.
RNA extraction, reverse transcription andRT‑qPCR analysis
e first leaves of pgl-sd, B73, Zheng58, Qi319 plants at
V2 stages were harvested for total RNA extraction by
Table 3 Primers used in the study
a start position of forward primer on the chromosome 1 based on RefGen_v5; bstart position of reverse primer
NAME Forward primer Reverse primer Anealing
Temp.
(oC)
Type F Start Pos.aR start Pos.bGene
gy489 ACA TCT TTC GTT CGT AGA CCGT CCG ACT CTT CTG ACC CAG TCC 55 IDP 192,081,138 192,081,405
gy490 ACT CCG CT T CAT CGA CTC AT TTG GCA CTC AAT CAC CAA AA 55 IDP "204,958,969 204,959,142
gy496 AAG CTC GAC TCT TTT GGT TC CTC TTT TCA TTT ACC CCT GCTAC 55 SSR 193,783,975 193,784,132
gy502 CTG CAC AAG AAC GTG AGT GA AGT CAT CAA AAG ATC TAC ATGC 55 SSR 199,513,745 199,513,899
gy521 ATC TTA ATC TTG TGC GGG TTCA CAA AAT TTT AAC AAC ACC TCC CCT 55 SSR 173,334,992 173,335,251
gy524 AAA ATA CCA GCA TGA GGG AT GTG AAC AAA TTA ACT AAT GAAAC 55 SSR 175,789,629 175,789,904
gy527 ATC ATG ACA AAA GGC AAG TGAC GGC CCA AAA CAC AGT AGA ACC 55 SSR 179,920,357 179,920,590
gy541 GAC ATT TAA GGT GCC CAC GA CTC AAG AAA GGT TAA ACG GG 55 SSR 182,124,940 182,125,239
gy544 AGC AAT TAG TTT ACA AAT TTCCA CCT TTC TGT TTA CTT CGC CTG 55 IDP 177,015,314 177,015,562
gy545 TGG AGA AGT TAG CCA CAA CC TAC ATG TTT GTG GCT TGA ACT 55 IDP 177,299,288 177,299,483
gy546 CTA GCA AAA TTG TAG ATG CAC AAG AGA CAA TCT ATG GCT T 55 IDP 178,330,761 178,331,000
gy547 TCA TGG ACT ATG GTG TGA CGA TCG ATC GGA CAA TTA TGG AC 55 IDP 178,476,920 178,477,058
gy548 TTG TGC TCC ATA TCC TGT CC TT T CGT CAG CGA TCT ACC TC 55 IDP 178,487,771 178,487,962
gy549 CCA ATA GCT TGT AAT GGT T TAT CGT CTC TAC AAG TCC T 55 IDP 178,599,823 178,599,969
gy550 GAT GCA AAA TTT CGC CGA T ATA TAG CTA GCC AAC AAA GGG 55 IDP 178,338,656 178,338,868
gy553 ACT CGC AAA GAT TTC CTG CCA TAG ATC CCT AGC TCC 55 IDP 178,411,535 178,411,726
gy554 ACA TAG TCC ATA ATT GTC CGAT TAG CGT TAA ACC AAC TAC CAG 55 IDP 178,477,034 178,477,154
gy562 TTG GAG TAT AAA TTA AAA GACTA ATC GAA AAG AGA ATT GAT T 55 IDP 178,399,186 178,399,334
gy555 CGA GAT CAT CGG AGG AAT CCTG GCC AGC GTG TCC AGC ACC GTC 60 Zm00001eb031850
gy556 GAG TTG CCA CAC ACT AAC CAA TCA CAA ACC CAA CTA ACA GCA 60 Zm00001eb031860
gy557 CGA TCC GTG GGG CTA TAC AGACA ATC CCA GAT CCT TTA CGT GTGA 60 Zm00001eb031870
gy559 AAT AGC TTT CAA GAG TGC CTA ATC CTC TCC GAT TGT AGC AAG 60 Zm00001eb031890
gy560 ATC CAA GTC AGG CGG CCA TGA ACA TCT TCA CCC ATG ACG CACAC 60 Zm00001eb031900
gy565 CCT CCT CCA GCT GTA GCC TCA GCT GTC GGA CGA GGA GCT GAAC 60 Zm00001eb031880
gy573 TGA CCT TCG CT T CTA TAC TGTCT TCA GAT GTC CAG CAC CGT CGAT 66 178,395,427 178,399,988
gy580 TGA CCT TCG CT T CTA TAC TGTCT TCA GAT GTC CAG CAC CGT CGAT 57 178,393,468 178,393,571
gy586 TGA CCT TCG CT T CTA TAC TGTCT TCA GAT GTC CAG CAC CGT CGAT 57 178,417,639 178,417,793
gy598 TCC CCG TTC CAA TCC ATG TTC CCA CCC TGC CAT GCA CGC CGC AAA CTA TAC CG 68 178,334,160 178,478,070
gy599 TGA CCT TCG CT T CTA TAC TGTCT TCA GAT GTC CAG CAC CGT CGAT 60 178,335,787 178,477,075
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 12 of 14
Yaoetal. BMC Plant Biology (2023) 23:360
using a Aidlab’s plant RNA extraction kit RN09 (Aidlab,
Beijing, China). RNA was reverse transcribed to cDNA
using Accurate Biology’s RT Kit (AG accurate Biology,
Changsha, China). RT-qPCR was performed with a SYBR
Green Real-Time PCR Master Mix (Accurate Biology,
Changsha, China) using e Applied Biosystems 7500
Real-Time PCR System (ermFisher Scientific) fol-
lowing the comparative CT experiment protocol of the
manufacturer. Experiments were performed with three
independent RNA samples and three technical replicates
and folypolyglutamate synthase (FPGS) was used as the
reference gene [34]. Relative gene expression was calcu-
lated as described by Livak and Schmittgen [30].
