Genomic and epigenomic variation in Psidium species and their outcome under the yield and composition of essential oils

Abstract

Diploid and polyploid species derived from the euploid series x = 11 occur in the genus Psidium, as well as intraspecific cytotypes. Euploidy in the genus can alter the gene copy number, resulting in several “omics” variations. We revisited the euploidy, reported genomic (nuclear 2C value, GC%, and copy number of secondary metabolism genes) and epigenomic (5-mC%) differences in Psidium, and related them to essential oil yield and composition. Mean 2C values ranged from 0.90 pg (P. guajava) to 7.40 pg (P. gaudichaudianum). 2C value is intraspecifically varied in P. cattleyanum and P. gaudichaudianum, evidencing cytotypes that can be formed from euploid (non-reduced) and/or aneuploid reproductive cells. GC% ranged from 34.33% (P. guineense) to 48.95% (P. myrtoides), and intraspecific variations occurred even for species without 2C value intraspecific variation. Essential oil yield increased in relation to 2C value and to GC%. We showed that P. guajava (diploid) possesses two and P. guineense (tetraploid) four copies of the one specific TPS gene, as well as eight and sixteen copies respectively of the conserved regions that occur in eight TPS genes. We provide a wide “omics'' characterization of Psidium and show the outcome of the genome and epigenome variation in secondary metabolism.

Full text

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Genomic and epigenomic
variation in Psidium species
and their outcome under the yield
and composition of essential oils
Matheus Alves Silva
1, Fernanda Aparecida Ferrari Soares
2, Wellington Ronildo Clarindo
2,
Luiza Alves Mendes
3, Luziane Brandão Alves
1, Adésio Ferreira
1 &
Marcia Flores da Silva Ferreira
1*
Diploid and polyploid species derived from the euploid series x = 11 occur in the genus Psidium, as
well as intraspecic cytotypes. Euploidy in the genus can alter the gene copy number, resulting in
several “omics” variations. We revisited the euploidy, reported genomic (nuclear 2C value, GC%,
and copy number of secondary metabolism genes) and epigenomic (5-mC%) dierences in Psidium,
and related them to essential oil yield and composition. Mean 2C values ranged from 0.90 pg (P.
guajava) to 7.40 pg (P. gaudichaudianum). 2C value is intraspecically varied in P. cattleyanum and
P. gaudichaudianum, evidencing cytotypes that can be formed from euploid (non-reduced) and/or
aneuploid reproductive cells. GC% ranged from 34.33% (P. guineense) to 48.95% (P. myrtoides), and
intraspecic variations occurred even for species without 2C value intraspecic variation. Essential
oil yield increased in relation to 2C value and to GC%. We showed that P. guajava (diploid) possesses
two and P. guineense (tetraploid) four copies of the one specic TPS gene, as well as eight and sixteen
copies respectively of the conserved regions that occur in eight TPS genes. We provide a wide “omics’’
characterization of Psidium and show the outcome of the genome and epigenome variation in
secondary metabolism.
e neotropical and monophyletic genus Psidium, family Myrtaceae, contains approximately 92 species1, hich
are taxonomically classied in four sections: Psidium (10 species), Obversifolia (six species), Apertiora (31
species) and Mitranthes (26 species)2. ~ 60 Psidium species occur in Brazil distributed in all biomes, in several
phytogeographic domains, representing great diversication3. Psidium guajava L. (guava tree, Psidium section)
is the well-known species of the genus due to its relevance for fruit production4 and medicinal value5,6. Other
Psidium species, popularly called “araçás”, are potential genetic resources for breeding programs and medicinal
purposes7, as well as they are relevant for taxonomic, ecological and evolutive studies in this genus2.
Besides diploid species, in Psidium occur polyploid species with dierent ploidy levels commonly derived
from the basic chromosome number x = 11. One of the consequences of euploidy in Psidium is the interspe-
cic and intraspecic variation of the nuclear 2C value. Previously, we found that the increase (2C = 0.93pg
– 2C = 5.12pg) of the nuclear 2C value is outcome of the increase in ploidy level (diploid with 2C = 0.93pg
– 2C = 0.98pg to octoploid with 2C = 5.12pg) in seven Psidium species8. In addition to interspecic variation,
genomic dierences have been identied for P. cattleyanum. is species is considered a polyploid complex
due to genetic diversity (intraspecic variation) of the 2n chromosome number and, consequently, the ploidy
level (triploid 2n = 3x = 33 to duodecaploid 2n = 12x = 132 chromosomes), nuclear 2C value (tetraploid with
2C = 2.17pg to duodecaploid with 2C = 5.64pg), and number of the CMA3 + /DAPI-, 18S rDNA and 5S rDNA
sites (triploid with three sites to duodecaploid with 12, 12 and 10 sites)9.
Despite the 2n chromosome number importance for ploidy level determination (euploidy condition), previous
data indicate that the nuclear 2C value is an indicator of higher ploidy level in Psidium8,9. us, nuclear 2C value
OPEN
1Departamento de Agronomia, Centro de Ciências Agrárias e Engenharias, Universidade Federal do Espírito Santo,
Alto Universitário, s/n, Guararema, Alegre, ES 29500-000, Brazil. 2Departamento de Biologia Geral, Universidade
Federal de Viçosa, Av. Peter Henry Rolfs, s/n, Campus Universitário, Viçosa, MG 36570-900, Brazil. 3Departamento
de Química, Universidade Federal de Viçosa, Av. Peter Henry Rolfs, s/n, Campus Universitário, Viçosa,
MG 36570-900, Brazil. *email: marcia.ferreira@ufes.br
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can be used especially when dealing with a large number of individuals. Nuclear 2C value is mainly measured
using ow cytometry, which is widely used because it is fast, accurate and reproducible10.
