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Chinese Herbal Medicines 11 (2019) 267–274

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Chinese Herbal Medicines

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Original Article

Parasitic relationship of Cistanche deserticola and host-plant Haloxylon ammodendron based on genetic variation of host

∗ Liang Shen a,b, Rong Xu a, , Sai Liu a, Chang-qing Xu a, Fang Peng a, Xiao-jin Li c, ∗ Guo-qiang Zhu c, Cai-xiang Xie a, Jun Zhu c, Tong-ning Liu d, Jun Chen a, a Institute of Medicinal Plant Development, Chinese Academy of Medical Science & Peking Union Medical College, Beijing 100193, China b Beijing Museum of Natural History, Beijing Academy of Science and Technology, Beijing 10 0 050, China c Xinjiang Institutes of Traditional Chinese Medica and National Medica, Urumqi 830 0 02, China d Ningxia Yongning Plantation of Herba Cistanche, Yinchuan 750100, China

a r t i c l e i n f o a b s t r a c t

Article history: Objective: Cistanche deserticola is a famous and endangered medicinal plant that is parasitic upon Haloxy- Received 5 May 2018 lon ammodendron with rather low parasitic rates . It is important to ﬁnd high aﬃnity germplasms for

Revised 13 January 2019 increasing the survival of C. deserticola . However, little is known in genetic variation and high affinity Accepted 16 April 2019 populations of H. ammodendron in China. Available online 6 June 2019 Methods: In this study, 98 accessions of H. ammodendron seeds were collected from five regions covering Keywords: almost the entire natural distribution of H. ammodendron in China. Their genetic variations were analyzed Cistanche deserticola Y. C. Ma using AFLP and ITS by the maximum parsimony method, and a dendrogram was constructed using the genetic structures unweighted pair-group method with arithmetic average (UPGMA). The parasitic rates of C. deserticola on Haloxylon ammodendron (C. A. Mey.) Bunge different accessions of H. ammodendron were calculated in the field experiment. parasitic rates Results: Both AFLP and ITS methods consistently revealed that there was a high level of genetic diversity in the natural populations of H. ammodendron . Hierarchical population structure analysis uncov- ered a clear pattern that all populations were grouped into three main clusters, and eight populations from eastern region were genetically clustered together. These regions were significantly differentiated ( P < 0.05), 13.10% of variation occurred among populations, and 86.90% within populations was revealed by analysis of molecular variance (AMOVA). The populations of Inner Mongolia had the highest parasitic rates followed by Ganjiahu Reserve and Yongning Plantation for the top three, which were not completely related to the genetic variation. Conclusion: Genetic characteristics of H. ammodendron in China were clarified and the order of affinity of different populations was given, which were primers for discovering high affinity germplasms. ©2019 Tianjin Press of Chinese Herbal Medicines. Published by Elsevier B.V. All rights reserved.

1. Introduction Genetic variation is one of the premiers for conserving host and parasite, with view to screening out of high affinity germplasms Cistanche deserticola Y. C. Ma is a rare and famous medicinal ( Bay et al., 2017; Cortanˇ & Tubi c,´ 2017 ). Several molecular markers plant which mainly parasitizes on roots of Haloxylon ammoden- including isozyme, ISSR, RAPD and AFLP have been used to study dron (C. A. Mey.) Bunge, a xerophytic desert shrub (Amaranthaceae) genetic variation of H. ammodendron populations ( Sheng, Zheng, ( Akhani, Edwards, & Roalson, 2007; Toghraie, 2012 ). Due to a low & Pei, 2005; Zhang, Dong, Wei, & Hu, 2006; Wang et al., 2009 ; parasitic rate upon host H. ammodendron, C. deserticola is an en- Shen et al., 2014 , 2015 ) . However, population samples in those dangered species that are deteriorated by land reclamation, over- studies were not enough for revealing genetic spectrum and par- cutting, and overstocking ( Wang et al., 2015; Xu et al., 2009 ). In asitic rates in the unclear field. It is necessary to collect more pop- China, H. ammodendron has been reduced from 110 0 0 0 km 2 in the ulations to represent entire H. ammodendron germplasms in China 1960s to 65 300 km 2 ( Guo et al., 2005 ) . It is important to find the and investigate the parasitic rates of different populations. host-plant germplasms with high parasitic rates for the survival of In recent years, AFLP markers have been used to detect the ge- C. deserticola . netic variation and structure of many species ( Shen et al., 2014; Zhao, Yin, Xu, & Wang, 2012, Głebocka ˛ & Pogorzelec, 2017 ). The AFLP technique provided genetic information on multiple loci in a ∗ Corresponding authors.

