Genet Resour Crop Evol

https://doi.org/10.1007/s10722-019-00765-x (0123456789().,-volV)
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NOTES ON NEGLECTED AND UNDERUTILIZED CROPS

Phenotypic diversity analysis of the fruit of Amomum tsao-ko

Crevost et Lemarie, an important medicinal plant
in Yunnan, China
Zhang Wei . Lu Bingyue . Meng Hengling . Wei Xiang . Yang Zhiqing .
Yang Shengchao

Received: 25 October 2018 / Accepted: 5 March 2019

Ó Springer Nature B.V. 2019

Abstract Amomum tsao-ko (Zingiberaceae) is a
commonly used spice and medicinal plant. Morpho-
logical variations exist in the fruits of A. tsao-ko. In
this study, we compared the phenotypic traits of fruits
collected from nine regions in Yunnan Province,
China. Based on the mean square values of fruit
weight, number of ridges, seed weight, pericarp
thickness, and seed diameter, the fruits of A. tsao-ko
in Yunnan were grouped into three categories, corre-
sponding to different regions in Yunnan. A principal
component analysis revealed that the number of
ridges, number of seeds per fruit, and vertical fruit
diameter are the most variable parameters. Consider-
ing the genetic improvement potential at the regional
level, A. tsao-ko in the regions of Gongshan, Teng-
chong, Jinping, Lushui, and Pingbian is the most
efficient for breeding new cultivars, as the plants
growing in these regions have a heavier fruit weight,
larger fruit diameter, larger transverse diameter,
greater number of ridges, greater seed number per
Z. Wei  L. Bingyue  M. Hengling  W. Xiang
Honghe University, 661100 Mengzi, People’s Republic of
China
Y. Zhiqing  Y. Shengchao (&)
National and Local Joint Engineering Research Center on
Germplasms Innovation and Utilization of Chinese
Medicinal Materials in Southwest China, Yunnan
Agriculture University, 650201 Kunming, People’s
Republic of China
e-mail: zw_biology2@126.com
fruit, heavier seed weight, moderate pericarp thick-
ness, and larger seed diameter. Based on the selected
area, the nested square analysis showed that the fruit
vertical diameter, fruit diameter, and the number of
seeds per fruit in the region are greater than the
regional mean square, and the differences reached an
extremely significant level as well. Thus, these three
traits can be selected as genetic materials in this region
for breeding.
Keywords Amomum tsao-ko  Properties of fruit 
Phenotypic analysis
Introduction
Amomum tsao-ko Crevost et Lemarie (Zingiberaceae)
is a medicinal and edible plant commonly known as
black cardamom (Lim 2013; National Pharmacopoeia
Committee 2015). A. tsao-ko grows in moist, broad-
leaved forests in tropical and subtropical regions of
Southeast Asia. It is native to Vietnam, but has been
introduced to the subtropical and tropical regions of
Yunnan, Guangxi, and Guizhou Province, China. The
cultivation history of A. tsao-ko in Yunnan has been
more than 300 years. Amomum tsao-ko in Yunnan is
mainly distributed in the south, southeast, and south-
west in Honghe, Wenshan, and Xishuangbanna (The
Kunming Institute of Botany 1997), which account for