In addition to the nuclear 2C value, the AT/GC base composition can also be measured by ow cytometry,
expanding the genomic data11. AT/GC base composition knowledge allows inferences about the genome struc-
ture and dynamic. So, AT/GC base composition must be incorporated into “omics” data, and consequently used
in taxonomic, systematic and evolutive studies. Psidium guajava genome has GC = 39.5%12, and P. cattleyanum
transcriptome has ~ 49% in the yellow and red morphotypes13.
Polyploidy has been considered one of the main genomic changes that results in genetic8 and epigenetic
modications14 inuencing the population genetic structure, ecological niche dierentiation, diversication and
speciation in plants15. About it, Psidium is one outstanding example of the polyploid impact in speciation and
geographic distribution8,15. Euploidy (autopolyploidy, true allopolyploidy or segmental allopolyploidy) plays a
central role in shaping and restructuring plant genomes15. Regardless of the genomic origin, one euploidy out-
come is the increase of the gene copy number, which probably results in phenotypic changes and/or new traits.
Also furthering phenotypic variations, genomic changes occur (“genomic shock”) aer the euploidy, such as
aneuploidy, structural chromosome rearrangements, mobile elements activation or silencing, and DNA sequence
change16. us, the euploidy and its outcomes are sources of evolutionary novelties. Duplicate genes can follow
an evolutive path from an initial state of complete redundancy, in which a copy is probably disposable, to a stable
situation17,18 – neutral theory. Still, they may include new gene functions and expression patterns. e duplicated
genes can maintain their original or similar function, undergo diversication in function or expression patterns,
or a copy can be silenced by mutations or epigenetic mechanisms19. Many duplicate genes can have loci in tandem
in the genome or occur regionalized (within a few Mbp)20,21. Additionally, phylogenetic evidence links genome
increase (nuclear 2C value) to the increase in the overall percentage of global 5-methylcytosine (5-mC%). 5-mC
is an important epigenetic chemical change in DNA that promotes heterochromatinization (chromatin compact
level increase) and, consequently, gene expression control22,23.
Leaf essential oils, characteristic of the Myrtaceae family secondary metabolism, are rich in terpene and
exhibit quali- and quantitative variations reported24. Terpenes play ecological roles, and essential oils are eco-
nomically exploited25–27 for their biological and phytotherapeutic activities28. Our research group evidenced that
seven P. cattleyanum plants exhibited dierent nuclear 2C values (2C = 3.20pg – 2C = 6.03pg), seven monoter-
penes and eight sesquiterpenes in essential oils. From these data, the P. cattleyanum plants were discriminated in
three cytotypes (nuclear 2C value) related to three chemotypes (monoterpene and sesquiterpene compounds).
P. cattleyanum plants with the relatively lower nuclear 2C values (2C = 3.23pg – 2C = 4.71pg) produced a lower
amount of essential oils composed mostly of hydrogenated monoterpenes. Dierently, the plants with relatively
higher nuclear 2C values (2C = 5.81pg and 2C = 6.03pg) produced a higher amount of essential oils composed
mostly of hydrogenated sesquiterpenes, as trans-caryophyllene and alpha-humulene24.
We aimed to revisit the 2n chromosome number (euploidy), measure the nuclear 2C value, GC% and 5-mC%,
and determine the copy number of TPS genes in Psidium species of three sections (Psidium, Apertiora and
Obversifolia). In addition, we correlated these genomic and epigenomic data with the yield and composition of
the essential oils, evidencing the outcomes of the genetic and epigenetic dierences in this phenotype.
Results
Intraspecic and interspecic variation of nuclear 2C value and GC%. e nuclear 2C value of
each Psidium access ranged from 0.90pg (P. guajava) to 7.40pg (P. gaudichaudianum, Supplementary TableS2).
We noticed, for the rst time, the nuclear 2C value for P. acidum, P. gaudichaudianum, P. friedrichsthalianum,
P. macahense and P. r uf um . Psidium guajava, P. oblongatum and P. macahense presented the lowest 2C values.
Psidium guajava and P. oblongatum are diploids (2n = 2x = 22 chromosomes). erefore, possibly P. macahense
also has the same 2n chromosome number and ploidy level, since this species has closer mean nuclear 2C value
(2C = 0.93pg) than P. guajava (2C = 0.96pg) and P. oblongatum (2C = 0.99pg).
Interspecic variation in nuclear genome size was conrmed by mean nuclear 2C values of the Psidium species
(Table2). Considering the lowest mean 2C value = 0.93pg for P. macahense and highest 2C = 4.99pg for P. gau-
dichaudianum, we realize a variation equivalent to 2C = 4.06pg more nuclear DNA. Additionally, the individual
nuclear 2C values also show the interspecic variation in nuclear genome size, reaching 2C = 6.50pg more nuclear
DNA, since one access of P. guajava has 2C = 0.90pg and one access of P. gaudichaudianum has 2C = 7.40pg.
In addition to interspecic variation, the individual values point to intraspecic variation of the nuclear 2C
value, including among relatives. Accesses belonging to P. guajava (2C dierence = 0.13pg among individuals),
P. guineense (2C = 0.20pg) and P. acidum (2C = 0.08pg) showed less variation of the nuclear 2C value. ese
nuclear 2C values dierences are lower than the 1Cx value of P. guajava (0.475pg) and P. oblongatum (0.490pg)
determined considering the basic chromosome number (x = 11) of the genus Psidium and the ploidy level of
these species (2n = 2x = 22 chromosomes – diploid8). erefore, the intraspecic variation found among accesses
of P. guajava, P. guineense and P. acidum is probably a consequence of secondary metabolites that interfere with
the intercalation of the propidium iodide uorochrome to DNA in the staining step for nuclear suspension
preparation for ow cytometry.
