Sequence characteristics and genetic diversity analysis of the ITSu1−4 gene region
The ITSu1−4 gene region of the Pepper population showed high success rates for PCR amplification and sequencing using a single primer pair specific (100%). The sequences characteristics of the gene region are presented in Table 1 shows that the genetic distances of the ITSu1−4 gene region of the total of 39 individuals ranged from 0.000 to 0.155 (mean = 0.033) (Table 1)
Table 1
The characteristics based on of ITSu1−4 gene region of Pepper populatation
Regions gene
|
PCR success
(%)
|
Sequencing success
(%)
|
Total aligned length (bp)
|
Number of monomorphic sites
|
Variable sites
(%)
|
Intraspecific distance
(mean)
|
ITSu1−4
|
100
|
100
|
667–685
|
517–535
|
21.898
|
0.000-0.155 (0.033)
|
Table 2
Nucleotide components of ITSu1−4 gene region of Pepper population
Individuals
|
Compute nucleotide composition (%)
|
Total
(bp)
|
T(U)
|
C
|
A
|
G
|
G + C
|
HUIB_PN27
|
19.970
|
28.912
|
19.672
|
31.446
|
60.358
|
671
|
HUIB_PN29
|
19.940
|
28.720
|
19.940
|
31.399
|
60.119
|
672
|
HUIB_PN38
|
19.940
|
28.720
|
19.940
|
31.399
|
60.119
|
672
|
HUIB_PN42
|
19.970
|
28.912
|
19.821
|
31.297
|
60.209
|
671
|
HUIB_PN43
|
19.970
|
28.912
|
19.672
|
31.446
|
60.358
|
671
|
HUIB_PN45
|
19.970
|
28.912
|
19.672
|
31.446
|
60.358
|
671
|
HUIB_PN47
|
19.821
|
29.061
|
19.672
|
31.446
|
60.507
|
671
|
HUIB_PN35
|
19.970
|
28.763
|
19.672
|
31.595
|
60.358
|
671
|
HUIB_PN52
|
19.970
|
28.763
|
19.821
|
31.446
|
60.209
|
671
|
HUIB_PN54
|
19.821
|
29.210
|
19.821
|
31.148
|
60.358
|
671
|
HUIB_PR48
|
19.640
|
29.085
|
21.739
|
29.535
|
58.621
|
667
|
HUIB_PR41
|
19.640
|
29.085
|
21.739
|
29.535
|
58.621
|
667
|
HUIB_PH46
|
18.394
|
31.825
|
18.832
|
30.949
|
62.774
|
685
|
HUIB_PH30
|
18.394
|
31.825
|
18.832
|
30.949
|
62.774
|
685
|
HUIB_PN21
|
19.970
|
28.763
|
19.672
|
31.595
|
60.358
|
671
|
HUIB_PN56
|
19.970
|
28.763
|
19.672
|
31.595
|
60.358
|
671
|
HUIB_PN55
|
19.970
|
28.763
|
19.821
|
31.446
|
60.209
|
671
|
HUIB_PN10
|
19.374
|
29.210
|
19.970
|
31.446
|
60.656
|
671
|
HUIB_PN20
|
20.119
|
28.614
|
19.821
|
31.446
|
60.060
|
671
|
HUIB_PN50
|
19.970
|
29.210
|
19.225
|
31.595
|
60.805
|
671
|
HUIB_PN69
|
19.970
|
28.763
|
19.821
|
31.446
|
60.209
|
671
|
HUIB_PN70
|
19.672
|
29.210
|
21.311
|
29.806
|
59.016
|
671
|
HUIB_PN84
|
19.672
|
29.508
|
20.268
|
30.551
|
60.060
|
671
|
HUIB_PN87
|
19.970
|
28.763
|
19.821
|
31.446
|
60.209
|
671
|
HUIB_PN89
|
19.523
|
28.912
|
19.970
|
31.595
|
60.507
|
671
|
HUIB_PN91
|
19.851
|
28.955
|
19.851
|
31.343
|
60.299
|
670
|
HUIB_PN93
|
19.672
|
28.614
|
20.417
|
31.297
|
59.911
|
671
|
HUIB_PN95
|
19.523
|
29.061
|
20.119
|
31.297
|
60.358
|
671
|
HUIB_PN96
|
19.970
|
28.763
|
19.821
|
31.446
|
60.209
|
671
|
HUIB_PN97
|
19.970
|
28.763
|
19.821
|
31.446
|
60.209
|
671
|
HUIB_PN101
|
19.970
|
28.763
|
19.970
|
31.297
|
60.060
|
671
|
HUIB_PN102
|
20.119
|
28.614
|
19.821
|
31.446
|
60.060
|
671
|
HUIB_PN105
|
19.821
|
29.061
|
19.672
|
31.446
|
60.507
|
671
|
HUIB_PN113
|
19.970
|
28.763
|
19.821
|
31.446
|
60.209
|
671
|
HUIB_PN114
|
19.821
|
29.061
|
19.672
|
31.446
|
60.507
|
671
|
HUIB_PN115
|
19.523
|
29.061
|
19.821
|
31.595
|
60.656
|
671
|
HUIB_PN116
|
19.672
|
29.061
|
19.821
|
31.446
|
60.507
|
671
|
HUIB_PD36
|
20.597
|
28.209
|
24.627
|
26.567
|
54.776
|
670
|
HUIB_PN34
|
19.970
|
28.763
|
19.821
|
31.446
|
60.209
|
671
|
Avg.