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over 90% of the yield in China, with the planting areas
expanded to 133,330 hectares.
As a precious medical and perfume material, A.
tsao-ko has important value and significance in daily
life and has even broad exploitation and utilization
prospects. Many clinical and animal trials have shown
that A. tsao-ko has a variety of medicinal usages
including the treatment of abdominal distension, liver
protection, and the inhibition of cancer cells (Moon
et al. 2005; Yu et al. 2008, 2010). Additionally, the
volatile oil from A. tsao-ko has strong antioxidant
activity and participates in the process of apoptosis,
thus can be a promising potential medicinal resource
(Li et al. 1998; Yang et al. 2010). In addition, A. tsao-
ko has great ecological and environmental value in the
mountainous areas in Yunnan. A. tsao-ko is a crop that
has relatively high economic value. Typically, A. tsao-
ko fruit can be harvested in 3 years after planting, with
the maximal yield at the seventh year and A. tsao-ko
can produce fruits for 20 years.
The yield of A. tsao-ko is determined by the number
of panicles, flowers, fruits, fruit-setting percentage,
and fruit weight, of which the most important feature
is the fruit weight. Fruit characteristics influence the
fruit weight, which is always used as an important
objective trait for breeding (Lei et al. 2013). At
present, most studies on A. tsao-ko are focused on the
pharmacological action (Yang et al. 2009a, b; Rahman
et al. 2017), cultivation, and chemical composition
(Yang et al. 2008; Cui et al. 2017), while few studies
have been conducted on the fruit characteristics. A
previous research on A. tsao-ko divided A. tsao-ko
fruits in Xishuangbanna into four categories based on
fruit shapes: oval, round, spindle, and oblong fruit
shape (Ma et al. 2008). The chemical compositions of
volatile oils in the four groups of A. tsao-ko are
different (Ma et al. 2008).
The morphological characteristics of A. tsao-ko
fruits is related to the yield and quality. The fruit
length and fruit aspect ratio can be used as a visual
quality evaluation index. Generally, large, reddish-
brown A. tsao-ko fruits that have strong smell are of
high quality of volatile oil (Ma et al. 2008). Fruit
characteristics also have important significance and
practical value on breeding. The study of fruit traits
can reveal the genetic diversity of its phenotype and
lay the foundation for future breeding.
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Genet Resour Crop Evol
Materials and methods
Plants
Amomum tsao-ko was collected from nine regions in
Yunnan Province in January, 2018 (Table 1). Thirty
fruits with different fruit shapes were collected from
one to three populations in each region. The fruits
were stored in a shady and airy place. Data were
collected in February, 2018.
Survey of phenotype of Amomum tsao-ko fruits
We employed normal statistical methods to investi-
gate eight traits of the A. tsao-ko fruits collected from
the above districts: fruit length, fruit diameter, single
fruit weight, seed mass weight, seed number of each
fruit, seed diameter, pericarp thickness, and number of
ridges. Length and diameter were measured using a
Vernier caliper with a measurement accuracy of
0.02 mm. Fruit and seed weight was measured using
a 1/10,000 analytical balance with a measurement
accuracy of 0.1 mg.
A single factor variance analysis of the traits of A.
tsao-ko fruits was carried out. We calculated the
coefficient of variation of the measurement results
from the nine regions.
r2T=S
VST ¼  100%
r2T=S þ r2S
A population nested analysis of variance was
carried out according to Wanchun Gu and the linear
model was:
Yijk ¼ l þ Si þ TðiÞj þ eðiÞjk
where, Yijk = region i, region j, and the measurement
of k; l = total mean, and Si= population effect
(stable); T(i)j = region effect within the group (ran-
dom); e(i)jk = experimental error.
The inter-regional phenotypic differentiation coef-
ficient was calculated according to Song Ge. The
formula used was:
r2T=S
VST ¼  100%
r2T=S þ r2S
Genet Resour Crop Evol

Table 1 Characteristics of Region (County) Locatity (Village) Longitude Latitude Altitude/m

the Amomum tsao-ko
collection place Lushui Shuihe hamlet of Luzhang E98°450 N25°540 1581
0
Gutanhe hamlet of Luzhang E98°45 N25°560 1578
Shangjiang E98°500 N25°410 1077
Fugong Maji E98°500 N27°230 1325
Shiyueliang E98°520 N27°150 1366
0 0
Gongshan Puladi E98°46 N27°35 1452
Puladi E98°460 N27°350 1497
Puladi E98°460 N27°350 1391
Tengchong Zhonghe E98°240 N24°680 1503
Houqiao E98°140 N25°120 1925
0 0
Luchun Qimab E102°18 N22°40 1743
Yuanyang Xiaoxinjie E103°010 N23°000 1663
Shangxincheng E102°550 N23°110 1648
Jinping Adebo E103°130 N22°520 1616
Adebo E103°130 N22°520 1638
Pingbian Heping E103°690 N22°970 1410
0 0
Maguan Miechang E103°56 N22°48 1484
Gulinqing E103°540 N22°450 1397

