1. Introduction

Chinese Medicine

2151-1918

Scientific Research Publishing

10.4236/cm.2014.52015

CM-47462

Articles

MEDICINE & HEALTHCARE

DALP Analysis on Genetic Diversity of Panax notoginseng

Xiuming

Cui

¹^*Hui

Xiao

²Jingjing

Yang

¹Tina

Dong

³Karl

Wah-Keung Tsim

³Yuqin

Sun

²Yan

Zhu

Wenshan Sanqi Research Institute, Wenshan College, Wenshan, China

College of Life Science and Technology, Kunming University of Science and Technology, Kunming, China

Department of Biology and Biotechnology Research Institute, The Hong Kong University of Science and Technology, Hong Kong, China

* E-mail:sanqi37@vip.sina.com(XC);

20052014

050212312914 April 201421 May 2014 3 June 2014

2014

This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/

Panax notoginseng (Burk) F.H. Chen is one of the most famous Chinese traditional medicinal plants, which belongs to Panax genus under Araliaceae family. At the present, Panax notoginseng cultivated is a mix colony. Using DALP makers, this article studied on genetic diversity of cultivated populations of Panax notoginseng in Wenshan County of Yunnan province and Napo country of Guangxi province. And the results showed that there were 260 polymorphic loci detected from the total of 292 in 13 populations, and there was great part of genetic diversity found between populations and the genetic differentiations were lower within populations. So there is broad prospect in good species breeding. And it can provide basic information for resource protection and sustainable use of Panax notoginseng.

<i>Panax notoginseng</i> Genetic Diversity DALP

1. Introduction

Panax notoginseng (Burk) F. H. Chen is one of the most famous Chinese traditional medicinal plants, which belongs to Panax notoginseng under Araliaceae family. Wenshan prefecture is the major producing area, and it is about 400 years that notoginseng was cultivated [1] . In the process of cultivation and selection, some excellent genetic resource was lost. To protect that genetic resource and provide basic information for sustainable use of Panax notoginseng, we must strengthen the basic genetic research of Panax notoginseng. And it has to deeply understand the genetic differentiation of Panax notoginseng. Molecular maker is the most direct and accurate method for genetic diversity analysis, so we used DALP molecular maker in genetic diversity.

2. Materials and Methods2.1. Materials

The three-year panax notoginseng’ s leaves were collected from Yanshan base in Wenshan county of Yunnan province and Jingxi country of Guangxi province, except yellow panax notoginseng and purplish panax notoginseng’ s leaves that were collected from Zhela base of Wenshan County, Yunnan province. The geographical location and the habitat overview of the population is showed in Table 1.

2.2. Methods

Using CTAB to extract Genomic DNA from leaves of Panax notoginseng. Detecting DNA concentration is diluted and preserving in −20˚C. Getting 8 primers from 40 random primers that can get clear bands and react stably; they are PS01-PR02, PS02-PR01, PS04-PR01, PS04-PR02, PS05-PR02, PS08-PR01, PS09-PR01and PS10-PR01 (the primers’ sequence is in Table 2).

Table 1

. The habitats and localities of materials

Code	Sampling position	Population	Altitude (m)	Longitude	Latitude
WM WZ WL WD	Matang Zhuilijie Laohuilong Dongshan	PA PB PL PI	1470 1430 1450 1760	104˚5' 204˚24' 104˚36' 104˚58'	23˚49' 23˚37' 23˚46' 23˚54'
YC YZ YA YP	Chongka Zhela Aolongke Panlong	PD PC PE PJ	1498 1580 1600 1510	104˚25' 104˚35' 105˚6' 104˚31'	23˚32' 23˚62' 23˚6' 23˚54'
MR MB	Renhe Bazhai	PG PH	1600 1480	106˚7' 104˚7'	23˚4' 23˚0'
QB	Niejiao	PF	1992	103˚87'	23˚54'
GNP	Napo	PK	1100	105˚57'	23˚18'

“Code” is the abbreviation of “sampling position”, for example, WM = Wenshan matang, YC = Yanshan chongka; PA—PL is the randomizing ID of the population.

Table 2

. The number and sequence of primers

Primer	No.	Sequence (5'-3')
Selective primers Selective primers Selective primers Selective primers Selective primers Selective primers Selective primers Selective primers Selective primers Selective primers Reverse primers Reverse primers	PS01 PS02 PS03 PS04 PS05 PS06 PS07 PS08 PS09 PS10 PR01 PR02	5'-GTTTTCCCAGTCACGACAGC-3' 5'-GTTTTCCCAGTCACGACGAC-3' 5'-GTTTTCCCAGTCACGACACG-3' 5'-GTTTTCCCAGTCACGACCAG-3' 5'-GTTTTCCCAGTCACGACCAC-3' 5'-GTTTTCCCAGTCACGACTCAG-3' 5'-GTTTTCCCAGTCACGACCCAG-3' 5'-GTTTTCCCAGTCACGACGC-3' 5'-GTTTTCCCAGTCACGACCTAG-3' 5'-GTTTTCCCAGTCACGACCGC-3' 5'-AACAGCTATGACCATGA-3' 5'-TTTCACACAGGAAACAGCTATGAC-3'