E-mail addresses: [email protected] (R. Xu), [email protected] (J. Chen). single assay without prior sequence knowledge at a relatively low https://doi.org/10.1016/j.chmed.2019.04.006 1674-6384/© 2019 Tianjin Press of Chinese Herbal Medicines. Published by Elsevier B.V. All rights reserved. 268 L. Shen, R. Xu and S. Liu et al. / Chinese Herbal Medicines 11 (2019) 267–274

cost with the requirement of small quantities of plant materials ering almost the entire distribution of the species in China. The ( Joyce et al., 2014 ). Another DNA sequence, from the internal tran- results will contribute to the accessions of H. ammodendron with a scribed spacer (ITS) region of the nuclear genome, is commonly high affinity with C. deserticola. applied in species identification, genetic variation and phylogenetic evolutionary studies ( Chen et al., 2010 ). ITS sequences are prone 2. Materials and methods to neutral mutations without strong constraints, leading to a relatively fast evolutionary rate and making the region appropriate for 2.1. Plant materials phylogenetic and evolutionary studies ( Hebert, Cywinska, & Ball, 2003 ). Assessing the evolutionary phylogenetic of H. ammodendron A total of 98 accessions of H. ammodendron were collected using AFLP jointly with ITS sequence can provide a valuable ge- from 14 populations from five regions including Xinjiang, Gansu, netic complementary information about host-plant identification, Ningxia, Qinghai, and Inner Mongolia, located between longitudes genetic variation and structure of a population. 82 °E to 106 °E and latitudes 36 °N to 44 °N, approximately covering In this study, the genetic variation and parasitic relationship of the main area of the species in China ( Fig. 1 , Table 1 ). Reserves H. ammodendron and C. deserticola were analyzed, and 14 popu- of Ganjiahu (Tuoli, Huyang and Guanfengtai), Tsaidam (Bolanggou, lations of H. ammodendron from five regions including Xinjiang, Huaitoutala and Zhaohe) and Inner Mongolia (Urat Reserve, Or- Gansu, Ningxia, Qinghai, and Inner Mongolia were collected, cov- dos Reserve and Alxa Reserve) were national nature reserves, and

Fig. 1. Geographical distribution of ﬁve growing regions [XG (XGT, XGH and XGG); QT (QTB, QTH and QTZ); GL (GLG, GLH and GLS); IM (IMUB, IMOK and IMAJ); NY (NYG and NYY)] of H. ammodendron sampled in this study. Populations were represented by black dots and their locations were given in Table 1 . Map was plotted using software ArcGIS 10.2 (ESRI, Redland, CA. URL http://www.esri.com/ ).

Table 1 Samples of Haloxylon ammodendron used in this study.

No Region codes Population codes Location of populations Longitude / °E Latitude / °N Altitude / m Samples No.

1 XG XGT Ganjiahu Reserve Tuoli Xinjiang 82.59 44.76 277.40 7 2 XG XGH Ganjiahu Reserve Huyang Xinjiang 83.43 44.91 254.60 7 3 XG XGG Ganjiahu Reserve Guanfengtai 83.41 44.88 249.50 7 4 QT QTB Tsaidam Reserve Bolanggou 97.74 36.60 2902.40 7 5 QT QTH Tsaidam Reserve Huaitoutala 96.55 37.09 3123.60 7 6 QT QTZ Tsaidam Reserve Zhaohe 97.67 36.77 2960.30 7 7 GL GLG Liangucheng Reserve Qinfeng 102.95 38.61 1366.30 7 8 GL GLH Liangucheng Reserve Huaer 102.45 38.83 1354.90 7 9 GL GLS Liangucheng Reserve Xuebai 102.99 38.74 1379.00 7 10 IM IMUB Inner Mongolia 105.80 41.23 1068.60 7 Urat Reserve 11 IM IMOK Inner Mongolia 107.61 40.37 1084.00 7 Ordos Reserve 12 IM IMAJ Inner Mongolia 105.77 39.61 1048.00 7 Alxa Reserve 13 NY NYG Yongning Plantation Guangxia 106.08 38.24 1124.20 7 14 NY NYY Yongning Plantation Yuquan 106.02 38.22 1130.90 7