where, r2T/S refers to inter-regional variance compo-
nent; r2S refers to intra-regional variance component;
VST refers to phenotypic differentiation coefficient
indicating the percent of inter-regional variation
against the total genetic variation.
A nested variance analysis, principal component
analysis, NMDS analysis, and cluster analysis were
conducted on the averages of the measured characters.
These analyses were completed using SPSS15.0 and
the PAST program (Hammer et al. 2001). Addition-
ally, the cluster analysis illustrated the quantitative
characteristics of A. tsao-ko from the nine regions.
Data were normalized and clustered by UPGMA
(Unweighted Pair Group Method with Arithmetic
Mean) to produce a dendrogram.
Results
Frequency distribution of fruit-types of A. tsao-ko
in different regions
According to the record in the Chinese Pharma-
copoeia, the fruit-type of A. tsao-ko is elliptic.
However, our field survey within different regions of
Yunnan Province revealed that in addition to the long
elliptic form, there are also cone-shaped, spindle-
shaped, and spheroidal fruits (Fig. 1). The frequencies
of distinct shapes in different regions are variable
(Fig. 2). The cone-shaped fruit type was mostly
distributed in Gongshan, where no spindle-shaped
fruits were observed. In Luchun, the spindle-shaped
fruits were also hardly detected. However, all four
fruit types were detected in the other seven regions.
Polymorphic analysis of Amomum tsao-ko fruit
types in different regions
The polymorphism index is commonly accepted to
represent community diversity. Comparison of poly-
morphism indices of A. tsao-ko fruit types among
different regions revealed that the highest polymor-
phism index was detected in Pingbian followed by
Tengchong, Lushui, and Yuanyang. This indicates that
the genetic variation of A. tsao-ko is abundant in these
regions (Table 2). The lowest polymorphism index
was found in Gongshan, indicating a low diversity
compared with those found in other regions.

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Fig. 1 The distinct types of A. tsao-ko fruit shapes: elliptic (a), spheroidal (b), cone-shaped (c), and spindle-shaped (d)
Fig. 2 The frequencies of
distinct shapes in different
regions
Variation of phenotypic traits
between and within regions
Amomum tsao-ko has capsule fruits that are reddish-
brown when they are ripe. The cortex of A. tsao-ko
fruit is equipped with three blunt arrises, reddish-
brown to grey-brown surface, grooves, a round
projection at the top, and a stem or stem scar at the
bottom. The cortex is extreme tough yet tears easily on
the longitudinal. There is a yellowish-brown dia-
phragm outside the cortex and the diaphragm divides
the seeds into three sections. The seed is coniform
polytope, colored red-brown, and covered with a gray
membranous cuticle.
Coefficient of variation (CV) was used to evaluate
the variability of statistical data, suggesting intrinsic
characteristics and differences between individuals in
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Genet Resour Crop Evol
different regions. The fruit type of A. tsao-ko and the
CV among regions were shown in Fig. 3. Among the
different characteristics in fruit types of A. tsao-ko, the
total seed weight had the highest CV (26.82%),
followed by the number of seeds (26.78%), and the
fruit weight (23.84%). The CV of the vertical and
transverse diameter were relatively small with values
of 10.79% and 10.28%, respectively. The lowest CV
identified was for seed diameter, which had an average
value of 9.94% (Table 3). These results indicate that
seed diameter and vertical and transverse fruit diam-
eter remain relatively stable.
The phenotypic variation among different fruit
types in certain regions was variable. The transverse
diameter of fruits in Maguan and the pericarp thick-
ness in Fugong showed the greatest variation (5.57%
in cone-shape to 42.31% in spindle-shape, 15.71% in
Genet Resour Crop Evol

Table 2 The comparative study of polymorphism index of Amomum tsao-ko types in different areas
Producing area (County) Site (Village) Polymorphism index Average number