The PCR system for DALP analysis was as follows: dd H₂O 6.5 uL, 2.5 mmol/L dNTP 1 uL, 25 mmol/L Mg²⁺ 2 uL, 10 × PCR buffer 2.5 uL, 50 - 100 ng template DNA 2 uL, 5 pmol/L selective primer 1 uL, 5 pmol/L reverse primer 3 uL, 1U Taq polymerase in 20 uL reaction system. And the PCR program was as follow: pre- denaturation 5 min at 94˚C, denatured 30 s at 94˚C, annealed 30 s at 50˚C, 1 min at 72˚C, 12 cycles, and denatured 30 s at 94˚C, annealed 30 s at 50˚C, 30 s at 72˚C 28 cycles, then extend at 72˚C, 4˚C hold.

Using 0/1 matrix and POPGENE1.32 to calculate the Nei’s genetic diversity index (H), Shannon information index (I), percentage of polymorphic bases (PPB), genetic differentiation (Gst), diversity index within population (Hs), total genetic diversity (Hs & Ht), the Nei’s genetic distance (D) and genetic identity (I). Then the populations were calculated by the MEGA software.

3. Results

1% Agar gel electrophoresis showed that the bands of 192 samples’ DNA extracted by CTAB method were the highest purity, best quality, clear and less fragments (As shown in Figure 1). The test results of DALP analysis as shown in Figure 2 to Figure 5.

A total of 292 clear bands were amplified from 8 selected DALP primers, 260 (89.05%) of which were polymorphic. The PPB (79.08), Shannon index (0.2817) and the Nei’s genetic diversity index (0.1172) of popula-

Figure 1

Total DNA extracted by CTAB method

Figure 2

The PCR amplified results of Panax notoginseng cultivated in Wenshan prefecture by primer ps05-pr1

Figure 3

The PCR amplified results of Panax notoginseng cultivated in Guangxi province by primer ps05-pr1

Figure 4

The PCR amplified results of yellow Panax notoginseng by primer ps05-pr1

tions cultivated in Zhela (PC) were highest in Wenshan’s populations. And The PPB (40.92), Shannon index (0.0868) and the Nei’s genetic diversity index (0.0606) of populations cultivated in Bazhai (PH) were lowest in Wenshan’s populations. And there were big difference between the highest one and the lowest one. Overall, the PPB, Shannon index and the Nei’s genetic diversity index of Wenshan’s populations were lower than Guangxi’ s populations’ which had more genetic diversity. And the PPB, Shannon index and the Nei’s genetic diversity index of Panax stipuleanatus which was contrast were next to Guangxi’s populations (The results shown in Table 3).

Based on the proportion of genetic diversity level in Ht, Ht-Hs and Gst, we know that genetic differentiation between 10 populations cultivated in Wenshan was 0.7732. And it showed that there was 23.38% molecular variation only within populations and there was 77.32% molecular variation between populations, so there was more variation between populations. Genetic differentiation idex (0.1696) of the PK population cultivated in Napo Guangxi province was lowest. There was 83.04% molecular variation within population and only 16.96% molecular variation between populations. The degree of genetic diversity within population was lower than the total degree in 11 populations, and it showed that genetic diversity mainly existed between populations. Based on Wright (1931) [2] , the migration number of every populations (Nm*) was over 1, which suggested that there existed sufficient genetic exchanges to prevent the genetic differentiation made by genetic drift among population. The sufficient genetic exchange number was inversely proportional to genetic differentiation level among populations in PD population of Longka and PK population of Napo, which’s Nm* was over 1 and the genetic differentiation was low (The results shown in Table 4).

The analysis results of genetic distance showed that the genetic distance of every populations ranged from 0.0714 to 0.2408 (Table 5). The distance ranged from PB population to PC population was only 0.01. And genetic distance of Wenshan populations was close expecting PG. More distance excited between PK and other populations, especially PA which was over 0.1694. The cluster analysis showed that PB and PC classified one group, PD, PE and PF classified one group, PH classified with PI and PJ, and PG and PK were separate ones.