|
19.795
|
29.047
|
20.031
|
31.128
|
60.174
|
671.513
|
The PCR products of the ITSu1− 4 gene region were sequenced on ABI PRISM® 3100 Avant Genetic Analyzer (Applied Biosystems) by the dideoxy terminator method. The results of the ITSu1− 4 gene region were 667 bp (HUIB_PR41 and HUIB_PR48), 670 bp (HUIB_PN36 and HUIB_PN91), 672 bp (HUIB_PN29 and HUIB_PN38), 685 bp (HUIB_PN46 and HUIB_PN30), and 671 bp for the remaining individuals Peper (Table 1). The BLAST result on NCBI was used to verify and compare with the sequences of the genus Peper showed that the nucleotide sequences obtained were highly similar to species of the Piper nigrum (accession number: MH493477-MH493487, KF924121, KF924111), Piper retrofractum (accession number: MH493562), Piper hancei (accession number: EF450274) and Piper divaricatum (accession number: DQ868714) ranging from 96 to 100%. The percentage of occurrence of each type of nucleotide in the ITSu1− 4 gene region showed that Guanidin (G) accounts for the highest proportion, ranging from 26.567 to 31.595% (mean = 31.128%), followed by Cysteine (C) accounting for 28.209 to 28.955 % (mean = 29.047%) and the lowest was Timin (Uracin) accounting for 18.394 to 20.119% (mean = 19.795%).
Table 3
The results of DNA Polymorphism based on ITSu1−4 gene region of Pepper population
Regions gene
|
n
|
S
|
Eta
|
h
|
Hd
|
k
|
Pi (x10− 3)
|
Rm
|
ITSu1−4
|
31
|
150
|
169
|
23
|
0.947
|
20.352
|
30.930
|
18
|
Note- n: Number of samples; S: Number of variable sites; Eta: Total number of mutations; h: Number of Haplotypes; Hd: Haplotype (gene) diversity; Pi: Nucleotide diversity (per site); k: Average number of nucleotide differences; Rm: Minimum number of recombination events |
The percentage (G + C) contained in the ITSu1−4 gene region was the highest at 60.805% (HUIB_PN50) and there is a difference between different Pepper individuals, the differences ranged from 54.776–60.805% and reached an average of 60.174% (Table 2). All of the new sequences have been deposited in GeneBank under the following accession numbers: MZ636718 to MZ636756.
The results presented in Table 3 show that the nucleotide sequence of the ITSu1−4 gene region have contains 150 different nucleotide positions between 31 studied pepper individuals. Of these, there are 53 singleton variable sites (with 52 singleton variable sites containing two variants: 17, 21, 31, 36, 37, 41, 50, 63, 75, 130, 141, 146, 147, 148, 164, 165, 167, 173, 195, 224, 301, 318, 343, 367, 392, 395, 410, 420, 431, 432, 439, 451, 462, 463, 468, 469, 484, 487, 506, 507, 509, 538, 588, 597, 598, 601, 616, 622, 663, 670, 672, 677 and 1 singleton variable sites containing three variants: 262) and 97 Parsimony informative sites (with 79 Parsimony informative sites containing two variants: 10, 22, 47, 59, 64, 65, 74, 85, 86, 89, 91, 94, 97, 106, 111, 115, 121, 128, 129, 150, 168, 179, 180, 181, 186, 189, 192, 194, 201, 202, 211, 213, 229, 231, 241, 265, 351, 379, 386, 390, 402, 405, 411, 413, 424, 428, 436, 438, 440, 441, 444, 447, 448, 455, 461, 480, 497, 518, 523, 536, 539, 557, 569, 570, 574, 575, 587, 590, 593, 604, 606, 627, 630, 642, 650, 651, 655, 685, 690 và 18 Parsimony informative sites containing three variants: 108, 187, 188, 220, 352, 396, 414, 422, 430, 434, 452, 491, 510, 525, 558, 584, 632, 671), and ITSu1−4 gene region without the coding region assignation protein. The ITSu1−4 gene region contains 169 (S) total number of mutations, with the average number of nucleotide differences (k = 20.352), the nucleotide diversity coefficient accounts for 30.930 x 10− 3 (Pi), created 23 types of haplotype (h) in 31 individuals with haplotype diversity coefficient accounting for 0.947 (Hd). The individuals in the pepper population with large differences based on the ITSu1−4 gene region showed up to 18 minimum recombination events predicted to occur in this population when analyzed on DNAsp 6.0 software. All indicators were processed with statistical significance p < 0.05 (Table 3).