Lushui Dishuihe hamlet, Sanhe town,Luzhang 1.111 0.943

Gutanhe hamlet, Sanhe town, Luzhang 0.904
Binggong hamlet, Shangjiang 0.814
Fugong Shiyueliang 0.731 0.807
Maji 0.883
Gongshan Puladi 0.194 0.536
Puladi 0.640
Puladi 0.773
Tengchong Gaotian hamlet, Zhonghe 0.922 0.972
Houqiao 1.022
Lvchun Qimaba 0.781 0.781
Yuanyang Xiaoxinjie 0.936 0.931
Shangxincheng 0.928
Jinping Adebo 1.197 0.971
Adebo 0.745
Pingbian Heping 1.007 1.007
Maguan Miechang 0.868 0.882
Gulinjing 0.897

Fig. 3 PCA and NMDS analysis of phenotypes in A. tsao-ko. square represents the type of cone-shape, and the triangle
Annotation: The PCA analysis is on the left; the NMDS analysis represents the type of spheroidal-shape
is on the right. The dots represent the type of elliptic-shape, the

cone-shape to 51.80% in spindle-shape; with a range fruits collected from Jinping with a range from 7.26%
of 36.74% and 36.09%, respectively). The smallest in spheroidal type to 9.24% in cone-shape fruits and
variations were found in the transverse diameter of

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 Genet Resour Crop Evol

Table 3 CV of phenotypic characters of Amomum tsao-ko

Area Fruit- Vertical Transverse Weight Weight of Number Seed Pericarp Curve Average
types diameter diameter total seed of seed diameter thickness number

Lushui Elliptic 13.04 10.07 25.12 26.50 25.94 11.46 25.08 17.92 19.39
Cone 12.63 11.01 19.51 22.19 25.19 12.86 18.63 17.42 17.43
Spindle 9.65 7.74 16.19 17.59 17.76 11.04 19.00 17.31 14.53
Spheroidal 11.90 10.58 22.94 24.74 26.26 8.96 19.86 15.07 17.54
Fugong Elliptic 8.34 10.22 22.29 28.21 26.04 8.61 18.70 27.18 18.69
Cone 10.04 7.27 20.83 22.67 19.01 11.23 15.71 15.28 15.25
Spindle 12.30 10.02 29.54 34.57 34.48 10.76 51.80 31.56 26.87
Spheroidal 10.13 8.97 21.48 23.27 16.44 8.04 17.57 27.14 16.63
Gongshan Elliptic 15.98 12.97 30.17 31.07 33.32 8.21 27.89 22.31 22.74
Cone 7.23 6.94 16.19 18.69 17.29 10.65 17.65 9.74 13.04
Spindle 0 0 0 0 0 0 0 0 0
Spheroidal 16.50 11.77 27.25 29.89 36.17 10.48 18.41 21.80 21.53
Tengchong Elliptic 14.99 14.24 32.75 35.64 37.34 10.01 18.67 15.12 22.34
Cone 8.41 10.32 21.38 22.49 19.21 10.23 22.77 17.06 16.48
Spindle 7.35 6.56 18.11 23.61 25.22 9.69 14.82 14.78 15.01
Spheroidal 10.13 8.83 27.74 29.81 28.84 7.54 19.43 17.61 18.74
Luchun Elliptic 13.42 9.17 25.93 28.98 33.87 10.56 23.40 17.79 20.39
Cone 18.44 11.89 32.75 42.07 43.36 9.65 17.77 26.23 25.27
Spindle 0 0 0 0 0 0 0 0 0
Spheroidal 13.43 10.86 39.96 44.31 49.64 13.54 19.90 16.97 26.07
Yuanyang Elliptic 15.69 9.11 30.76 36.18 33.0.9 16.29 16.67 30.92 22.23
Cone 13.99 12.70 28.89 30.75 27.99 9.20 15.37 31.69 21.32
Spindle 9.04 12.67 27.97 31.72 36.64 10.61 17.69 32.89 22.40
Spheroidal 13.79 10.83 29.47 33.67 39.98 13.94 21.64 27.31 23.82
Jinping Elliptic 8.82 8.89 23.16 26.08 18.89 10.34 18.59 19.83 16.82
Cone 10.14 9.24 24.14 25.56 28.50 6.29 13.54 24.02 17.67
Spindle 10.14 7.51 20.26 22.51 14.65 6.82 11.00 23.87 14.59
Spheroidal 8.10 7.26 20.32 21.95 23.69 8.22 17.07 21.53 16.02
Pingbian Elliptic 13.16 9.33 22.71 28.11 23.93 11.06 20.84 16.49 18.20
Cone 7.33 10.21 25.63 26.73 20.53 13.89 14.77 13.47 16.57
Spindle 9.57 15.92 23.71 31.28 33.64 14.97 15.34 22.80 20.90
Spheroidal 9.57 9.38 29.44 32.13 33.86 10.63 21.87 14.88 20.22
Maguan Elliptic 16.13 11.89 28.58 31.76 30.51 12.99 20.49 22.41 21.84
Cone 7.34 5.57 16.17 16.99 22.87 9.22 13.40 22.96 14.31
Spindle 9.23 42.31 18.33 20.14 23.21 9.27 20.63 20.34 20.43
Spheroidal 13.34 14.54 40.33 45.29 44.24 11.19 17.46 22.63 26.13
Average 10.81 10.47 23.88 26.86 26.81 9.95 18.42 19.89