We also observed intraspecic variation of the nuclear 2C value among individuals, as well as in the rela-
tives of P. myrtoides, P. cattleyanum and P. gaudichaudianum. e nuclear 2C value dierence was: 2C = 0.40pg
between individuals of P. myrtoides, 2C = 5.03pg of P. cattleyanum, and 2C = 2.76pg of P. gaudichaudianum. For
these species, the nuclear 2C value dierences are close to or higher than the reference 1Cx value (1Cx = 0.475pg
P. guajava—1Cx = 0.490pg P. oblongatum) at the basic chromosome number x = 11 of Psidium. erefore, these
values indicate that individuals of these species have dierent 2n chromosome numbers among each other, pos-
sibly arising from numerical chromosomal changes (euploidy and/or aneuploidy).
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According to the mean nuclear 2C values (Table2) and the data reported for diploid and polyploid Psidium
species (Table1, http:// ccdb. tau. ac. il/ search/, https:// cvalu es. scien ce. kew. org/ search/ angio sperm), we suggest
that accesses of the species P. acidum, P. rufum, P. friedrichsthalianum and P. gaudichaudianum are potential
polyploids. us, individuals of these species probably have more than 2n = 2x = 22 chromosomes. However,
chromosome number counting should be conducted to conrm the ploidy level.
Six groups were identied in relation to the individual nuclear 2C value (Table3). Group I comprises the
individuals with the smallest nuclear 2C value (2C = 0.90pg to 2C = 1.10pg), which include the diploid species
Table 1. Section, habit, Brazilian region of occurrence, phytogeographic domain, chromosome number and
ploidy level reported for Psidium species.
Specie and Section2Habit3Occurrence3Phytogeographic domain3Chromosome number and ploidy
level
Psidium acidum (DC.) Landrum (sec-
tion Psidium)Tre e North, Southeast, South Amazon –
P. cattleyanum Sabine (section Obver-
sifolia)Tre e Northeast, Southeast, South Caatinga, Cerrado, Atlantic Forest 46, 55, 58 and 8229; 33 (3x), 44 (4x), 55
(5x), 66 (6x), 77 (7x), 88 (8x), 99 (9x),
110 (10x) and 132 (12x)9
P. guajava L. (section Psidium)Tre e North, Northeast, Center-West, South-
east, South Amazon, Caatinga, Cerrado, Atlantic
Forest 22 (2x)29,30
P. guajava L. x P. guineense Sw – – – –
P. guineense Sw. (section Psidium) Shrub, Tree North, Northeast, Center-West, South-
east, South Amazon, Caatinga, Cerrado, Atlantic
Forest 44 (4x)30
P. myrtoides O.Berg (section Apertiora)Tre e North, Northeast, Center-West, South-
east, South Caatinga, Cerrado, Atlantic Forest 66 (6x)8; 88 (8x)31
P. gaudichaudianum Proença & Faria Tree Southeast Atlantic Forest -
P. friedrichsthalianum (O.Berg) Nied
(section Psidium)Shrub, Tree North Amazon 44 (4x)31
P. macahense O.Berg (section Aperti-
ora)Shrub Southeast Atlantic Forest -
P. oblongatum O.Berg Tree Southeast Atlantic Forest 22 (2x)8
P. ru fu m Mart. ex DC. (section Aper-
tiora)Tree Northeast, Center-West, Southeast,
South Cerrado, Atlantic Forest –
Psidium sp. – – – –
Table 2. Nuclear DNA content (2C value) and percentage of GC bases (GC%) of Psidium accesses and half-
sibling family (MI).
Access
Valor 2C (pg) CG%
Mean ± SD Minimum and maximum value Mean ± SD Minimum and maximum value
P. macahense 0.93 – 37.56 –
P. guajava 0.96 ± 0.030 0.90–1.10 37.97 ± 1.092 36.16–40.74
P. oblongatum 0.99 – 39.81 –
P. guajava x P. guineense 1.90 ± 0.000 1.90–1.90 35.64 ± 0.506 35.07–36.08
P. guineense 1.90 ± 0.078 1.80–2.00 36.86 ± 3.535 34.33–43.07
P. acidum 2.04 ± 0.043 2.00–2.08 40.29 ± 0.356 40.04–40.54
P. myrtoides 2.95 ± 0.090 2.72–3.12 39.62 ± 2.015 37.63–48.95
P. cattleyanum 3.92 ± 0.618 2.00–7.03 40.40 ± 2.815 36.37–48.91
P. ru fu m 4.23 – – –
Psidium sp. 4.70 ± 0.411 1.92–4.94 38.98 ± 1.858 35.90–40.92
P. friedrichsthalianum 4.72 – 38.59 –
P. gaudichaudianum 4.99 ± 0.477 4.64–7.40 39.67 ± 0.723 38.38–40.48
P. guajava-MI 06 0.96 ± 0.022 0.92–1.00 37.91 ± 0.899 36.27–39.29
P. guajava-MI 05 0.97 ± 0.033 0.95–1.03 38.59 ± 1.140 37.32–40.33
P. myrtoides-MI 08 2.93 ± 0.099 2.72–3.12 43.83 ± 0.599 38.71–48.95
P. myrtoides-MI 07 3.00 ± 0.060 2.82–3.09 39.38 ± 0.900 38.37–40.49
P. cattleyanum-MI 03 3.67 ± 0.195 3.34–4.27 39.94 ± 1.295 37.99–41.46
P. cattleyanum-MI 02 3.82 ± 0.215 3.45–3.98 – –
P. cattleyanum-MI 01 3.86 ± 0.663 3.29–5.68 – –
P. cattleyanum-MI 04 4.16 ± 0.161 3.63–4.38 41.91 ± 0.962 41.23–42.59
P. gaudichaudianum-MI 09 4.99 ± 0.485 4.64–7.40 39.72 ± 0.800 38.38–40.48
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P. guajava, P. oblongatum, and the P. machaense that, based on the 2C and 1Cx values, possible is a diploid spe-
cies. Group II (2C = 1.80pg—2C = 2.08pg) has the tetraploid species P. guineense and P. acidum, as well as all
14 P. guajava × P. guineense hybrids, one individual of P. cattleyanum and seven individuals of Psidium sp. e
evaluated P. guajava × P. guineense hybrids have 2C = 1.90pg, exhibiting the same nuclear 2C value (Table2).
