Phylogenetics and diversity analysis of Pennisetum species using Hemarthria EST‐SSR markers

Abstract

Expressed sequence tag‐simple sequence repeat (EST‐SSR) markers are widely applied in plant molecular genetics studies due to their abundance in the genome, codominant nature, and high repeatability. To study the genetic diversity of 35 accessions and transferability of EST‐SSR markers in cross‐species applications, 21 primer pairs previously developed in Hemarthria were amplified across Pennisetum species. A total of 148 polymorphisms were generated with an average of 7.05 bands per locus. The mean values of the genetic parameters polymorphism information content, number of bands, effective number of bands, Nei's gene diversity, and Shannon's information index were 0.8430, 2.0000, 1.7640, 0.4247 and 0.6132, respectively. Cluster analysis of 35 accessions divided them into three clusters, which were consistent with dendrograms of 14 species. The among‐population component explained most of the genetic diversity (66%); the remaining variation (34%) occurred within populations. This study will provide more available EST‐SSR markers for Pennisetum species, and facilitate studies on germplasm collection and utilization of Pennisetum species.

Full text

ORIGINAL ARTICLE
Phylogenetics and diversity analysis of Pennisetum species
using Hemarthria EST-SSR markers
Sifan Zhou
1
|
Chengran Wang
1
|
Guohua Yin
2
|
Yu Zhang
3
|
Xiaoyun Shen
4,5
|
Kayla K. Pennerman
2
|
Jianbo Zhang
6
|
Haidong Yan
1
|
Chenglin Zhang
1
|
Xinquan Zhang
1
|
Shuping Ren
1
|
Tianfeng Guo
1
|
Yan Peng
1
|
Xiao Ma
1
|
Wei Liu
1
|
Yanhong Yan
1
|
Linkai Huang
1
1
Department of Grassland Science, Faculty
of Animal Science and Technology, Sichuan
Agricultural University, Chengdu, China
2
Department of Plant Biology, Rutgers, The
State University of New Jersey, New
Brunswick, New Jersey
3
IRTA, Centre de Recerca en Agrigen
omica
(CSIC-IRTA-UAB), Barcelona, Spain
4
State Engineering Technology Institute for
Karst Desertification Control, Guizhou
Normal University Guiyang, Guiyang, China
5
School of Life Science and Engineering,
Southwest University of Science and
Technology, Mianyang, China
6
Sichuan Academy of Grassland Science,
Chengdu, China
Correspondence
Lin-Kai Huang, Department of Grassland
Science, College of Animal Science and
Technology, Sichuan Agricultural University,
Chengdu, Sichuan 611130, China.
Email: huanglinkai@sicau.edu.cn
Funding information
National Project on Sci-Tec Foundation
Resources Survey; Sichuan Province
Breeding Research Grant; Modern
Agricultural Industry System Sichuan Forage
Innovation Team
Abstract
Expressed sequence tag-simple sequence repeat (EST-SSR) markers are widely
applied in plant molecular genetics studies due to their abundance in the genome,
codominant nature, and high repeatability. To study the genetic diversity of 35
accessions and transferability of EST-SSR markers in cross-species applications, 21
primer pairs previously developed in Hemarthria were amplified across Pennisetum
species. A total of 148 polymorphisms were generated with an average of 7.05
bands per locus. The mean values of the genetic parameters polymorphism informa-
tion content, number of bands, effective number of bands, Nei’s gene diversity, and
Shannon’s information index were 0.8430, 2.0000, 1.7640, 0.4247 and 0.6132,
respectively. Cluster analysis of 35 accessions divided them into three clusters,
which were consistent with dendrograms of 14 species. The among-population com-
ponent explained most of the genetic diversity (66%); the remaining variation (34%)
occurred within populations. This study will provide more available EST-SSR markers
for Pennisetum species, and facilitate studies on germplasm collection and utilization
of Pennisetum species.