the ridge number in Lushui with a range from 15.07% in the pericarp thickness of spindle-shaped fruits, with
in spheroidal type to 17.92% elliptic type fruits. a range from 11.00 to 51.80%. The transverse diameter
The phenotypic variations between the regions also had great variations, with a range from 6.56 to
were also variable. The greatest difference was found 42.32%. In addition, the seed number in spheroidal

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Genet Resour Crop Evol

fruits varied from 16.44 to 49.64%. The range in the
total weight of spindle-shaped fruits was 25.08%, with
minimal and maximal values of 16.99% and 42.07%.
The range of the total number of a single spindle-
shaped fruit was 26.07%, with minimal and maximal
values of 17.29% and 43.36%. The smallest change in
CV was found in the pericarp thickness of spheroidal
fruits (4.8%). The range of transverse diameter of
elliptic fruits was 5.35%, while the range of the
vertical diameter of spindle-shape fruits was 4.94%.
There is abundant genetic diversity in A. tsao-ko
distributed in different regions of Yunnan Province.
The phenotypic variation between and within regions
was different, with the latter predominant. There were
also differences in phenotypic variation of each trait
between and within regions.
Variation of A. tsao-ko between and within regions
The nested variance analysis could reveal the variation
of A. tsao-ko between and within regions, and further
determine the variation (Table 4). The variances of
fruit weight, number of ridges, seed weight, pericarp
thickness, and seed diameter between regions was
greater than within regions. The variance of pericarp
thickness within regions reached a remarkable level.
No significant difference was detected in the variance
of seed diameter. The variance of the number of seeds
in each fruit, and the vertical and transverse diameter
was greater within regions than between regions,
reaching a remarkable level.
Based on the results of nested variance analysis, the
variance component of the germplasm resources
between and within regions was calculated (Table 4).
According to the variance components of eight traits
between and within regions, variance within regions
accounted for 58.25%, variance within regions was
38.79%, and 2.96% of variance resulted from random
errors. The phenotypic differentiation coefficient of
different phenotypes within regions was different, of
which seed diameter, number of ridges, pericarp
thickness, and the weight of fruits and seeds had the
greatest differentiation coefficient. The vertical and
transverse fruit diameter had a relatively low coeffi-
cient. The mean phenotypic differentiation coefficient
in the nine regions was 60.48%, indicating that
39.52% of the phenotypes resulted from variation
among populations.
PCA and NMDS analyses of phenotypes in A. tsao-
ko
The main element analysis showed that the phenotypic
characteristics are integrated into three principal
components: the vertical diameter, the number of
seeds, and the number of ridges, of which component
one accounts for a proportion of 44.22%, component
two accounts for 23.42%, and component three