Table 4
Neutrality tests results based on ITSu1−4 gene region of Pepper population
Regions gene
|
Fu’s
|
Tajima’s
|
Fu and Li’s
|
S
|
Fs
|
D
|
P
|
D*
|
P
|
F*
|
P
|
ITSu1−4
|
-0.209
|
-1.824
|
Statistical significance: *, P < 0.05
|
-1.205
|
Not significant, P > 0.10
|
-1.699
|
Not significant, 0.10 > P > 0.05
|
0.684
|
Note: D, Tajima’s statistic [Error! Reference source not found.]; Fs, Fu’s statistic [19]; D* and F*, Fu and Li’s statistics [21], S, Strobeck's S statistic [29] |
The mismatch distribution of pairwise nucleotide differences among ITSu1−4 sequences among all individuals in the population exhibited a smooth unimodal distribution characteristic of a large population expansion [17; 18]. Study-wide site frequency spectra reveal an excess of singleton mutations when compared with expected frequencies under neutrality and stable population size (Fig. 1).
Fu’s Fs were negative (Fs = -0.209) which occurs when an excess of rare haplotypes is present and suggests that either population expansion or genetic hitchhiking has taken place [19]. The values of D, Fu and Li’s D* and F* were negative as well (D = -1.824; P < 0.05, D* = -1.205; not significant, P > 0.10 and F* = -1.699; not significant, 0.10 > P > 0.05), indicating an excess of recently derived haplotypes and suggesting that either population expansion or background selection has occurred [20; 21]. Strobeck’s S, the probability of obtaining equal or fewer haplotypes based on gene frequency and mutation rate was high in between individuals in a population (S = 0.684). These results are consistent with deviation from neutrality due to either selection or population expansion (Table 4).
Phylogenetic analysis
Figure 2. Haplotype network of Pepper population based on ITSu1−4 gene region of the nucleus.
An ITSu1−4 haplotype network was constructed using statistical parsimony with a 95% connection limit (parsimony cutoff = 7 mutational steps) (Fig. 2). The evolutionary history based on ITSu1−4 gene region allele frequencies was inferred by using the Maximum Likelihood method and Tamura-Nei model [22], in which a constant rate of evolution across individuals in the population is assumed and genetic distances between individuals in the population are relative. The tree with the highest log likelihood (-2082.26) is shown. Evolutionary analyses were conducted in MEGA X [22]. The results presented in Figs. 2 and 3 show that have the 3 groups are present. Group I include: 1 individual of species Piper divaricatum (HUIB_PD36), group II include: 4 individuals of species Piper retrofractum (HUIB_PR41 and HUIB_PR48) and Piper hancei (HUIB_PH30 and HUIB_PH46), and group III include: 26 for the remaining individuals of species Piper nigrum (Figs. 2 and 3). The highest value of Fst is shown in the pairs of groups II and III (Fst = 0.151), and the lowest is in groups II and I (Fst = 0.015), between different groups, the Fst value based on ITSu1−4 haplotype is also different, frequencies ranged from 0.015 to 0.151 (Table 5).
Table 5
The FST values of the between group mean distance
Group
|
n
|
Group I
|
Group II
|
Group III
|
Group I
|
1
|
**
|
0.015
|
0.023
|
Group II
|
4
|
0.097
|
**
|
0.023
|
Group III
|
26
|
0.144
|
0.151
|
**
|
Note- n: Number individual; Data allow the diagonal is Fst ; indicators were processed with statistical significance p < 0.05 |