KEYWORDS
EST-SSR, genetic diversity, Hemarthria,Pennisetum, transferability
1
|
INTRODUCTION
The genus Pennisetum Rich comprises approximately 140 species
(Brunken, 1977), and is one of the most important genera of Pani-
coideae (Gramineae) (Dahlgren, Clifford, & Yeo, 2012). The cereal
crop is farmed on over 27 million hectare across the globe with a
utilization period of 4–10 years depending on cultivation and man-
agement conditions (Jalaja, Maheshwari, Naidu, & Kavi, 2016). Pen-
nisetum species are becoming increasingly popular in many countries
owing to their pleasing aesthetics, strong stress resistance, and low
maintenance costs (Contreras, Owen, Hanna, & Schwartz, 2013). In
tropical and subtropical regions, they are a source of grain, fodder
and raw materials for papermaking, weaving and housing. They are
especially important as staple crops in dry and arid regions where
maize and wheat are not viable (Manwaring, Bligh, & Yadav, 2016).
The species of this genus are tolerant to drought and other stresses
and may be good sources for drought resistance in traditional and
molecular breeding. However, genetic resources for this genus are
Received: 11 August 2017
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Revised: 29 March 2018
|
Accepted: 24 May 2018
DOI: 10.1111/grs.12208
Grassland Science. 2018;1–10. wileyonlinelibrary.com/journal/grs ©2018 Japanese Society of Grassland Science
|
1

low; relationships within the genus Pennisetum have been rarely
inferred using molecular approaches.
To date, DNA-based molecular markers including restricted frag-
ment length polymorphisms (RFLP) (Karl & Avise, 1992), random
amplified polymorphic DNA (RAPD) (Sekino, Hamaguchi, Aranishi, &
Okoshi, 2003), amplified fragment length polymorphism (AFLP) (Yu &
Guo, 2003), simple sequence repeat (SSR) (Liu & Cordes, 2004), inter-
simple sequence repeat (ISSR) (Bornet & Branchard, 2001), and single
nucleotide polymorphism (SNP) (Germer, Holland, & Higuchi, 2000)
have been used extensively in genetic studies. Compared with them,
Expressed sequence tag-simple sequence repeat (EST-SSR) molecular
markers are widely and increasingly used because of their co-dominant
inheritance, high abundance, and enormous extent of allelic variation
(Agarwal, Shrivastava, & Padh, 2008). They have the potential to facili-
tate evolutionary analyses in a wide variety of taxa and may be the
best way forward for the analyses with limited resources (Ellis &
Burke, 2007). Moreover, they are ideal molecular markers to catalog
useful gene functions and find correlations between related species
with the increased availability of expressed sequence tags and tran-
scriptomic data on public databases. Senthilvel et al. (2008) developed
58 EST-SSR primer pairs of pear millet, 21 polymorphic EST-SSRs, and
6 genomic SSR markers were mapped using existing mapping popula-
tions. Rajaram et al. (2013) developed 99 EST-SSR primers pairs based
on transcriptomic data, and constructed linkage maps of pearl millet
based on EST-SSR, genomic SSR, and sequence-tagged site markers.
However, studies on Pennisetum by EST-SSR markers are still scarce,
and more EST-SSR markers need to be developed.
Considering that grass genomes have co-evolved and share large-
scale synteny (Yu, La Rota, Kantety, & Sorrells, 2004), it should be possi-
ble to use the genome sequence based on EST-SSR markers from field
crops such as Hemarthria for genome analyses in Pennisetum as both
genera belong to the subfamily Panicoideae (Chemisquy, Giussani, Scata-
glini, Kellogg, & Morrone, 2010; Connor, 2005). The main objective of
this study was applying EST-SSR primers from Hemarthria to Pennisetum
and studying the genetic diversity and phylogenetics of Pennisetum.
2
|
MATERIALS AND METHODS
2.1
|
Plant materials
Thirty-five Pennisetum accessions (bulked samples with three plants
per accession) were used in this study (Table 1). Among them, 13
accessions were obtained from the National Herbal Gene Bank of
China (NHGBC), and the rest were from the National Plant Germ-
plasm System (NPGS) of the United States Department of Agricul-
ture. All these individuals were grown in Agrostology Testing Sites at
Sichuan Agricultural University, Sichuan, China. The leaves were col-
lected in October 2015 and stored in allochroic silica gel for use.
2.2
|
DNA extraction
DNA was extracted using a TIANGEN genomic DNA extraction kit
(TIANGEN Biotech, Beijing, China) from 30 mg of young dried leaves
of each accession. The quality and concentration of the extracted
DNA were determined through NanoDrop 2000 spectrophotometer
(Thermo Scientific, Wilmington, USA). The DNA was diluted to
20 ng/llinddH
2
O and stored at 4°C for subsequent use.