Table 4 variance analyzes of phenotypic characters of Amomum tsao-ko within and across regions
Phenotype F value Variance component Phenotypic differentiation
coefficient (%)
Across Within Across Within Random
regions regions regions regions error

Fruit weight 26.85** 9.99** 70.96 26.40 2.6 72.88

**
Fruit length 13.71 320.59** 4.09 95.61 0.3 4.10
** **
Fruit diameter 18.02 58.72 23.17 75.54 1.29 23.48
Number of ridges 35.48** 7.54** 80.59 17.14 2.27 82.46
Seed number of each fruit 18.59** 20.07** 46.87 50.61 2.52 48.08
Seed mass weight 23.13** 8.87** 70.10 26.87 3.03 72.28
Pericarp thickness 11.19** 2.57* 75.80 17.43 6.77 81.30
Seed diameter 19.28** 0.142 94.41 0.7 4.90 99.27
Average 58.25 38.79 2.96 60.48
Annotation: *a = 0.05,**a = 0.01

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accounts for 8.72%. The total proportion of the three
components is 76.36% ridges (Fig. 3a).
The non-metric multidimensional scaling (NMDS)
ordination produced a linear and non-metric fit and
low stress values, illustrating differentiation. The
NMDS bi-plot indicated that fruit shape can be split
into three distinct types: spheroidal-shape, spindle-
shape, and elliptic-shape, while cone-shape, elliptic-
shape, and spindle-shape have overlapping sections,
suggesting that they may act as the transitional type of
elliptic-shape and spindle-shape (Fig. 3b).
Cluster analysis of phenotypes in A. tsao-ko fruits
The germplasm resources in the nine regions can be
divided into three groups (Fig. 4). The first group
included the germplasm resources in Yuanyang,
Maguan, and Fugong. Fruit similarities from these
regions included relatively low fruit and seed weight,
moderate vertical diameter, number of seeds in each
fruit, pericarp thickness, small transverse fruit diam-
eter, and low ridges number. The second group
included the germplasm resources in Luchun, which
had a moderate fruit weight and seed weight, trans-
verse diameter of fruit, ridges number, seed diameter,
relatively small vertical fruit diameter, small number
of seeds in each fruit, and relatively thick pericarp.
The third group included the germplasm resources in
Gongshan, Tengchong, Jinping, Lushui, and Pingbian,
Fig. 4 Cluster analysis of phenotypes in A. tsao-ko fruit. (1)
Lushui, (2) Fugong, (3) Gongshan, (4) Tengchong, (5) Luchun,
(6) Yuanyang, (7) Jinping, (8) Pingbian, (9) Maguan
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Genet Resour Crop Evol
which had a high fruit and seed weight, larger
transverse and vertical diameter, larger number of
ridges and seeds, and moderate pericarp thickness and
seed diameter.
Discussion
Classification and selection of A. tsao-ko fruit
variation
Variation is the basis of plant genetic improvement.
Over the course of plant breeding, new germplasm or
strains can be achieved by screening variants. Amo-
mum tsao-ko has a long history of cultivation.
However, limited research has been conducted on
the classification of A. tsao-ko germplasm resources.
In an early study, A. tsao-ko was divided into four
types based on fruit shapes: spindle, oval, spherical,
and spherical-similar (Ma et al. 2008). There are
obvious differences in fruit setting rate, aroma, and
yield among these four fruit types. Our current study
suggests that it is more appropriate to divide A. tsao-ko
into oval, near-spherical, spindle, and conical fruit
shapes. The morphological characteristics of oval
fruits are fruits with a long longitudinal diameter with
round ends. The morphological characteristics of near-
spherical fruits are fruits with similar transverse and
longitudinal diameters. The characteristics of spindle-
shaped fruit are fruits with a longitudinal diameter that
is long with sharp ends. A conical fruit has a large
longitudinal diameter; however, there is a marked
difference between the two ends: one is sharp and the
other is rounded (Ma et al. 2008).
The chemical constituents of A. tsao-ko cultivated
in Xishuangbanna suggest that the plants grown here
can be divided into four types: oval, spherical, spindle,
and oblong (Ma et al. 2008). The former three types
are consistent with our study. However, we found that
there was little difference between oblong and oval
fruit shapes. Therefore, conical (one end is sharp and
the other is round) is more accurate to describe the
fourth type of fruit shapes. Because no special research
has been conducted on variety breeding, the four fruit
types typically appeared randomly throughout the
range of cultivation.
In this study, among the frequencies of fruit