Note: XG, Xinjiang Ganjiahu; QT, Qinghai Tsaidam; GL, Gansu Liangucheng; IM, Inner Mongolia; NY, Ningxia Yongning. L. Shen, R. Xu and S. Liu et al. / Chinese Herbal Medicines 11 (2019) 267–274 269

Liangucheng reserve (Qinfeng, Huaeryuan and Xuebai) and Yongn- cessions was based on Jaccard’s genetic distance matrix of acces- ing plantation (Guangxia and Yuquan) were man-made in the 1960 sion; Data were subjected to genetic analysis in GenAlEx package and 1998, respectively. Seeds and seedlings of H. ammodendron at ( Peakall & Smouse, 2012 ). Yongning plantation were mainly portion of Inner Mongolia. The A Bayesian approach (Markov Chain Monte Carlo Algorithm) accessions of H. ammodendron were identified by Professor Jun was applied to detect the underlying population genetic structure Chen, and the voucher specimens (H2015-1) were deposited at the among a set of accessions ( Wolf, Anselin, & Arribas, 2018 ). The National Medicinal Plant Seed Resource Library of the Institute of most likely number of clusters was estimated according to the Medicinal Plant Development, Chinese Academy of Medical Sci- model value ( K ) based on the second order rate of change, fol- ences (Beijing), China. lowing the method described by Sorkheh, Dehkordi, Ercisli, Hege- dus, and Halász (2017) . Five STRUCTURE runs were performed 2.2. DNA extraction and PCR for each K , ranging from 1 to 15, independently, with a burn- in length of 10 0 0 0 followed by 100 000 runs at each K , and a Seven accessions of each population were randomly selected higher run from the five runs was chosen. Other parameters were for the germination tests. After 3 − 5 d of incubation at 25 °C set to default values. To test the correlation between genetic and at illumination incubator, total genomic DNA was extracted from geographic distances among populations, a Mantel test was per- 0.50 g young stem tissue following the Cetyl Trimethyl Ammo- formed using TFPGA 1.3, and 10 0 0 permutations were computed nium Bromide (CTAB) DNA extraction procedure ( Porebski, Bailey, ( Miller, 1997 ). & Baum, 1997 ). DNA concentration and quality were assessed using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilming- 2.4. ITS analysis ton, DE, USA) and on a 1.2% agarose gel. AFLP primers were screened on five accessions using six Sequences were aligned, edited, and the G + C content of primers for each candidate (Sangon Biotech, China; Table 2 ). DNA each sequence calculated using Codon Code Aligner software 3.7.1 amplification was performed using a Bio-Rad PCR (Bio-Rad Labora- (Codon Code Corporation, MA, USA). The genetic structure of tories, Alfred Nobel Drive Hercules, CA94547, USA). Electrophoresis H. ammodendron was analyzed using AMOVA in Arlequin 3.0 on a 1% agarose gel alongside a 100 bp DNA ladder (Takara Bio- ( Excoffier, Laval, & Schneider, 2005 ). The Wright fixation index tec, Japan) in TAE allowed the size of polymorphic fragments to be level was obtained based on the five regional accessions using estimated ( Głebocka ˛ & Pogorzelec, 2017 ). DNA fragments were se- the formula Fst = 1/(1 + 2 Nm ) ( Wright, 1969 ), where Fst was used quenced using GeneMapper (Applied Biosystems by Thermo Fisher to measure genetic differentiation among populations, and Nm Scientific). was the immigration numbers of the accessions in a generation Primers for ITS application were designed based on preciously ( Crema, Kandler, & Shennan, 2016 ). Genetic variation and emu- reported sequences: forward primer ITS 5F and reverse primer lation rates of H. ammodendron were analyzed using DnaSP v.4.0 ITS 4R ( Joyce et al., 2014 ). PCR products were purified follow- ( Saitou & Nei, 1987 ). MEGA 3 DNA sequence analysis software was ing the TianGen manufacturer’s protocol and directly subjected used to generate genetic distances for phylogenetic trees using the to sequencing using an ABI 3730XL sequencer platform. The ITS maximum parsimony method and bootstrapping with 10 0 0 repli- sequence for H. ammodendron determined in the present study cates ( Kumar, Tamura, & Nei, 2004 ). Arlequin provides methods to were deposited in GenBank (GenBank accession No. KP318519 to analyze patterns of genetic variation within and between popula- KP318650). tion samples ( Tajima, 1989 ).