2.3
|
Primer selection and PCR amplification
Atotalof49Hemarthria EST-SSR primers (Huang et al., 2016), which
were developed by our research team and synthesized by Shanghai
Sangon Biological Engineering Technology and Services (Shanghai,
China), were screened and selected to genotype the 35 Pennisetum
accessions. Polymerase chain reaction (PCR) was performed in a final
reaction volume of 15 llcontaining30ngtemplate,0.4lMeachof
forward and reverse primers, 7.5 llPCRmix(1.5mMMg
2+
included,
Tiangen), 1.5 U Taq polymerase and ddH
2
O. All PCR reactions were
performed using the following thermal profile: initial denaturation at
94°C for 5 min followed by 35 cycles of denaturation at 94°C for 30 s,
annealing at 58°C for 45 s and extension at 72°C for 1 min, with a final
extension period of 7 min at 72°C. Amplified products were separated
on 8% polyacrylamide electrophoresis gels using 1X TBE buffer. DNA
fragments were visualized and photographed with Microtek Bio-6000.
2.4
|
Data analysis
Given the co-dominant nature of SSR markers, strong clear allelic bands
with the same mobility based on a molecular DNA marker (50-bp lad-
der; Tiangen) (Williams, Kubelik, Livak, Rafalski, & Tingey, 1990) were
scored manually as either present (1) or absent (0) and compiled into a
“bp”type original matrix to estimate the number, range, and distribution
of amplified bands, and ultimately determine the variation level in thez
accessions and species. Total number of bands and number of polymor-
phic bands were visually obtained from the gel image and the percent-
age of polymorphic bands was calculated. Additionally, the information
content per EST-SSR locus was estimated by calculating the polymor-
phism information content (PIC) using the formula described by Nei
(1973): PICi¼1PP2
ij ,whereP
ij
was the frequency of the jth allele
for the ith locus, which must be summed across all the bands. The aver-
age PIC of each primer was measured as PIC
a
=∑PIC
i
/N,whereNwas
the number of polymorphic bands per primer. Furthermore, the effi-
ciency of the SSR was assessed by employing the marker index (MI),
estimated as the average band informativeness (Ibav) for the polymor-
phic markers multiplied by the effective multiplex ratio (EMR),
MI =Ibav 9EMR (Powell et al., 1996). The Ibav was calculated with
the formula: Ibav =1/n∑1-(2|0.5-p
i
|), where nwas the total number of
amplification sites and p
i
represents the proportion of the ith amplifica-
tion site. The EMR was the average number of polymorphic bands
(Archak et al., 2003).
Genetic analyses including observed number of bands, effective
number of bands, Nei’s gene diversity (H), and Shannon’s information
index (I) for each population were estimated using POPGENE v.1.3.2
(Yeh, 1997) with a model for dominant markers and individuals. The
data input files for POPGENE were prepared with the DCFA1.1
(Zhang, 2001). Alternatively, an analysis of molecular variance
2
|
ZHOU ET AL.

(AMOVA) with the program GenAlEx v6.5 was conducted to deter-
mine the variance within and among species through ɸ
PT
(analogous to
F
st
) (Peakall & Smouse, 2012) using 9999 random repetitions.
Dendrograms were constructed with POPGENE v. 1.31 using the
unweighted pair-group method of cluster analysis and arithmetic aver-
ages (UPGMA) based on the matrix of Nei’s genetic diversity. The phy-
logenetic tree was subsequently visualized with the software program
FIGTREE (Hampl, Pavl

ıcek, & Flegr, 2001) with a bootstrap of 9999
replicates. Principal coordinate analysis (PCoA) was carried out with
NTSYS-pc v2.21 (Exeter Software, New York, NY, USA) to provide a
graphical representation of the genetic relationships among the
Pennisetum accessions studied. Population structure was estimated by
the software STRUCTURE version 2.2 (Pritchard, Stephens, & Donnelly,
2000), using a burn in length of 50,000, run length of 100,000, and a
model allowing for admixture and testing for K from 1 to 14.