variation in each region, the frequency of oval-shaped
fruits was the highest; the frequency of elliptical-
Genet Resour Crop Evol
shaped fruits was the second highest, but the ratio was
much lower than that of the oval fruits. Additionally,
the ratio of the spherical-shaped and spherical-similar-
shaped fruits was lower than the other two types. The
distribution of the four fruit shapes in the population
was relatively consistent. In Luchun and Gongshan,
there were no spindle-shaped fruits, but the distribu-
tion of the four fruit types in the population was
relatively consistent. The NMDS analysis showed that
the oval-shaped, spindle-shaped, and spherical-simi-
lar-shaped fruits were three independent types, while
the conical-shaped, elliptical-shaped, and spindle-
shaped fruits overlapped, which might indicate that
the latter shapes are intermediate between oval-shaped
and conical fruits. The appearance of these types of
distributions and the intermediate types may be due to
the influence of human activity in different regions.
Amomum tsao-ko fruit diversity and selection
Population resistance and low fruit-setting rate are
relevant technical issues in the cultivation of A. tsao-
ko. A previous study showed that geographical region
is associated with genetic differentiation (Zhang
2013). The present study compared the fruit diversity
of phenotypic traits in different regions throughout
Yunnan Province, China. Pingbian has the highest
diversity index; Tengchong, Lushui, and Yuanyang
also had high diversity index, indicating that A. tsao-
ko growing in these four regions has rich genetic
variation.
The nested analysis revealed that the mainland
interval variance is the most prominent variance,
which accounts for 58.25% of the total variance
(including variance of fruit traits in mainland species,
and area and types of variation among individuals
within species). This shows that for the genetic
improvement of A. tsao-ko at the regional level,
selection of new varieties is feasible and effective.
The classification and selection of A. tsao-ko fruit
phenotypic traits
Fruit shape index is mainly decided by additive gene
effects. When the fruit shape index of broad sense and
narrow sense heritability is high, it is less affected by
the environment. Therefore, if the early generations
are chosen according to phenotypes, effective breed-
ing can lead to progenies featuring these traits.
Our results showed that the A. tsao-ko variation
between regions is greater than within regions.
Therefore, genetic improvement at the regional level
is the most effective method for breeding new
varieties. The mean variance of traits (including the
weight of the fruit, the number of ridgelines, the
weight of seed group, the thickness of seed capsule,
and the diameter of the seeds) between regions is
higher than those within the regions. Thus, germplasm
resources from Gongshan, Tengchong, Jinping,
Lushui, and Pingbian should be used for breeding,
especially in increasing fruit longitudinal and trans-
verse diameter and the number of seeds per fruit.
Conclusions
This study showed a direct comparison of A. tsao-ko
diversity in fruit morphological traits. Results showed
that the number of ridges, number of seeds per fruit,
and vertical fruit diameter were the most varied traits.
Thus, these three traits can be selected from genetic
materials in the corresponding regions for breeding.
Furthermore, our study helped explain discrepancies
observed among the population structures and char-
acteristics obtained with the four fruit types. The
obtained results enriched the fundamental understand-
ing of trait selection and structure in A. tsao-ko.
Acknowledgements This work was supported by the Project
of Young and Middle-aged Talent of Yunnan Province (Grant
No. 2017HB076) and by National Natural Science Foundation
of China (NSFC) (Grant Nos: 31460380, 81560615).
Author’s contribution Yang SC, Zhang W, Lu BY conceived
and designed the experiments; Zhang W, Meng HL and Wei X
performed the experiments; Yang SC and Yang ZQ analysed the
data; Zhang W, Meng HL and Yang ZQ wrote the manuscript.
Compliance with ethical standards
Conflict of interest The authors declare they have no conflicts
of interest.
References
Cui Q, Wang LT, Liu JZ, Wang HM, Guo N, Gu CB, Fu YJ
(2017) Rapid extraction of Amomum tsao-ko essential oil
and determination of its chemical composition antioxidant
and antimicrobial activities. J Chromatogr B Anal Technol
Biomed Life Sci 1061–1062:364–371
123