A total of 150 clear bands were amplified from 8 screened DALP primers, 120 (80%) of which were polymorphic in 96 samples from Wenshan, Guangxi, yellow Panax notoginsseng, purple Panax notoginseng and Panax stipuleanatus. From the Table 6, the genetic diversity analysis showed that PPB (81.82), Shannon index

Figure 5

The PCR amplified results of purple Panax notoginseng by primer ps05-pr1

Table 3

. Genetic diversity of 11populations in Panax notoginseng

Population	Total number of loci	No. of polymorphic loci	PPB	na^*	ne^*	The Nei’s genetic diversity H^*	Shannon index I^*
PA PB PC PD PE PF PG PH PI PJ PK stipuleanatus	110 148 120 135 128 80 117 113 127 138 110 140	84 105 94 73 80 37 47 45 55 82 90 112	76.36% 70.92% 79.08% 54.54% 62.5% 46.35% 73.65% 40.92% 43.65% 60% 81.82% 80%	1.3818 1.2364 1.2636 1.1818 1.2091 1.1545 1.2455 1.1364 1.1455 1.2 1.8182 1.8	1.3055 1.1891 1.2109 1.1455 1.1674 1.1255 1.1964 1.1091 1.1164 1.16 1.396 1.4168	0.1697 0.1051 0.1172 0.0808 0.0928 0.0692 0.1091 0.0606 0.0646 0.0889 0.2442 0.2544	0.243 0.1504 0.2817 0.2466 0.264 0.0989 0.1562 0.0868 0.0926 0.1273 0.3782 0.3896

Table 4

. Analysis on genetic differentiation among populations

Population	Ht	Hs	Dst	Gst	Nm^*
PA SD PB SD PC SD PD SD PL SD PF SD PG SD PH SD PI SD PJ SD PK SD	0.1534 0.0395 0.0955 0.0304 0.1091 0.0344 0.0693 0.0220 0.0773 0.0278 0.0659 0.0229 0.1102 0.0386 0.0534 0.0185 0.0614 0.0229 0.0864 0.0309 0.2478 0.0312	0.0750 0.0132 0.0455 0.0094 0.0455 0.0094 0.0477 0.0109 0.0273 0.0061 0.0318 0.007 0.0250 0.0057 0.0295 0.0066 0.0227 0.0052 0.0273 0.0061 0.2058 0.022	0.0784 0.0263 0.05 0.021 0.0636 0.0248 0.0216 0.0111 0.05 0.0217 0.0341 0.0159 0.0852 0.0329 0.0239 0.0119 0.0387 0.0177 0.0591 0.0248 0.042 0.0092	0.5111 0.5238 0.5833 0.3115 0.6471 0.5172 0.7732 0.4468 0.6296 0.6842 0.1696	0.4783 0.4545 0.3571 1.1053 0.2727 0.4667 0.1467 0.6190 0.2941 0.2308 2.4476

^*: Ht = Total gene diversity; Hs = Gene diversity within population; Gst = The coefficient of gene differentiaton; SD = Standard deviation; ^*Nm = estimate of gene flow from Gst or Gcs. E.g., Nm = 0.5(1 - Gst)/Gst; See McDermott and McDonald, Ann. Rev. Phytopathol. 31:353-373 (1993).

Table 5

. The Nei’s genetic similarity (above the diagonal) and the genetic distance D (below the diagonal)

pop	PA	PB	PC	PD	PE	PF	PG	PH	PI	PJ	PK
PA PB PC PD PE PF PG PH PI PJ PK	^**** 0.0714 0.0827 0.1306 0.1303 0.1561 0.2094 0.1517 0.1247 0.1127 0.2408	0.9311 ^**** 0.0514 0.0459 0.0459 0.078 0.1658 0.1055 0.1179 0.1237 0.2114	0.9206 0.9499 ^**** 0.0678 0.0678 0.1119 0.1809 0.0810 0.1007 0.1192 0.1902	0.8779 0.9551 0.9345 ^**** −0.017 0.0397 0.1632 0.1095 0.1267 0.1583 0.1921	0.8779 0.9551 0.9345 1.0179 ^**** 0.0396 0.1633 0.1097 0.1266 0.1583 0.1924	0.8555 0.9250 0.8941 0.9611 0.9611 ^**** 0.1355 0.1424 0.1372 0.1756 0.1811	0.8111 0.8472 0.8345 0.8494 0.8494 0.8732 ^**** 0.1267 0.1711 0.1919 0.1934	0.8593 0.8999 0.9222 0.8963 0.8963 0.8673 0.8810 ^**** 0.0580 0.0955 0.1594	0.882 0.888 0.904 0.881 0.881 0.871 0.842 0.943 ^**** 0.047 0.193	0.8934 0.8837 0.8877 0.8536 0.8536 0.8390 0.8254 0.9089 0.9536 ^**** 0.2138	0.786 0.809 0.826 0.825 0.825 0.834 0.824 0.852 0.824 0.807 ^****

Table 6

. The genetic variation among populations

Population	Total number of loci	PPB	na^*	ne^*	H^*	I^*
Wenshan Guangxi YELLOW PURPLE stipuleanatus	110 98 133 102 118	70 81.82 50.91 37.27 79.6	1.7 1.8182 1.5091 1.3727 1.8	1.3182 1.396 1.1774 1.1585 1.4168	0.1965 0.2442 0.1166 0.1029 0.2544	0.3063 0.3782 0.1893 0.1633 0.3896

^*: PPB = The percentage of polymorphic loci is; ^*na = Observed number of alleles; ^*ne = Effective number of alleles [Kimura and Crow (1964)]; ^*h = Nei’s (1973) gene diversity; ^*I = Shannon's Information index [Lewontin (1972)].