3
|
RESULTS
3.1
|
Polymorphism of EST-SSR markers
From a total of 49 Hemarthria-derived EST-SSRs, 21 primer pairs
were selected based on clean amplification from the primer
TABLE 1 35 accessions of Pennisetum species used in the analysis
Code Species Accession Collection location Source Species classification
1Pennisetum purpureum Guiminyin China, Guangxi NHGBC S1
2Pennisetum americanum Ningza No. 3 China, Jiangsu NHGBC S2
3Pennisetum purpureum TLG 2 China, Taiwan NHGBC S1
4Pennisetum hybridum Hybrid Giant Napier China, Taiwan NHGBC S3
5Pennisetum purpureum Guimu No.1 China, Guangxi NHGBC S1
6Pennisetum purpureum 061023003 Costa Rica NHGBC S1
7Pennisetum purpureum CX061023015 China, Hainan NHGBC S1
8Pennisetum purpureum 070117004 China, Guangdong NHGBC S1
9Pennisetum hybridum 061031001 Costa Rica NHGBC S3
10 Pennisetum mezianum PI214061 India NPGS S4
11 Pennisetum setaceum PI269235 India, Delhi NPGS S5
12 Pennisetum setigerum PI271527 India NPGS S6
13 Pennisetum orientale PI271596 India NPGS S7
14 Pennisetum setaceum PI300087 United States NPGS S5
15 Pennisetum unisetum PI304750 South Africa, KwaZulu-Nata NPGS S8
16 Pennisetum squamulatum PI319196 Kenya NPGS S9
17 Pennisetum bambusiforme PI390766 Peru NPGS S10
18 Pennisetum setaceum PI410311 South Africa, Cape Town NPGS S5
19 Pennisetum setaceum PI410312 South Africa, Cape Town NPGS S5
20 Pennisetum setaceum PI410315 South Africa, Limpopo NPGS S5
21 Pennisetum villosum PI410318 South Africa, Cape Town NPGS S11
22 Pennisetum setaceum PI414074 United States, Minnesota NPGS S5
23 Pennisetum villosum PI414689 United States, Maryland NPGS S11
24 Pennisetum flaccidum PI434640 Pakistan NPGS S12
25 Pennisetum sp.PI478473 Bolivia, Chuquisaca NPGS S13
26 Pennisetum flaccidum PI601630 United States, North Carolina NPGS S12
27 Pennisetum alopecuroides PI656417 China NPGS S14
28 Pennisetum hybridum Bangde No. 1 China, Guangxi NPGS S3
29 Pennisetum setigerum PI271145 India NPGS S6
30 Pennisetum setaceum PI364993 South Africa, Limpopo NPGS S5
31 Pennisetum orientale PI600996 United States NPGS S7
32 Pennisetum purpureum Mott China, Guangxi NHGBC S1
33 Pennisetum purpureum Zise China, Guangxi NHGBC S1
34 Pennisetum hybridum Pennisetum sinese China NHGBC S3
35 Pennisetum hybridum Nutrifeed Australia NHGBC S3
ZHOU ET AL.
|
3

prescreening (Table 2), demonstrating that microsatellite primers
developed from Hemarthria species are frequently (42.86%) transfer-
able to the Pennisetum genus. These EST-SSRs were subsequently
tested for their ability to detect polymorphism in 35 accessions.
Polymorphic bands, polymorphic rate and PIC values per primer pair
are presented in Table 3.