Hammer O, Harper D, Ryan P (2001) PAST: paleontological
statistics software package for education and data analysis.
Palaeontol Electron 4(1):1–9
Lei E, Tian XJ, Yang RP, Su YL, Lu HJ, Liu YH (2013) Study of
yield and yield components of Amomun tsas-ko. Southwest
China J Agric Sci 06:2493–2496
Li ZQ, Luo L, Dai WH, Huang R, Zhang YQ (1998) Chemical
constituents of the essential oil on Amomum tsao-ko from
Yunnan Province. Acta Bot Yunnanica 20:119–122
Lim TK (2013) Edible medicinal and non-medicinal plants.
Springer, Netherlands, pp 813–817
Ma J, Zhng LX, Peng JM (2008) Chemical composition of
Amomum tsao-ko in Xishuangbanna. Chin Tradit Pat Med
30(8):1192–1194
Moon SS, Cho SC, Lee JY (2005) Tsaokoarylone, a cytotoxic
diarylheptanoid from Amomum tsao-ko fruits. Bull Korean
Chem Soc 26(3):447–450
National Pharmacopoeia Committee (2015) Pharmacopoeia of
People’s Republic of China. Chemical Industry Press,
Beijing, p 409
Rahman MR, Lou Z, Yu F, Wang P, Wang H (2017) Anti-
quorum sensing and anti-biofilm activity of Amomum
tsaoko (Amommum tsao-ko Crevost et Lemarie) on food-
borne pathogens. Saudi J Biol Sci 24(2):324
The Kunming Institute of Botany (1997) The flora of Yunnan.
The Science Publishing Company, Beijing, pp 618–639
Yang Y, Yan RW, Cai XQ, Zheng ZL, Zou GL (2008) Chemical
composition and antimicrobial activity of the essential oil
of Amomum tsao-ko. J Sci Food Agric 88(12):2111–2116
123
Genet Resour Crop Evol
Yang X, Küenzi P, Plitzko I, Potterat O, Hamburger M (2009a)
Bicyclononane aldehydes and anti-proliferative constituents
from Amomum tsao-ko. Planta Med 75(05):543–546
Yang YW, Liu XL, Pu CX, Yang JL, Qian ZG (2009b) Study on
quantitative multistate character of fruit and seed of
Amomun tsao-ko. J Chin Med Mater 32(008):1184–1187
Yang Y, Yang Y, Yan RW, Zou GL (2010) Cytotoxic, apoptotic
and antioxidant activity of the essential oil of Amomum
tsao-ko. Bioresour Technol 101(11):4205–4211
Yu L, Shirai N, Suzuki H, Hosono T, Nakajima Y, Kajiwara M,
Takatori K (2008) Effect of lipid extracted from tsao-ko
(Amomum tsao-ko Crevost et Lemaire) on digestive
enzyme activity, antioxidant activity, plasma and liver
lipids, and blood glucose levels of mice. J Nutr Sci Vita-
minol 54(5):378–383
Yu L, Shirai N, Suzuki H, Sugane N, Hosono T, Nakajima Y,
Kajiwara M, Takatori K (2010) The effect of methanol
extracts of tsao-ko (Amomum tsao-ko Crevost et Lemaire)
on digestive enzyme and antioxidant activity in vitro, and
plasma lipids and glucose and liver lipids in mice. J Nutr
Sci Vitaminol 56(3):171–176
Zhang YH (2013) The constituent research on three medicinal
materials from Yunnan. Yunnan University Of traditional
Chinese medicine, Kunming
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