(0.3782) and the Nei’s genetic diversity index (0.2442) of Guangxi population were highest; the ones of Wenshan population were next to Guangxi population. And PPB (37.27), alleles number (1.3727), Shannon index (0.1633) and the Nei’s genetic diversity index (0.1029) of purple panax notoginseng were minimum value in all populations, shows the amount of genetic variation is the lowest.

Based on the proportion of genetic diversity level in Ht, Ht-Hs and Gst, we know that species genetic differentiation was 0.5182. There was 48.12% molecular variation existed within populations, and more molecular (51.82%) variation existed between populations. In different population level, there was only 16.96% molecular variation existed among populations, and 83.04% one existed within populations in Guangxi’s populations. There was 21.37% molecular variation existed among populations and 78.63% molecular variation existed in population in yellow fruit notoginseng. There was 14.76% molecular variation existed among populations, and 85.24% existed in population in Panax stipuleanatus. In the five populations, the genetic differentiation mainly existed among populations. Based on Wright (1931) [2] , the migration number of every populations (Nm^*) was over 1, which suggested that there existed sufficient genetic exchanges to prevent the genetic differentiation made by genetic drift among population. The sufficient genetic exchange number was inversely proportional to genetic differentiation level among populations in all populations and the genetic differentiation was lowest, except Wenshan’s population (Table 7).

The analysis results of genetic distance showed that the genetic distance of every populations ranged from 0.137 to 0.259 (Table 8). The distance ranged from Panax notoginseng to Panax stipuleanatus was farthest, which was over 0.5154. And genetic distance of Panax notoginseng cultivated in Wenshan was close to Guangxi one. The distance of yellow fruit Panax notoginseng was close to purple fruit Panax notoginseng which was 0.5001.

And The cluster analysis using UPGMA method showed that Wenshan’s Panax notoginseng and Guangxi’s classified one group, yellow fruit Panax notoginseng and purple fruit Panax notoginseng classified one group, Panax notoginseng was Separate clustering (Figure 6).

4. Discussion

Intraspecific genetic variation decides the evolution trend of species [3] . The more genetic diversity, the more extensive evolution [4] . So it is the basic for sustainable use of genetic resources that maintain the intraspecific

Table 7

. The genetic diversity analysis of populations

Population	Ht	Hs	Gst	Nm^*
Wenshan SD Guangxi SD Yellow fruit SD Purple fruit SD stipuleanatus SD	0.1963 0.0325 0.2478 0.0312 0.1181 0.0235 0.0899 0.0224 0.2549 0.0301	0.0946 0.0099 0.2058 0.022 0.0929 0.0137 0.071 0.0145 0.2173 0.0227	0.5182 0.1696 0.2137 0.2197 0.1476	0.4649 2.4476 1.8393 1.8712 2.887

Table 8

. The Nei’s genetic similarity (above the diagonal) and the genetic distance D (below the diagonal)

pop	Wenshan	Guangxi	Pingbian	Purple fruit	Yellow fruit
Wenshan Guangxi Pingbian Purple fruit Yellow fruit	^**** 0.1370 0.2590 0.1511 0.1381	0.8720 ^**** 0.1831 0.1839 0.1636	0.7718 0.8327 ^**** 0.2656 0.2576	0.8598 0.8320 0.7667 ^**** 0.0331	0.8710 0.8491 0.7729 0.9674 ^****

Figure 6

The UPGMA clustering dendrogram of populations

genetic variation. The results of DALP analysis showed that Panax notoginseng and its allied species were abundant in genetic diversity which had strong evolution potential. Based on Ruozhu QU [5] , the stable group had high Gst value, but the unstable one had low Gst value. The genetic differentiation index of Wenshan’s populations and Guangxi’s ones were over 0.3439, which showed that more genetic variation existed among populations, so they had great genetic differentiation and stable population. Many researches showed that the genetic diversity of endangered species was lowed, and the lack of diversity was the important reason to endanger [6] - [11] . So for Panax notoginseng, we must protect its genetic diversity at first, to build the resource garden and breed good varieties. The study provides basic information for resource pretection and sustainable use of Panax notoginseng.

Acknowledgments

We wish to thank Mr. X. M. Cui who is my tutor for thesis guide. This work is supported by Wenshan Sanqi Research Institute in Yunnan province.

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