TABLE 2 Expressed sequence tag-simple sequence repeat primers used in this study
Primers Primer ID Forward/Reverse primer (50?30) Repeat motif T
a
(°C) Expected size (bp)
HSSR2 CL22Contig3 F:TCACTCTGTGCGAGGAGTC (CGC)5 52 297
R:ATGGACATCAACTAAGTCACC
HSSR3 CL54Contig1 F:AACTCCTCTCCATCTGTTCTC (GC)6 56 210
R:GAACCCTAGCTTTGCACAC
HSSR4 CL66Contig1 F:GTAACTGGTTCTCCTGCTTCT (TGC)5 56 256
R:ACTAGGTTACTGCTGCTTGTG
HSSR6 CL965Contig1 F:ATCACATACCTCGTGGACTC (GGC)5 52 269
R:CTAGCAGACGCATCATCTATC
HSSR7 CL994Contig1 F:AAGTACGGGTCCTCCTCTAC (GGC)6 52 208
R:GAGAACCAGCTGAGCCTAAC
HSSR11 CL2144Contig1 F:GAGCTTCTCCTTCTCCAACT (GGC)5 56 264
R:ATCACCTTCGCTATCGTGT
HSSR12 CL2205Contig1 F:AGTTTGATACACCTGCATGAC (AC)7 56 150
R:GTAATCTTGTGTGTCCGTTTG
HSSR14 CL2526Contig1 F:TAATGTAGCACTTGTCGAGGT (CGC)5 56 274
R:CAGATGCTCTTCAAGACCTG
HSSR17 CL3075Contig3 F:CTTCAGATAGCATTGAGGTTG (CAT)5 56 101
R: CAGCCAGCCTTTTATAGTATG
HSSR25 CL5320Contig1 F:GGATCCCTCTCTTACTCCTTC (GAG)5 56 191
R:AGAGGGAGAGAGGTCCATAG
HSSR27 CL5536Contig3 F:TTCTCTTCTCCAGTACACCTC (GAG)5 56 197
R:ACTTCTGAAAAACGAACTGC
HSSR28 CL5542Contig1 F:GGTTTTGGTAAGATAGGAGGA (AT)6 54 135
R:ACCAAGACCCTACAACAAACT
HSSR34 CL6426Contig1 F:GGTGGTACTACCGCTACCTAT (GCCG)5 54 268
R:AGTAACACAGCAGCAGAACAT
HSSR46 CL16253Contig1 F:CTGGATCGTGTGGTGGTA (CGG)5 52 137
R:CAGAACCCGAGAAAGTAAGAA
HSSR50 CL17085Contig1 F:CTCTTCCCTCTCTCCCTCT (CGC)6 54 123
R:ATACTCCTCTCTGGCTACTCC
HSSR51 CL17142Contig1 F:GTCGTCCTCCTTGAACAG (TGG)5 54 257
R:ACTACTCCTCGCTCAACATCT
HSSR52 CL17301Contig1 F:CACCTCAGTACACCTTGGAG (CA)6 56 173
R:GTCCTCATCAGAGTCGTCAT
HSSR53 CL17319Contig3 F:CAGCAGTAAGGAACAAAGAGA (CAA)5 56 179
R:CAGATATTGCTTCCAAGACAG
HSSR54 CL17615Contig1 F:GACTCTTCATCCTCCTCCTC (CGG)5 52 280
R:CGTTAATGCTCTCCTGGTTA
HSSR56 CL17649Contig1 F:CGTGATTAGAGAAGGAGATCG (GGC)5 56 166
R:GAGTTGACGGGTATGACG
HSSR58 CL18880Contig1 F:CCGTACACCATCTCGTACA (TGC)5 52 281
R:GTATTGTCGTGCCTGTTCA
Note.T
a
: annealing temperature of primer pair.
4
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ZHOU ET AL.

A total of 148 bands, all of them polymorphic, were yielded by the
21 EST-SSR markers in the 35 Pennisetum accessions. The number of
polymorphic bands per locus ranged from four (HSSR11) to nine
(HSSR12 and HSSR52) with an average of 7.0 bands per locus. The PIC
of the EST-SSRs varied from 0.7045 (HSSR3) to 0.921 (HSSR2) with an
average of 0.8217 for this genus. All 21 primers showed a high polymor-
phism level (PIC >0.5). The EST-SSR efficiency was measured by calcu-
lating the Ibav, EMR and MI. The average Ibav, EMR and MI values
were 0.6695, 7.05, and 4.72, respectively, indicating a distinctive and
highly efficient nature of the EST-SSRs with using in Pennisetum genus.
Nei’s gene diversity and I were 0.4247 and 0.6132, respectively.
Although the application of these markers to Hemarthria enabled
the identification of 241 different bands (Huang et al., 2016), the PA
and PIC values determined in the case of Pennisetum (100.0000%
and 0.8430%, respectively) were higher than those observed in this
genus (95.6006% and 0.7230%).
3.2
|
Genetic diversity of 35 Pennisetum accessions
The genetic diversity of 35 accessions was studied by estimating
the following parameters: observed number of bands, effective
number of bands, H and I. The average values were 2.0000,
1.7640, 0.4241 and 0.6124, respectively.