2.3. AFLP analysis 2.5. Analysis of parasitic rates

The resultant genetic binary data matrix of AFLP was analyzed Parasitic rates were surveyed at the plantation of C. deserticola using POPGENE (version 1.32) ( Yeh, Yang, Boyle, Ye, & Mao, 1997 ). in Yongning, Ningxia Hui Autonomous Region (38 °31 ʹN, 106 °17 ʹE, The percentage of polymorphic bands ( PPB ), number of alleles ( Na ), 620 m), China. The plantation is a tract of sand with weeds, and effective number of alleles ( Ne ), Nei’s genetic diversity ( h ), Shan- it has served as a cultivation area of C. deserticola since 1998. In non’s information index of diversity ( I ), Nei’s coefficient of genetic this study, the area of the test plot was 600 square meters, 80 differentiation ( Gst ), and gene flow ( Nm ) were employed to calcu- seedlings of H. ammodendron of one year old were used to inves- late genetic variation parameters of the populations ( Nei, 1978 ). tigate the parasitic rate of C. deserticola which came from five re- Analysis of molecular variance (AMOVA) was used to deduce the gions, respectively. All samples were transplanted in seeding field genetic structure and variability among and within populations us- of H. ammodendron in April 2013, two young seedlings of H. am- ing GenAIEx version 6.5 ( Peakall & Smouse, 2012 ). The variance modendron were planted in the plastic crate, the row spacing of H. components were tested statistically by nonparametric randomiza- ammodendron was 2 m, and the spacing of each plant was 0.2 m. tion tests, using 999 permutations. Genetic similarity and the ge- The seeds of C. deserticola were collected from the same plant, netic distance matrix were constructed from the resultant binary and 50 granule seeds were scattered in a block of organic fertil- data matrix using UPGMA in the NTSYS-PC program (version 2.0) izer (6 × 6 cm), and the seed piece of C. deserticola was placed in ( Rohlf, 1997 ). Principle coordinate analysis (PCoA) of population ac- the two flanks of plastic crate. The plastic crate of H. ammodendron

Table 2 Six AFLP primers used in this study. Adapter names Primer names Forward primer sequence (5 −3 ) Reverse primer sequence (5 −3 )