is mean value is equivalent to the tetraploid genomic origin. Dierent progenies are possible considering the
P. guajava × P. guineense crossing: (a) allotriploid hybrids (~ 2C = 1.43pg) from the fusion of reduced reproduc-
tive cells of the two species, (b) allotetraploid hybrids (~ 2C = 1.91pg) generated from the fusion of non-reduced
reproductive cells of P. guajava and reduced P. guineense, (c) allopentaploid hybrids (~ 2C = 2.38pg) formed by
fusing reduced reproductive cells of P. guajava and non-reduced P. guineense, and (d) allohexaploid hybrids
(~ 2C = 2.85pg) arising from the fusion of non-reduced reproductive cells of the two species. erefore, probably
the P. guajava × P. guineense hybrids with 2C = 1.90pg are the result of the fusion of non-reduced reproductive
cells of P. guajava and reduced P. guineense. In addition, we found a P. guajava × P. guineense hybrid (Hib_11)
mixoploid with 30% of cells with 2C = 0.95pg (similar to diploid P. guajava) and 70% of cells with 2C = 1.90pg
(similar to tetraploid P. guineense) (Supplementary TableS2).
Group III (2C = 2.72—2C = 4.38pg) contains most of the individuals evaluated, including the species P.
cattleyanum, P. myrtoides, P. rufum and one Psidium sp. Due to the variation in 2n chromosome number and
ploidy level of P. cattleyanum and P. myrtoides (Table1), the group III includes possibly hexaploid and octaploid
accesses. Group IV (2C = 4.47pg—2C = 5.19pg) has individuals from P. cattleyanum, P. gaudichaudianum, P.
friedrichsthalianum and three Psidium sp, which are probably octaploids (Table1), as well as the only indi-
vidual from group V of P. cattleyanum. Group VI includes the three individuals with the highest 2C values
(2C = 6.83pg—2C = 7.40pg), two from P. cattleyanum and one from P. gaudichaudianum. Probably the accesses
in group VI have a ploidy level higher than octaploid (2n = 8x = 88 chromosomes).
Psidium cattleyanum showed the highest intraspecic variation of the nuclear 2C value, exhibiting individuals
in ve of the six groups. Dierently, less intraspecic variation was conrmed for the diploid species P. guajava
(all individuals in group I), for the tetraploids P. guineense and P. acidum (group II) and for the hexaploid P.
myrtoides (group III, Table3).
Table 3. Grouping by Tocher’s method (optimized) of nuclear DNA content (2C value) (pg) and percentage of
GC bases (GC%) of Psidium accesses. Aci = P. acidum; Cat = P. cattleyanum; Gua = P. guajava; Hib = P. guajava x
P. guineense; Gui = P. guineense; Myr = P. myrtoides; Gau = P. gaudichaudianum; Fri = P. friedrichsthalianum;
Mac = P. macaense; Obl = P. oblongatum; Ruf = P. r uf um ; Psi = Psidium sp. Superscript value in
parentheses = number of individuals of the species present in the group. Color representations = Half-sib families
01
, 02, 03, 04, 05, 06, 07, 08, 09
(separately accounted).
Group Accesses Mean value Minimum and maximum
value
2C value
IGua(6), Gua(17), Gua(31), Mac(1), Obl(1) 0.96 0.90 – 1.10
II Aci(4), Cat(1), Hib(14), Gui(7), Psi(7) 1.94 1.80 – 2.08
III Cat(19), Cat(11), Cat(5), Cat(30), Cat(30), Myr(6), Myr(18),
Myr(30), Ruf(1), Psi(1)
3.51 2.72 – 4.38
IV Cat(5), Gau(1), Gau(28),Fri(1), Psi(3) 4.88 4.47 – 5.19
VCat(1) 5.68 -
VI Cat(2), Gau(1) 7.09 6.83 – 7.40
CG%
IGui(1) 34.33 -
II Hib(2), Gui(2) 35.29 35.07 – 35.37
III Cat(1), Gua(2), Gua(1), Hib(2), Gui(1), Psi(1) 36.15 35.90 – 36.37
IV Cat(3), Cat(1), Gua(3), Gua(7), Gua(18), Myr(1), Mac(1), Psi(2) 37.40 36.72 – 38.17
VCat(12), Cat(1), Gua(2), Gua(8), Gua(6), Myr(4), Myr(11),
Myr(1), Gau(1), Gau(1), Fri(1), Psi(1)
38.92 38.29 – 39.62
VI Aci(2), Cat(5), Cat(2), Gua(1), Gua(3), Myr(1), Myr(7), Gau(4),
Obl(01)
40.14 39.67 – 40.74
VIICat(6), Cat(2), Cat(2), Gui(1), Myr(2), Psi(1) 43.80 40.92 – 48.95
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e GC% values of the evaluated 137 individuals ranged from 34.33% for an individual of P. guineense to
48.95% for an individual of P. myrtoides. e largest intraspecic variations (~ 10%) were in P. myrtoides and P.
cattleyanum, which are species that also show nuclear 2C value intraspecic variation. For the other species, the
GC% ranged from 0.50% for P. acidum to 8.74% for P. guineense. e variation in the two half-sib families of P.
myrtoides was 2.12% (Family 7) and 10.24% (Family 8). Within families of P. cattleyanum, a small variation was
observed (1.36% for Family 7 and 3.47% for Family 3), despite intraspecic nuclear genome size variation in the
species of up to 2C = 5.03pg. e intraspecic variation of GC% was ~ 3.00% for the two families of P. guajava,
and of ~ 2.10% P. gaudichaudianum (Table3).
Seven groups were obtained from comparative GC% analysis. Psidium cattleyanum, P. guajava, P. guineense,
P. myrtoides, P. gaudichaudianum, P. guajava × P. guineense hybrids, and Psidium sp. showed individuals in at
least two groups, evidencing GC% intraspecic variation. Groups III—VI consisted of diploid and polyploid
species. Only one family of P. cattleyanum showed greater stability being allocated only to group VII (Table3).