The UPGMA dendrogram based on EST-SSRs to study the
genetic relationships of the 35 accessions revealed the formation
of three major clusters (Figure 1). Sixteen accessions, including
seven species (Pennisetum setaceum,Pennisetum orientale,Pennise-
tum unisetum,Pennisetum villosum, etc.), number codes (Table 1):
11, 13, 14, 15, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31 and 35
were clearly divergent from the rest and closely clustered into
group I. Fourteen accessions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 16, 28, 32,
33 and 34 formed the second group. Accessions of the same spe-
cies clustered together, which indicated that genetic background
had greater influence than collection place. Majority of them were
Pennisetum purpureum,Pennisetum americanum and their hybrids,
which indicated the close relationship of these species. Finally,
group III comprised five accessions: 10, 12, 17, 27 and 29. More-
over, bootstrapping was calculated and indicated in the dendrogram
to determine the accuracy and stability of the hierarchical cluster-
ing results. The information on genetic diversity and relationship
among breeding materials is essential for plant breeders to effi-
ciently improve species.
TABLE 3 Polymorphic comparison of
35 Pennisetum accessions and 44
Hemarthria germplasm resources (Huang
et al., 2016) by Hemarthria Expressed
sequence tag-simple sequence repeat
primers
Primers
Pennisetum Hemarthria
NTA NPA PA (%) PIC NTA NPA PA (%) PIC
HSSR2 8 8 100 0.9462 16 16 100 0.8769
HSSR3 8 8 100 0.7155 13 11 84.62 0.6652
HSSR4 8 8 100 0.9351 11 11 100 0.5924
HSSR6 8 8 100 0.8913 12 12 100 0.6999
HSSR7 6 6 100 0.8753 13 13 100 0.6668
HSSR11 4 4 100 0.8332 16 15 93.75 0.6775
HSSR12 9 9 100 0.7831 11 11 100 0.7139
HSSR14 7 7 100 0.8666 8 7 87.5 0.7767
HSSR17 7 7 100 0.8331 ————
HSSR25 5 5 100 0.8405 18 18 100 0.7535
HSSR27 6 6 100 0.8367 ————
HSSR28 7 7 100 0.8175 7 6 85.71 0.9496
HSSR34 7 7 100 0.8468 19 19 100 0.8871
HSSR46 7 7 100 0.8586 14 14 100 0.857
HSSR50 8 8 100 0.8464 15 15 100 0.6765
HSSR51 5 5 100 0.7973 10 10 100 0.5904
HSSR52 9 9 100 0.8231 13 10 76.92 0.6311
HSSR53 7 7 100 0.8639 ————
HSSR54 7 7 100 0.7874 14 14 100 0.7
HSSR56 8 8 100 0.8394 13 12 92.31 0.528
HSSR58 7 7 100 0.8655 18 18 100 0.7708
Total 148 148 ——241 232 ——
Average 7.0476 7.0476 100 0.8430 13.3889 12.8889 95.6006 0.7230
Note. NTA: number of total bands; NPA: number of polymorphic bands; PA: polymorphic bands (%);
PIC: polymorphic information content (%); —: not available or not applicable.
ZHOU ET AL.
|
5

3.3
|
Genetic relationship of 14 Pennisetum species
The analysis of the genetic diversity structure exhibited a typically
continuous organization of accessions, illustrating the relatively close
relationships of the Pennisetum species. To measure the diversity
among species, data of accessions belonging to the same species
were pooled. Supporting information Table S1 shows the pairwise
estimates of ɸ
PT
values between Pennisetum species. The mean ɸ
PT
value of 0.4499 indicated that a relatively high level of the gene
diversity was due to differentiation between species.
To better understand the genetic relationships among the 14
species, we clustered them into different defined groups based on
their genetic distances (Figure 2). On the basis of Nei’s genetic
diversity, 14 species could be grouped into three clusters. The small-
est of the three comprised three Pennisetum species: P. unisetum,
Pennisetum. sp. and P. villosum. The largest clade consisted of six:
P. bambusiforme,P. setigerum,P. squamulatum,P. americanum,P. hy-
bridum and P. purpureum. The remaining five species (P. setaceum,
P. alopecuroides,P. flaccidum,P. orientale, and P. mezianum) were
gathered into another cluster. This dendrogram was consisted with
the dendrogram of 35 accessions and the UPGMA result of 14 spe-
cies, confirming the reliability of our studies.