Measurement ofchlorophyll andcarotenoid contents
Because the population from which pgl-sd originated was
not available, plants from the B73/*2pgl-sd population were
used in the leaf pigment measurement as well as micros-
copy analysis and RNA-seq. Leaves of plants at V2-V4
stages (approximately 200mg fresh weight for each plant)
were cut into pieces and then submerged in a 10-ml solu-
tion of 2:1 (V/V) 95% acetone to ethanol for 48h at 26°C
under dark conditions. OD values of these extracts were
measured with Nanodrop2000 (ermFisher Scientific) at
663, 645, and 470nm, respectively. Contents of Chla, Chlb,
and Car were estimated as described by Guan etal.[14].
Transmission electron microscopy
Plants at V2 to V3 stages, which grew in a greenhouse,
were used for transmission electron microscopy analysis.
Sample sections were prepared according to the descrip-
tion of Guan et al. [14] and observed using a Hitachi
transmission electron microscope H-7500.
RNA‑Seq Analysis
Leaves of plants at growth stages from V2 to V4 were
harvested for RNA-Seq analysis. Six mutants or six wild
plants were pooled together as one biological sample for
one phenotype group and three independent samples were
prepared for each phenotype group. Sequencing was per-
formed at Novogene (Tianjin, China). Clean reads were
mapped to RefGen_v4 using Hisat [22]. DEGs between the
mutant and the wild group were determined with adjusted
P-value < 0.2 and fold change > 2 using the R package
DESeq2 [31]. GO enrichment analysis of DEGs was imple-
mented by using the R package clusterProfiler [50] with
maize-GAMER used as the GO annotation [49].
Statistical andgraph drawing soft
All data in the work were analyzed with R (https:// cran.r-
proje ct. org/) and visualized with R package ggplot2 [48]
except otherwise mentioned.
Abbreviations
BP Biological Process
Car Carotenoids
CC Cell Component
CDS Coding sequence
Chla Chlorophyll a
Chlb Chlorophyll b
Deg Degradation of periplasmic protein
DEG Differentially Expressed Gene
FPKM Expected number of fragments per kb of transcript sequences
per Mb sequenced
GO Gene Ontology
IDP Indel polymorphism
MF Molcular Function
pgl-sd Pale green leaf-shandong
ptPDC Plastid pyruvate dehydrogenase complex
ptPDC-E1-β Plastid pyruvate dehydrogenase complex E1 component
subunit beta
QTL Quantitative Trait Loci
RefGen_v3 B73 RefGen_v3
RefGen_v4 Zm-B73-REFERENCE-GRAMENE-4.0
RefGen_v5 Zm-B73-REFERENCE-NAM-5.0
RT-PCR Reverse transcription PCR
RT-qPCR Reverse transcription quantitative PCR
SNP Single nucleotide polymorphism
SV Large scale structure variant
Supplementary Information
The online version contains supplementary material available at https:// doi.
org/ 10. 1186/ s12870- 023- 04360-2.
Additional le1.
Additional le2.
Additional le3.
Acknowledgements
We thank the anonymous reviewer for his comments on the section of meth-
ods in our manuscript and the editors for their suggestions and corrections on
the English language use.
Authors’ contributions
CM, XL, HZ and GY conceived this research; GY, HZ and CM completed most
of experiments; BL, BC, JS, ZY, HG, and WC participated in the experiments;
GY wrote the manuscript; CM and HZ revised the manuscript; all authors
reviewed the this submission.
Funding
This work was supported by Science Fundation of Shandong Province for
Youth (ZR2020QC105)
Science Fundation of Shandong Province for Youth,ZR2020QC105,Bingying Leng
Availability of data and materials
All data generated or analyzed during this study are included in this published
article and its supplementary information files. The sequencing data are avail-
able at NCBI SRA database under the project of PRJNA823837.
Declarations
Ethics approval and consent to participate
Not applicable
Consent for publication
Not applicable
Competing interests
The authors declare that they have no competing interests
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Page 13 of 14
Yaoetal. BMC Plant Biology (2023) 23:360
Author details
1 Maize Research Institute, Shandong Academy of Agricultural Sciences,
Jinan 250100, China. 2 Key Laboratory of Biology and Genetic I mprovement
of Maize in Northern Yellow-Huai River Plain, Ministry of Agriculture, Jinan 250100,
China. 3 National Engineering Laboratory of Wheat and Maize, Jinan 250100,
China. 4 National Maize Improvement Sub-Center, Jinan 250100, China. 5 College
of Life Sciences, Shandong Normal University, Jinan 250014, China.
Received: 30 April 2022 Accepted: 21 June 2023
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