5-mC%, yield and chemical composition of the essential oil. We compiled in Supplementary
TableS3 the unpublished and published values, for each Psidium individual, of 5-mC%, yield, and the percentage
of the chemical compounds identied from the essential oil. 5-mC values varied between 16.34% (P. guajava)
to 33.30% (P. myrtoides), and between 0.20% (P. guajava) to 0.95% (P. cattleyanum) for yield of the essential oil.
A total of 56 compounds were identied for Psidium species. Of these compounds, 55 are chemically classied
between hydrocarbons and oxygenated mono- and sesquiterpenes.
Relationship of the mean nuclear 2C value, GC% and 5-mC% values, yield and chemical com-
position of the essential oil. e nuclear 2C and GC% values were positively correlated (correlation of
0.51). ese values also had positive correlation with 5-mC% (correlation of 0.35 and 0.36, respectively) and with
essential oil yield (correlation of 0.72 and 0.70, respectively). e variables related to genome (nuclear 2C value
and GC%) and to epigenome (5-mC%) correlated negatively with (E)-Nerolidol (correlation of − 0.64, − 0.45 and
− 0.55, respectively), β-Bisabolol (correlation of − 0.61, − 0.46 and − 0.35, respectively) and the group of oxygen-
ated sesquiterpenes (− 0.87, − 0.70 and − 0.46, respectively). e α-Pinene and the hydrocarbon monoterpene
group were positively correlated with nuclear 2C value (correlation of 0.54 and 0.49, respectively) and 5-mC%
(correlation of 0.60 and 0.53, respectively). e nuclear 2C value along with GC% correlated positively with the
β-caryophyllene (correlation of 0.70 and 0.65, respectively), α-Copaene (correlation of 0.57 and 0.56, respec-
tively) and the hydrocarbon sesquiterpene group (correlation of 0.42 and 0.52, respectively). ese genomic data
correlated negatively with 14-Hydroxy-epi-(E)-Caryophyllene (correlation of − 0.50 and − 0.46, respectively)
and Selin-11-en-4a-ol (correlation − 0.44 and − 0.38, respectively), which belong the oxigenated sesquiterpene
group. In addition, the β-Selinene and Hinesol correlated negatively with nuclear 2C value (correlation of − 0.35
and − 0.42 respectively).
TPS gene copy number. Copy number of TPS genes was determined in diploid P. guajava (2n = 2x = 22
chromosomes and 2C = 0.95pg) and tetraploid P. guineense (2n = 4x = 44 chromosomes and 2C = 1.90pg). We
expected the copy number to be exactly double in the tetraploid P. guineense. e genes showed distinct mark-
ing patterns when comparing the hybridization signal number in relation to the ploidy of the species. For the
specic region used, we detected two hybridization signals in P. guajava and four in P. guineense nuclei (Fig.1).
From the general primer of the conserved motives, the P. guajava nuclei showed four strong signals and four
weak signals. P. guineense nuclei exhibited eight strong and eight weak signals (Fig.1). e presence of weak
signals was considered as DNA sequences that have a relatively homology in relation to the probe, corresponding
to regions that had the same origin, but that accumulated dierences in the gene sequence. In addition, the weak
hybridization signals can be resulted by occurrence of a single gene copy. Dierently, the strong hybridization
signals correspond to gene copies in tandem repeats, which form clusters that amplify the uorescence signal.
erefore, we conrmed that the copy number of the TPS genes was directly related to the ploidy level of the
species with double of signals found in the tetraploid P. guineense. is result also shows the genome evolution
of these Psidium species.
Discussion
We report the nuclear 2C and GC% values, TPS gene copy number, as well as 5-mC% and essential oil yield and
composition of a considerable number of Psidium species and individuals. e GC% values, with the exception
of P. guajava12, are unpublished for all Psidium, as well as the nuclear 2C values for the P. acidum, P. gaudichau-
dianum, P. friedrichsthalianum, P. macahense and interspecic hybrids (P. guajava L. × P. guineense). 5-mC% are
also unpublished for P. cattleyanum, P. guineense, P. myrtoides, P. gaudichaudianum, P. friedrichsthalianum, P.
oblongatum and one access of the genus. In addition, the determination of the TPS gene copy number evidenced
other genomic outcomes of the polyploidy beyond the nuclear 2C and GC% interspecic variation. e results
bring advances about the structure, organization and evolution of the genome and epigenome of Psidium spe-
cies in inter- and intraspecic contexts, including analyses of related individuals. e genomic and epigenomic
data were contextualized with the yield and diversity of compounds in the leaf essential oils, which are rich in
mono- and sesquiterpenes that have ecological and economic importance for the Myrtaceae family.
Based on the previous study of the our research group and the basic chromosome set of Psidium (x = 11), the
1C value varied from 0.465pg (P. cauliforum—diploid species) to 0.640pg (P. longipetiolatum—octaploid spe-
cies), and the increase in ploidy culminated in the increase in nuclear DNA content8. Intraspecic variation in
2C value has been demonstrated for P. cattleyanum (2C = 2.00 to 2C = 7.03pg), corroborating reports of naturally
occurring individuals with varied chromosome numbers and ploidy levels for the species, which has cytotypes
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with 2n = 46, 55, 58 and 8229; 33 (3x), 44 (4x), 55 (5x), 66 (6x), 77 (7x), 88 (8x), 99 (9x), 110 (10x) and 132 (12x)9
(Table1). us, the 2n chromosome number variation in this species can also be supported by the 2C nuclear
value variation. Furthermore, we infer that euploidy occurs in the genus not only in P. cattleyanum, but also in
P. gaudichaudianum and P. friedrichsthalianum due to the amplitude of 2C nuclear value variation.
e oosphere and the reproductive nucleus of the pollen grain are usually reduced reproductive cells (n—
haploid). However, non-reduced and/ou aneuploid reproductive cells can be formed due to errors in anaphase
I or II during meiosis, by the non-disjunction of chromosomes32, and/or by the non-occurrence of cytokinesis
I or II. us, reproductive cells with dierent euploidy and/or aneuploidy can be generated. Individuals of P.