The within- and among-population (defined by species) compo-
nents of the total genetic variation were partitioned and evaluated
through AMOVA (Supporting information Table S2). The results
showed that the among-population component explained most of
the genetic diversity (66%), the remaining variation (34%) occurring
within populations. The mean F
st
value of 0.15 indicated a moder-
ated level of differentiation among accessions and a low level within
accessions.
The PCoA was performed to check the displacement of the
accessions and to further confirm the clustering pattern obtained
FIGURE 1 UPGMA dendrogram based on Nei’s genetic diversity between the 35 accessions. 1: Guiminyin; 2: Ningza No.3; 3: TLG 2; 4:
Hybrid Giant Napier; 5: Guimu No.1; 6: 061023003; 7: Pennisetum purpureum; 8: 070117004; 9: 061031001; 10: PI214061; 11: PI269235; 12:
PI271527; 13: PI271596; 14: PI300087; 15: PI304750; 16: PI319196; 17: PI390766; 18: PI410311; 19: PI410312; 20: PI410315; 21:
PI410318; 22: PI414074; 23: PI414689; 24: PI434640; 25: PI478473; 26: PI601630; 27: PI656417; 28: Bangde No.1; 29: PI271145; 30:
PI364993; 31: PI600996; 32: Mott; 33: Zise; 34: P.sinese; 35: Nutrifeed
6
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ZHOU ET AL.

from the dendrogram (Figure 3). The first two axes explained
57.62% of the cumulative molecular variance among accessions, with
PCo 1 accounting for 45.68% and PCo 2 for 11.94%. The estimated
population structure of the accessions was displayed by STRUC-
TURE plot (Figure 4). Admixture was observed among the acces-
sions; however, some of them (accessions 1, 3, 6 and 7 from S1;
accessions 4 and 9 from S3) were less than 25% admixture from
others, suggesting that little gene flow occurred between these and
other accessions.
4
|
DISCUSSION
Microsatellites are conserved among many plant species (Elazreg,
Ghariani, Chtourou-Ghorbel, Chakroun, & Trifi-Farah, 2011). In sev-
eral studies, PCR-based microsatellite markers developed for one
species could amplify DNA from a close relative, expanding the
applicability of the markers (Bertin, Zhu, & Gale, 2005; Luro et al.,
2008; Sudheer et al., 2011). In this study, 21 markers amplified mul-
tiple-band patterns similar to dominant markers rather than typical
EST-SSR banding patterns, which was consistent with previous stud-
ies on millet (Rajput, Plyler-Harveson, & Santra, 2014), ryegrass (Guo
et al., 2016), and zoysia grass (Cai et al., 2005). The percentage of
polymorphic loci for EST-SSR markers was 100% which was very
high as compared to the previous reports of 35% and 89% polymor-
phism for markers designed for Pennisetum accessions (Babu, Sun-
daramoorthi, Vijayakumar, & Ram, 2009; George, Prashanth, &
Parida, 2005). This may have been due to the rich geographical
diversity of accessions included, as well as the use of prescreened
highly informative primers. Loci polymorphism can be divided into
high, medium and low with PIC >0.5, 0.5 >PIC >0.25 and
FIGURE 2 UPGMA dendrogram based on Nei’s genetic between the 14 species. S1: P. purpureum; S2: P. americanum; S3: P. hybridum; S4:
P. mezianum; S5: P. setaceum; S6: P. setigerum; S7: P. orientale; S8: P. unisetum; S9: P. squamulatum; S10: P. bambusiforme; S11: P. villosum;
S12: P. flaccidum; S13: Pennisetum sp.; S14: P. alopecuroides
FIGURE 3 Principal coordinate analysis (PCoA) for the first and
second coordinates estimated for expressed sequence tag-simple
sequence repeat markers of 35 Pennisetum accessions
ZHOU ET AL.
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7

PIC <0.25 (Vaiman et al., 1994). For the 35 accessions, all of the 21
EST-SSR makers had PIC >0.5, indicating that they could provide
reliable information to the analysis of the relationship of the 35
accessions. Due to the high polymorphism and efficiency of EST-SSR
makers, they have been widely used for genetic mapping (Ferr~
ao
et al., 2015), evaluating genetic diversity (Wang et al., 2016), phylo-
genetic analysis (Wang et al., 2006), and many other research fields.
The clustering of the 35 accessions differed from with those by
Xie and Lu (2005) and Yao, Hong, and Zeng (2013), which can be
attributed to several causes. First, Xie and Lu (2005) and Yao et al.