EcoR I E32:EcoRI + AAC CTCGTAGACTGCGTACC GACTGCGTACCAATTCAAC EcoR I E33:EcoRI + AAG AATTGGTACGCAGTCTAC GACTGCGTACCAATTCAAG Mse I E43:EcoRI + ATA GACGATGAGTCCTGAG GACTGCGTACCAATTCATA Mse I M64:MseI + GAC TACTCAGGACTCAT GATGAGTCCTGAGTAAGAC E00 M65:MseI + GAG GACTGCGTACCAATTC GATGAGTCCTGAGTAAGAG M00 M86:MseI + TCT GATGAGTCCTGAGTAA GATGAGTCCTGAGTAATCT 270 L. Shen, R. Xu and S. Liu et al. / Chinese Herbal Medicines 11 (2019) 267–274 was covered in the suitable soil depth and watered after seed piece accessions of XG region, whereas QT region had the lowest genetic of C. deserticola added. We proposed that the parasitic relationship variation ( Pi = 0.01) ( Table 4 ). was established successfully when C. deserticola adhered to roots of H. ammodendron . The sampled accessions parasitized by C. de- 3.2. Genetic differentiation analysis serticola and those non-parasitized was investigated after 1.5 years later, and the host plants were counted after the parasite, which The relative magnitude of Gst among total populations was was observed to be attached to the roots ( Fig. 2 ). 0.08, indicating that 92.00% variance occurred within the populations. Nm within the five regions ranged from 1.08 (QT) to 3.31 3. Results (NY) indicating that gene flow was high in NY, but low in QT ( Table 3 ). AMOVA revealed that the proportion of variation was 3.1. Genetic variation analysis highly attributable to differences within populations (86.90%) and regions (90.43%), whereas only 13.10% and 9.57% occurred among In this study, six from 50 possible AFLP primer combinations populations and regions, respectively ( Table 5 ). were selected ( Table 2 ). The six AFLP primer combinations pro- ITS markers revealed the proportion of variation was lower duced 723 fragments in 98 accessions, of which 685 (94.74%) were among 14 populations (32.07%) and five regions (12.79%), whereas polymorphic ( Table 3 ). A relatively high level of genetic variation a higher level of genetic variation occurred within populations existed among the 14 populations ( h = 0.32, I = 0.49, PPB = 94.74%). (67.93%) and region (87.21%). A relatively high emulation rate The XG region had the highest level of genetic variation ( h = 0.29, (3.85) was observed in XG, whereas QT had the lowest emulation I = 0.43, PPB = 83.54%), whereas QT region had the lowest one rate (0.74). The results of the ITS analysis were similar to those of ( h = 0.21, I = 0.32, PPB = 64.32%). the AFLP markers ( Table 5 ), which indicated a high level of genetic The length of ITS sequences of H. ammodendron ranged from variability existed in H. ammodendron . Relationships between the 675 bp to 678 bp, and the GC content was approximately 60% in genetic and environmental data of the populations were further il- the 14 populations. The lengths of the ITS sequences and CG con- lustrated by Mantel test, and a significant correlation ( r = 0.91, P < tent were similar in the five regions. A relatively high level of ge- 0.05) between Nei’s distance and geographic distance for the five netic variation (nucleotide variation Pi = 0.51) occurred among the regions was detected.

Fig. 2. Field parasitic test procedure of C. deserticola and H. ammodendron. A: H. ammodendron planting, B: H. ammodendron harvest, C: C. deserticola parasitic on H. ammodendron, D: Larger individual of C. deserticola , E: XG germplasm parasitic test, F: IM germplasm parasitic test. L. Shen, R. Xu and S. Liu et al. / Chinese Herbal Medicines 11 (2019) 267–274 271

Table 3 Genetic variation statistics of analyzed Haloxylon ammodendron plants.

Codes Na ± SD Ne ± SD h ± SD I ± SD Np PPB /% Gst Nm

XGT 1.62 ± 0.49 1.42 ± 0.41 0.23 ± 0.21 0.35 ± 0.30 443 61.27 −− XGH 1.57 ± 0.51 1.37 ± 0.40 0.21 ± 0.21 0.31 ± 0.29 406 56.15 −− XGG 1.44 ± 0.50 1.28 ± 0.37 0.16 ± 0.20 0.24 ± 0.29 316 43.71 −− XG 1.85 ± 0.36 1.49 ± 0.35 0.29 ± 0.18 0.43 ± 0.24 604 83.54 0.20 1.99 QTB 1.44 ± 0.50 1.28 ± 0.37 0.16 ± 0.20 0.24 ± 0.29 316 43.71 −− QTH 1.39 ± 0.49 1.25 ± 0.36 0.14 ± 0.20 0.21 ± 0.28 275 38.04 −− QTZ 1.33 ± 0.47 1.20 ± 0.34 0.12 ± 0.18 0.17 ± 0.26 239 33.06 −− QT 1.65 ± 0.48 1.36 ± 0.37 0.21 ± 0.20 0.32 ± 0.28 465 64.32 0.32 1.08 GLG 1.46 ± 0.50 1.30 ± 0.38 0.17 ± 0.20 0.25 ± 0.29 325 44.95 −− GLH 1.56 ± 0.50 1.36 ± 0.40 0.21 ± 0.21 0.30 ± 0.29 401 55.46 −− GLS 1.60 ± 0.49 1.40 ± 0.39 0.23 ± 0.21 0.33 ± 0.31 426 58.92 −− GL 1.80 ± 0.40 1.47 ± 0.35 0.27 ± 0.18 0.41 ± 0.25 571 78.98 0.22 1.80 IMUB 1.60 ± 0.49 1.36 ± 0.38 0.21 ± 0.20 0.31 ± 0.29 432 59.75 −− IMOK 1.61 ± 0.49 1.37 ± 0.38 0.22 ± 0.20 0.33 ± 0.29 438 60.58 −− IMAJ 1.59 ± 0.49 1.38 ± 0.40 0.22 ± 0.21 0.32 ± 0.29 419 57.95 −− IM 1.78 ± 0.41 1.44 ± 0.36 0.26 ± 0.19 0.40 ± 0.26 560 77.46 0.13 3.25 NYG 1.55 ± 0.50 1.35 ± 0.39 0.20 ± 0.21 0.30 ± 0.29 394 54.50 −− NYY 1.55 ± 0.50 1.34 ± 0.38 0.20 ± 0.20 0.29 ± 0.29 391 54.08 −− NY 1.69 ± 0.46 1.41 ± 0.38 0.24 ± 0.20 0.36 ± 0.28 491 67.91 0.13 3.31 Total 1.99 ± 0.08 1.55 ± 0.32 0.32 ± 0.15 0.49 ± 0.18 685 94.74 0.08 6.13