cattleyanum and P. gaudichaudianum, including relatives, showed expressive variations in nuclear 2C value
(possibly reecting the 2n chromosome number) that may have resulted from the unilateral or bilateral fusion
of reduced and unreduced reproductive cells. e unreduced reproductive cells may come from one (unilateral)
or both parents (bilateral) in cross-fertilization or self-fertilization33. In addition, numerical chromosomal varia-
tions (euploidy and aneuploidy) can occur in cells of meristematic regions resulting in mixoploid tissues and/or
individuals. us, the male and/or female reproductive organs of the owers can have meiocytes with dierent
chromosome numbers compared to the sporophyte. In this context, it is important to highlight the occurrence
of a mixoploid individual (Hib_11), not yet reported in Psidium. e mixoploidy can compromise the stability
and fertility of plants in the eld and, thus, the use of these plants for breeding purposes is not very desirable34.
In general, polyploid species of Psidium present a greater geographical distribution compared to diploids8,
with the exception of P. guajava because it is widely exploited and cultivated and, therefore, present in the most
varied regions and biomes35. is fact was pointed out by our research group, considering species P. guajava,
P. guineense, P. myrtoides, P. cattleyanum, P. longipetiolatum, P. oblongatum and P. cauliorum8. In addition, the
geographical distribution of P. cattleyanum cytotypes is inuenced by the ploidy level. P. cattleyanum cytotypes
with higher ploidy levels were identied in regions where the environmental conditions are more adverse, with
higher temperatures, higher incidence of solar radiation and lower precipitation9,15,36. erefore, the polyploid
condition of the species studied here, may be favorable for expansion of their geographic distribution, both by
natural and anthropic action. Hence, exploitation and utilization of these natural resources is relevant for breed-
ing programs and for familiar production.
We veried inter- and intraspecic variations of GC% for the diploid and polyploid species. Although varia-
tions occurred, the overall mean 38.92% CG in Psidium is close to the mean value of 38.06% obtained by means
of 22 diploid Eucalyptus species and three of the genus Corymbia, also species of the Myrtaceae family37.
Due to 2C value and GC% variations in Psidium, especially in families, and their inuence on secondary
metabolism, we suggest, in a practical context, the individual pre-selection of plants to compose an experimental
project, breeding program, germplasm bank or cultivation. In this sense, we recommended that the pre-selected
accesses or individuals of Psidium should be vegetatively propagated, generating new individuals with the same 2n
chromosome number, 2C nuclear value and GC% (genomic stability). On the other hand, inter- and intraspecic
genomic diversity is important as a source of genetic resources for breeding.
We veried an increase in essential oil yield in Psidium due to the larger genome, evidencing the impact of the
genomic changes (2n chromosome number and 2C nuclear value) in the secondary metabolism, which is a trait
of ecological and economic importance. Experimentally, the tetraploid induction (2n = 4x = 72 chromosomes)
Figure1. Copy number of TPS genes.P. guajavapossesses one copy of the specic TPS gene (a), whileP.
guineenseshows two copies of this gene (b). From the probe in conserved motifs, we evidenced eight
uorescence signals in P. guajava(c) and sixteen in P. guineense(d), being that strong uorescence signals are
related to cluster genes. Based on these results, we showed the polyploidy impact in the gene copy number
inPsidium.
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in Lippia integrifolia (family Verbenaceae) increases the essential oil yield compared to diploids (2n = 2x = 36
chromosomes), in addition to larger leaves and trichomes, structures related to essential oil yield38. Additionally,
we showed by FISH that the polyploidy increases the copy number of the orthologs of two TPS genes related to
essential oil biosynthesis of Psidium species. erefore, the polyploidy, also evidenced by 2C nuclear value, aects
the essential oil yield in Psidium from the diploid species (P. guajava 2n = 2x = 22 chromosomes) and the hitherto
reported closest species P. guineense (2n = 2x = 44). e impact of the polyploidy in the essential oil traits can be
related with the diversication and size of TPS gene family in Psidium species. e evolution of the TPS genes in
the Myrtaceae family genomes have reported the largest TPS gene family in plants (Eucalyptus spp. having up to
100 genes)26,39 and occurrence of lineage-specic pathways and products. Although the essential oil of Psidium
species exhibits a great diversity in its chemotypes conditioned to environmental and genetic variations24,27,40,41,
the evolution of TPS genes in Myrtaceae neotropical fresh fruits remain unknown.
e increase of the values of 2C nuclear, CG% and 5-mC% was related to the decrease in (E)-Nerolidol and
β-Bisabolol. erefore, in addition to the genome eect (2n chromosome number, 2C nuclear value and GC%),
chemical changes of the cytosine (5-mC) also inuences composition of the essential oils. So, we showed the
inuence of the epigenetic control in the compound biosynthesis of the secondary metabolism in Psidium. e
higher abundance of oxygenated sesquiterpenes was related to the occurrence of smaller genomes, with lower
CG% and 5-mC%, indicating the genomic and epigenomic inuence in this chemical class. In previous studies,
the presence of oxygenated sesquiterpenes was clearly increased at the expense of hydrocarbons sesquiterpenes in
spring in P. guajava genotypes42. Together, these data, which were reported for the rst time, show the inuence
of genome and epigenome on essential oil yield and in specic compounds, suggesting for epigenetic control
for terpene in Myrtaceae.