(2013) generated the dendrograms based on RAPD and sequence
related amplified polymorphism (SRAP) markers, while we used EST-
SSR for analysis. The marker systems employed may affect the level
of polymorphism detected, which reinforces again the importance of
appropriately selecting the loci and covering the complete genome in
order to obtain a reliable estimation of the genetic diversity (Ye, Yu,
Kong, Wu, & Wang, 2005). In addition, the amount of samples and
molecular markers can also influence the results (Davierwala et al.,
2000). Xie and Yao used 19 RAPD markers for eight Pennisetum cul-
tivars and 38 SRAP markers for 19 Pennisetum cultivars, respectively,
while we applied 21 EST-SSR markers for 35 Pennisetum accessions.
Finally, the nomenclature commonly employed for the Pennisetum
genus is confusing and different species may share the same name.
The mean ɸ
PT
value of 14 Pennisetum species was similar to
those obtained from studies about analyzing walnut (Christopoulos,
Rouskas, Tsantili, & Bebeli, 2010) and bamboo (Barkley, Newman,
Wang, Hotchkiss, & Pederson, 2005), which were about 0.50 among
cultivars. The lowest ɸ
PT
value (0.0828) was found between P. hy-
bridum and P. purpureum, while the highest (0.8189) was between
P. mezianum and P. americanum. Moreover, P. hybridum showed the
closest identity with P. purpureum and P. americanum (ɸ
PT
values of
0.0828 and 0.2807 respectively), in accordance with this species
being the cross between them (Muldoon & Pearson, 1979). Accord-
ing to AMOVA analysis, most of the total diversity was accounted
by among-populations variation component (66%) and only 34%
gene differentiation existed within species. The analysis of the
genetic diversity structure exhibited an organization of accessions
which was typically continuous, and PCoA analysis based on Dice’s
similarity did not distinguish 14 Pennisetum species. These results
were the same as the cluster analysis obtained through UPGMA,
confirming the close relationship of these species.
Microsatellites are conserved in many plant species and can be
used in related species (Elazreg et al., 2011). As explained by Wang
et al. (2005), the transferability of EST-SSR markers depends on both
the genetic relatedness among the species examined (including dif-
ferences in DNA sequence, genome size and evolution rate) and the
PCR conditions used (such as the amount of template DNA or ion
concentration). The present study demonstrated that microsatellite
primers developed from Hemarthria are highly transferable for the
Pennisetum species, likely due to the relatedness of the genera
(Huang et al., 2014; Martel et al., 2004). The marker loci are proba-
bly homologous regions with similar alleles. Several studies have
been performed to assess genetic diversity in Pennisetum species
using molecular markers such as RAPDs, isozymes, ISSRs, AFLPs, and
SSRs (Azevedo et al., 2012; Babu et al., 2009; Bhandari, Sukanya, &
Ramesh, 2006; Harris, Anderson, & Malik, 2010; L
opez et al., 2014;
Lowe, Thorpe, Teale, & Hanson, 2003), and this study provided an
expanded set of transferable EST-SSR markers from other genus in a
genetic diversity study on Pennisetum collections.
5
|
CONCLUSION
To conclude, in this study, 21 pairs of markers were polymorphic
and could generated more than one band in 35 Pennisetum
accessions. It provided evidence for the potential transferability of
Hemarthria EST-SSRs to related Pennisetum species, showing that
species-related cross-amplification is a useful approach for the appli-
cation of microsatellite markers in this genus. Also, these primers are
now available and can be widely used to investigate the genetic
diversity and structure of the Pennisetum population, as well as for
future studies, such as germplasm conservation, gene mapping, and
marker-assisted selection.
ACKNOWLEDGMENTS
This work was supported by National Project on Sci-Tec Foundation
Resources Survey (2017FY100602), Sichuan Province Breeding
Research Grant and Modern Agricultural Industry System Sichuan
Forage Innovation Team.
ORCID
Linkai Huang https://orcid.org/0000-0001-7810-4852
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section at the end of the article.
How to cite this article: Zhou S, Wang C, Yin G, et al.
Phylogenetics and diversity analysis of Pennisetum species
using Hemarthria EST-SSR markers. Grassl Sci. 2018;00:1–10.
https://doi.org/10.1111/grs.12208
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