Note: Na , observed number of alleles; Ne , effective number of alleles; h , Nei’s gene diversity; I , Shannon’s information index; Np , number of polymorphic loci; PPB , percentage of polymorphic bands (%); Gst , coeﬃcient of gene differentiation; Nm , gene ﬂow. Codes used for populations were given in Table 1 .

Table 4 detected ( Fig. 3 B). PCoA was conducted to further understand the

Genetic variation of Haloxylon ammodendron in ﬁve regions. ecological distribution of total accessions ( Fig. 5 A), and the results Regions n TNS ENS Pi /% SMS Emulation rates /% were consistent with those of the UPGMA and STRUCTURE anal-

XG 21 677 622 0.51 26 3.85 ysis. Phylogenetic relationships among the ﬁve regions were also QT 21 677 665 0.01 5 0.74 illustrated by PCoA based on analysis of ITS sequences. The ﬁve re- GL 21 677 667 0.00 5 0.74 gions accessions were mixed together, and GL samples had lower IM 21 677 667 0.00 5 0.74 genetic variation ( Fig. 5 B). The membership assignment derived NY 14 677 615 0.11 11 1.63 from the PCoA of the ITS sequences had a little difference with Total 98 677 667 0.11 35 5.18 the UPGMA analysis of AFLP results. Note: n , number of population accessions; TNS, site of total RNA; ENS, site of effective nucleic acid; Pi , nucleotide variation; SMS, Single mutation site. 3.4. Genetic parasitic rates of H. ammodendron

Table 5

Analysis of molecular variance (AMOVA) within Haloxylon ammodendron Parasitic rates of different H. ammodendron germplasm were populations. calculated in the field experiment of Ningxia base ( Table 6 ). It showed that different H. ammodendron germplasm had significant Degree of Sum of Variance Percentage Significance effects on the parasitic rate of C. deserticola , and the parasitic Source of variance freedom squares component of variance/% ( P ) rate of H. ammodendron in Inner Mongolia (IM) had the high- AFLP Among 13 2262.39 13.40 13.10 < 0.05 est parasitic rate (58.78%), whereas Tsaidam (QT) was the low-

populations est (16.14%). The parasitism status of each sample was different Within 84 6667.29 88.90 86.90 < 0.01 populations after 1.5 years plantation, and some of C. deserticola had rotted Among 2 1075.33 9.89 9.57 < 0.05 ( Fig. 6 ). regions Within 84 7854.36 93.50 90.43 < 0.01

regions 4. Discussion ITS Among 13 73.29 0.57 32.07 < 0.01

populations 4.1. Genetic variation and structure Within 98 118.19 1.21 67.93 < 0.01 populations Among 2 20.72 0.21 12.79 < 0.01 In general, geographically wide dispersed species have regions higher proportions of polymorphic loci and genetic varia- Within 84 187.44 1.44 87.21 < 0.01

regions tion than geographically limited species (Husch et al., 2018). Sheng et al. (2005) reported that the PPB of H. ammodendron was 84.00%, whereas Zhang et al. (2006) reported that it was 89.23%. 3.3. Population structure and cluster analysis Table 6