Conclusion
From genome and epigenome to secondary metabolism, we provided data about the diversity of the Psidium
species. We characterize the Psidium germplasm in relation to the 2n chromosome number, 2C nuclear and GC%
values, TPS gene copy number and 5-mC%, generating knowledge about species previously studied and also
about others not yet evaluated. In addition, we also explore the secondary metabolism, evidence the phenotypic
divergences between Psidium species and individuals, and conrm our hypothesis about the inuence of the
genome and epigenome. erefore, this work provides an important characterization of the genus Psidium, bring-
ing information and evidence that can be incorporated in further studies, especially in phenotypic responses
related to characters of economic interest.
Material and methods
Plant material. We collected leaf samples from ten Psidium species: Psidium acidum (DC.) Landrum, P.
cattleyanum Sabine, P. guajava L., P. guineense Sw., P. myrtoides O.Berg, P. gaudichaudianum Proença & Faria,
P. friedrichsthalianum (O.Berg) Nied, P. macahense O.Berg, P. oblongatum O.Berg, and P. r uf um Mart. ex DC.
Leaves were also collected from hybrids of P. guajava x P. guineense. Individuals not identied by species were
kept and denominated as genus Psidium (Psi). Brazilian region of occurrence, phytogeographic domain and
2n chromosome number reported for the species are presented in Table1. e number of individuals of each
species for each analysis is presented in Supplementary TableS1. e localization of occurrence of each access,
individual identication and families are presented in Supplementary TableS2.
Nuclear 2C value and GC%. Young leaves from each germplasm (Supplementary TableS2) were used
for nuclear 2C value and GC% measurements. Solanum lycopersicum L., 1753, ‘Stupické’ was used as internal
standard (2C = 2.00pg)10. 2 cm2 leaf fragment from each Psidium germplasm and from the S. lycopersicum were
simultaneously chopped43 for about 30s in a Petri dish containing 0.5mL OTTO-I 44 modied for species of
the Myrtaceae family (0.1M citric acid, 0.5% Tween 20, 50µg mL-1 RNAse, 2mM dithiothreitol, and 7% poly-
ethylene glycol 2000 – PEG)37.Aer adding 0.5mL of the same buer, the resulted suspensions were incubated
for 3min, ltered on a 30μm diameter nylon lter (Partec) in a 2.0mL microtube, and centrifuged at 100 xg for
5min. e supernatant was discarded and 100 μL of the same buer was added to the pellet, which was homog-
enized in vortex and incubated for 10min. Subsequently, 0.5mL of modied OTTO-II staining buer (400mM
Na2HPO4H2O, 2mM dithiothreitol, 50µg mL-1 RNAse, and 75µg mL-1 propidium iodide (PI, excitation/emis-
sion wavelengths: 480–575/550–740nm) was added to the10,44. e suspensions were ltered through 20µm
nylon mesh (Partec) into tubes (Partec) and kept for 30min in the dark. en, the suspensions were analyzed
in a ow cytometer (BD Accuri C6 ow cytometer, Accuri cytometers, Belgium) equipped with a 488nm laser
source to promote emissions at FL2 (615—670nm) and FL3 (> 670nm). e uorescence peaks of the G0/G1
nuclei of each access and the standard were identied in the histograms using BD Accuri™ C6 soware. G0/G1
peaks with coecient of variation (CV) less than 5% were considered for nuclear 2C value measurement in pg
by the formula: nuclear 2C value of the access (pg) = [(mean G0/G1 peak channel of the access)*2.00pg S. lyco-
persicum]/(mean G0/G1 peak channel of S. lycopersicum).
For GC%, nuclear suspensions were generated following the procedure adopted to measure the nuclear 2C
value with some modications: (a) the OTTO I and II buers were not supplemented with RNAse, and (b)
the OTTO II buer was supplemented with 1.5μM of 4’,6-diamidino-2-phenylindole (DAPI, excitation/emis-
sion wavelengths: 320–385/400–580nm). e suspensions were analyzed with a Partec PAS ow cytometer
(Partec GmbH, Munster, Germany), equipped with an 388nm UV mercury arc lamp and a GG 435–500nm
band-pass lter. AT% was measured using the formula45%ATsample = %ATstandard*[(RDAPI/RPI)1/r], in which:
%ATS. lycopersicum = 64.50% 11,46; R = ratio of the uorescence intensity of the access/standard; r = 3 for DAPI46. From
the AT%, the GC% was calculated by the following formula: GC% = 100—AT%. e data corresponding to the
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nuclear 2C value and GC% of Psidium accesses were submitted to clustering by the Toucher method optimized by
Euclidean distance, in which the variables were separately evaluated (nuclear 2C value and GC%). e analyses
were conducted in the Genes computer program45.
Percentage of methylated cytosines (5-mC%) in the genome. e 5-mC% data of P. guajava
accesses were revisited from our previous study47. For P. cattleyanum, P. guineense, P. myrtoides, P. gaudichaudi-
anum, P. friedrichsthalianum and Psidium sp. the unpublished 5-mC% was measured based on the methodology
used for P. guajava47.
Yield and chemical composition of the essential oil. We revisited the data about yield and chemical
composition of the essential oil previously published by our research group for P. guajava40,42, P. guineense48 and
P. cattleyanum24. For the other accesses, the essential oil was extracted based on the methodology used for P.
guajava41. e identication and semi-quantication of the leaf essential oil compounds were performed using
gas chromatography with ame ionization detector (GC-FID QP2010SE, Shimadzu, Japan) and gas chromatog-
raphy coupled to mass spectrometry (GC–MS QP2010SE, Shimadzu, Japan). For these analyses, the following
conditions were adopted: the carrier gas used was He for both detectors with ow rate and linear velocity of
2.80mL min− 1 and 50.80cm sec− 1 (GC-FID) and 1.98mL min− 1 and 50.90cm sec− 1 (GC–MS), respectively;
injector temperature was 220°C at a split ratio of 1: 30; fused silica capillary column (30m × 0.25mm); Rtx-
5MS stationary phase (0.25μm lm thickness); the oven temperature had the following programming: initial
temperature of 40°C, which remained for 3min and then the temperature was gradually increased at 3°C min− 1
until it reached 180°C, remaining for ten minutes, with a total analysis time of 59.67min; the temperatures used
in the FID and MS detectors are 240 and 200°C, respectively. e sample used was drawn from the vial in a
volume of 1 μL of a 3% solution of essential oil dissolved in 95% hexane.