To further elucidate the relationship among populations, a den- Genetic parasitic rates of Haloxylon ammodendron in ﬁve regions. drogram was constructed used UPGMA cluster analysis, which indicated that the 14 populations were grouped into three clus- Regions n Parasitic rates /% ters ( Fig. 3 A). Cluster I (eastern region) consisted of populations XG 80 52.61 ± 10.71b from GL, IM, and NY, while clusters II (southern region) and III QT 80 16.14 ± 7.55a (western region) consisted of populations from QT and XG, respec- GL 80 21.27 ± 5.77a IM 80 58.78 ± 6.05b tively. Bayesian analysis inferred from STRUCTURE revealed that NY 80 47.53 ± 5.95b K peaked at K = 3 ( Fig. 4 ), and three cluster populations were 272 L. Shen, R. Xu and S. Liu et al. / Chinese Herbal Medicines 11 (2019) 267–274

Fig. 3. UPGMA (A) and STRUCTURE (B) dendrograms for Haloxylon ammodendron based on Nei’s distance as revealed using AFLP markers. Population codes used were given in Table 1 , I (eastern region), II (southern region), III (western region).

in China which included clay desert, gravel desert, saline land, and fixed and semi-fixed sands ( Shen et al., 2015 ). There is a strong correlation relationship between the level of genetic polymorphism and the degrees of environmental hetero- geneity and stress ( Shen et al., 2015 ). In this study, the population XG had the highest level of genetic variation, most likely ow- ing to the wide distribution of H. ammodendron in this region. This would have allowed germplasm resources to be exchanged with neighboring Kazakhstan through western borders ( Trapnell & Hamrick, 2005 ). QT (Southern region) showed the lowest level of genetic variation, most likely due to its isolated natural environ- ment and the genetic was difficult to exchange with other regions ( Guo et al., 2005 ). In addition, the clustered analysis and Mantel Fig. 4. Genetic structure of H. ammodendron populations inferred by Bayesian clus- tering of AFLP data. The most likely number of clusters based on K estimation test also revealed a significant correlation between genetic and ge- was three ( K = 3). ographic distances of H. ammodendron . Genetic material is the most important factor in determining Compared with the previous studies, our results based on almost the genetic variation of plant populations ( Husch et al., 2018 ). AFLP the entire distribution of H. ammodendron in China showed a sim- makers and ribosomal DNA region ITS1-5.8S-ITS2 was sequenced ilar but relatively higher PPB (94.74%), which also suggested that to assess the evolutionary phylogenetic of H. ammodendron , which a higher level of polymorphism existed in wild H. ammodendron could provide a valuable genetic complementary information for accessions. The high levels of polymorphism might be attributed to genetic variation and structure of a population. Both AFLP and the variation of habitats that H. ammodendron naturally occurred ITS markers revealed that H. ammodendron had more genetic

Fig. 5. UPGMA dendrogram of genetic data of Haloxylon ammodendron . A: Two-dimensional ordination of principal coordinate analysis of H. ammodendron populations used AFLP markers; B: Principal coordinate analysis of H. ammodendron populations obtained using pairwise, Nei’s genetic distance measurements from combined data of ITS markers. L. Shen, R. Xu and S. Liu et al. / Chinese Herbal Medicines 11 (2019) 267–274 273

Fig. 6. Growth status (A: 2 months, B: 4 months, C: 6 months, D: 8 months, E: 10 months, F: 14 months, G: 18 months, H: decayed C. deserticola ) of C. deserticola parasitic upon H. ammodendron after 1.5 years plantation. variation within populations than among populations. Similar find- able to meet the requirements of genetic cognition for germplasm ings were reported for the woody species Rhododendron in the resource screening. The main determining factor for the parasitism Qinling Mountain region in China ( Gst = 0.09) ( Zhao et al., 2012 ). should be the related genes for parasitism affinity. DNA sequencing Therefore, gene flow was higher among the interior of five regions method maybe more suitable for the high parasitic rate breeding of H. ammodendron . when the related genes have been reported in the future. The genetic structure of population reflects interactions among species with regards to their long-term evolutionary history, genetic drift, and natural selection ( Lowe, Kovach, & Allendorf, 4.3. Conservation and breeding considerations 2017 ). In the phylogenetic analysis of this study, populations were grouped into three clusters, eastern region (GL, IM, and NY), south- Successful management and preservation of a species depends ern region (QT) and western region (XG), respectively. The phylo- on a complete understanding of its genetic structure and variation genetic relationships comparison among different varieties could ( Prinz, Weising, & Hensen, 2013 ). Therefore, information on H. am- reflect their long-term evolutionary history, and would be a ref- modendron genetic structure is essential for designing appropriate erence for H. ammodendron selective breeding. However, phyloge- conservation strategies and finding the best or better germplasm netic analysis of accessions from other regions showed inconfor- accessions distribution for cultivating C. deserticola . Furthermore, mity between the two methods. The phylogram generated from our investigation showed that the parasitic rate of H. ammodendron ITS sequences did not correlate greatly with the AFLP results, this in Tsaidam was the lowest, whereas Inner Mongolia had the high- may be because AFLP data allows an assessment of population est parasitic rates, which could help us choose the proper source structure based on multiple loci, and it is the representative of for introduction. the entire genome, whereas ITS sequences are based on a single To preserve and apply valuable genetic resources of H. am- locus sequence variation data and its absence of heterogeneous modendron , different advices were recommended for specific re- sequences. gions according to the results of this study. Populations in Ganji- ahu of Xinjiang region presented the highest level of genetic vari- 4.2. Parasitic rate analysis ation and harbored the greatest distribution area, which could be used as source materials for restoration and introduction. As well, It’s significant to rank the C. deserticola parasitic rates on dif- more nature reserves should be established in this region. Qinghai ferent H. ammodendron germplasms in China for the first time. Tsaidam had the lowest levels of genetic variation and the most The parasitic rates on H. ammodendron from different regions were specific ecological habitat, therefore, in situ conservation should be varied from 16.14% in Tsaidam (QT) to 58.78% in Inner Mongolia adopted, and it was not a good introduction source of C. deserticola (IM), which were not mostly related to the genetic variation. How- host plants. The number of H. ammodendron populations in Inner ever, QT region had the lowest genetic variation and the lowest Mongolia has decreased dramatically, making it prone to extinction parasitic rates. This may be due to the high altitude of Qinghai- from random environmental fluctuations. As a result, implementa- Tibet Plateau where is the highest elevation distribution limit of tion of in situ and ex situ conservation would be necessitated im- H. ammodendron , and the long-term natural selection for the lower mediately. parasitic rates populations. The results will contribute to introduction breeding for select- Different parasitic rates of the trees from the controlled field ing introduction sources and better H. ammodendron germplasms trials reflect their genetic affinity with C. deserticola . However, the for C. deserticola. The further research could apply traditional and experimental samples maybe not enough to get a conclusion, the modern breeding methods to breed H. ammodendron varieties with deficient of the samples is caused by the low seedling rates and higher parasitic rate and increase the yield and quality of C. deser- survival rate of transplanting. As well, genetic variation may be un- ticola from the introduction accessions. 274 L. Shen, R. Xu and S. Liu et al. / Chinese Herbal Medicines 11 (2019) 267–274

Conﬂict of interest Nei, M. (1978). Estimation of average heterozygosity and genetic distance from a small number of individuals. Genetics, 89 (3), 583–590 . The authors declare that they have no competing interests. Peakall, R. , & Smouse, P. E. (2012). Genalex 6.5: Genetic analysis in excel. Population genetic software for teaching and research-an update. Bioinformatics, 28 (19), 2537–2539 .

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China ( 81773851 & U1403224 ), Fundamental Research Fundfor the Prinz, K., Weising, K., & Hensen, I. (2013). Habitat fragmentation and recent bot- tlenecks inﬂuence genetic diversity and differentiation of the Central Euro-

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