GC–MS analyses were performed in an electron impact equipment with an energy of 70eV; scanning speed
of 1000; scanning interval of 0.50 fragments.sec− 1 and detected fragments from 29 to 400 (m/z). GC-FID analy-
ses were performed by a ame formed by H2 and atmospheric air with a temperature of 300°C. Flow rates of
40mL min− 1 and 400mL min− 1 were used for H2 and air, respectively.
Identication of the essential oil compounds was performed by comparing the mass spectra in relation to
available in the spectrophotometer database (Wiley 7, NIST 05 and NIST 05s) and by the retention index (RI).
For the RI calculation, a mixture of saturated C7-C40 alkanes (Supelco, USA) submitted under the same chro-
matographic conditions as the OE was used and the adjusted retention time of each compound was obtained
using GC-FID. en, the calculated values for each compound were compared with those in the literature49–51.
Correlation analysis. 2C value, GC%, 5-mC%, yield and content of each compound present in the essential
oil were subjected to Pearson’s correlation. e analysis was conducted in the R environment39 using the package
“Agricolae” (https:// CRAN.R- proje ct. org/ packa ge= agric olae).
Terpene synthase gene (TPS) copy number in P. guajava and P. guineense. We showed the poly-
ploidy inuence in the copy number of the genes involved with essential oil synthesis, the terpene synthase genes
(TPS). ese genes encode enzymes that act in essential oil synthesis pathways27,39,52–54. For this, we used the
sequence of genes functionally characterized and involved in the synthesis of terpenes (TPS genes), which have
been described and available in database ID AB266390.1 and ID MK873024.1. rough the BLAST tool, the
similarity of these sequences was evaluated in relation to the TPS genes from the P. guajava genome annotation
(data of the research group). e alignments that presented a score of at least 80% were selected for the design
of the primers. From this, the primers were designed in the conserved motives of these TPS genes, consider-
ing mainly exon regions. Primers were designed and evaluated using the OligoIDTAnalyzer program (IDT).
We dened two pairs of primers: the rst (F 5’-GGT GGG ATG TCG ATG CTA AA-3’ and R 5’-CTC TTC CTC
CGT AAC TCT GTA TTG 3’) specic to one predicted TPS gene orthologue with an amplicon 500 pb; and a gen-
eral primer pair (F 5’-CGA TTC CGG CTA CTT AGA CATC-3’ and R 5’-GTT CTT CCA GCG TCC CAT ATAC-3’)
aligned to the conserved motifs in eight predicted TPS genes of P. guajava genome, corresponding to sequences
from 415 to 502 pb.
e DNA sequences of the putative TPS were amplied from P. guajava and P. guineense genomic DNA using
the primers. Amplication reaction consisted of 50ng genomic DNA, 200µM dNTPs, 0.5µM each R and F
primers, 1 U GoTaq enzyme (Promega), 1X GoTaq enzyme reaction buer and 1.8mM MgCl2. Amplication
conditions were initial denaturation at 95°C for 5min, followed by 30 cycles of 95°C for 1min, 58°C for 45s,
72°C for 1min and a nal extension at 72°C for 5min. e amplication products were evaluated on 1.5%
agarose gel and NanoDrop. en, DNA probes were generated for each putative gene by a second PCR reaction
on the same conditions described above, diering by the labeling with Tetramethyl-rhodamine 5-dUTP (Roche)
for the specic or ChromaTide Alexa Fluor 488–5-dUTP (Life Technologies) for the general. Fluorescent insitu
hybridization (FISH) was performed in slides containing isolated and preserved nuclei to detect the number of
hybridization signals corresponding to the TPS genes. Hybridization mix consisted of 50% formamide, 2X SSC
and 200ng of the probe. is mix was applied to the slide, which was covered with a coverslip, sealed with rubber
cement and kept at 37°C for 20h. Post-hybridization wash was in 2X SSC at 42°C for 20min. Slides were coun-
terstained with 4′,6-diamidino-2-phenylindole and analyzed on a photomicroscope Olympus BX60 equipped
with epiuorescence and an immersion objective 100x/A.N. 1.4. At least 20 nuclei were scrambled for each spe-
cies and for each gene using a 12-bit CCD digital video camera (Olympus) coupled to the photomicroscope and
a computer with a digitizer plate. Captured images were processed by Image ProPlus 6.1 (Media Cybernetics).
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Ethical approval. is article does not contain any studies with human participants or animals performed
by any of the authors.
Data availability
e datasets generated during and/or analysed during the current study are available from the corresponding
author on reasonable request.
Received: 6 June 2022; Accepted: 10 January 2023
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Author contributions
M.A.S.: Investigation, writing – original dra, Writing – review & editing. F.A.F.S.: Investigation, Writing –
original dra, Writing – review & editing. W.R.C.: Supervision, Conceptualization, Writing – original dra,
Writing – review & editing. L.A.M.: Investigation. L.B.A.: Investigation. A.F.: Supervision and Statistical Analysis.
M.F.S.F.: Supervision, Conceptualization, Writing – original dra, Writing – review & editing. All authors read
and approved the nal manuscript.
Funding
is work was supported by Conselho Nacional de Desenvolvimento Cientíco e Tecnológico (CNPq, Brasília
– DF, Brazil; grants 443801/2014-2 and 308828/2015–1), Fundação de Amparo à Pesquisa do Espírito Santo
(FAPES/VALE, Vitória – ES, Brazil; grant 75516586/16) and VALE. is study was nanced in part by the
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brazil (CAPES) – Finance Code 001.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 023- 27912-w.
Correspondence and requests for materials should be addressed to M.F.S.F.
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