青藏高原瀕危植物唐古特大黃的遺傳多樣性

王愛蘭,李維衛(wèi)

1 魯東大學(xué)生命科學(xué)學(xué)院, 煙臺 264025 2 魯東大學(xué)學(xué)報(bào)編輯部, 煙臺 264025

青藏高原瀕危植物唐古特大黃的遺傳多樣性

王愛蘭1,李維衛(wèi)2,*

1 魯東大學(xué)生命科學(xué)學(xué)院, 煙臺 264025 2 魯東大學(xué)學(xué)報(bào)編輯部, 煙臺 264025

唐古特大黃(Rheumtanguticum)是中國傳統(tǒng)的中藏藥材,近幾年由于生境的嚴(yán)重破壞,已瀕臨滅絕,并被列入瀕危植物名單。為了探索唐古特大黃物種瀕危的原因并保護(hù)其野生資源,本研究采集了9個(gè)居群87個(gè)個(gè)體的唐古特大黃樣本,基于該物種的葉綠體基因trnS-G序列對其進(jìn)行了遺傳多樣性研究。結(jié)果表明,唐古特大黃物種具有較高的遺傳多樣性水平(Ht=0.694),其中95.97%的遺傳分化來自于居群間(GST=0.960),4.03%的遺傳分化來自于居群內(nèi)(Hs=0.028)。AMOVA分析也顯示唐古特大黃居群間基因流較小(Nm=0.01),存在較高的遺傳分化(FST=0.9631)。唐古特大黃較高的遺傳多樣性水平可能與該物種較長的進(jìn)化史和生活史有關(guān),居群間較高的遺傳分化可能與高山地區(qū)特殊的地理環(huán)境和人類活動有關(guān)。根據(jù)研究結(jié)果,建議對唐古特大黃所有野生居群進(jìn)行就地保護(hù),同時(shí)收集種質(zhì)資源開展異地繁殖工作,以保護(hù)物種的遺傳多樣性,維持其進(jìn)化潛力。

唐古特大黃；trnS-G序列；遺傳多樣性；基因流

青藏高原是世界上海拔最高、面積最大的高原,具有豐富的物種多樣性,是全球25個(gè)生態(tài)熱點(diǎn)之一,有超過12000種植物生長在青藏高原及其鄰近地區(qū)[1- 2]。由于高海拔和干旱等極端環(huán)境的存在,導(dǎo)致該地區(qū)生態(tài)系統(tǒng)對環(huán)境變化及人類活動非常敏感[3],已有1000多種植物被列為瀕危物種[4]。過去,青藏高原地區(qū)居民依靠野生植物資源獲得收入,導(dǎo)致野生資源過分采挖,由于環(huán)境惡劣,致使這些物種很難恢復(fù),從而引起一系列生態(tài)問題。

唐古特大黃(RheumtanguticumMaxim. ex Balf.)是蓼科(Polygonaceae)大黃屬植物,主要分布在青藏高原地區(qū),其根入藥,具有通便、抗炎等功效,是傳統(tǒng)的中藏藥材[5]。由于青藏高原地區(qū)的野生資源品質(zhì)較高,出口到日本、韓國和其他地區(qū)[6]。近幾年由于經(jīng)濟(jì)利益的驅(qū)使導(dǎo)致人類過度采挖,使得唐古特大黃野生分布區(qū)急劇減少,因而被列入瀕危物種名單[4]。

根據(jù)實(shí)地調(diào)查,發(fā)現(xiàn)大黃野生資源已經(jīng)非常稀少,對其野生居群的保護(hù)迫在眉睫。居群遺傳多樣性研究對瀕危物種的保護(hù)具有重要意義[7- 9],相關(guān)研究工作也已逐步開展,如Chen等[10]、Hu等[11]和Wang等[12]分別利用SSR、ISSR和基因片段trnL-F對唐古特大黃進(jìn)行了遺傳多樣性的分析,迄今為止,尚未見利用trnS-G序列研究其遺傳多樣性的報(bào)道。葉綠體基因片段trnS-G序列因其具有較快的進(jìn)化速率和較高的遺傳變異等特點(diǎn),已被廣泛應(yīng)用到物種遺傳多樣性和種內(nèi)種間基因交流的研究中[13-14]。因此,研究選擇trnS-G序列對唐古特大黃物種多樣性水平及居群間的基因流進(jìn)行了研究。本研究重點(diǎn)探討以下問題：1)唐古特大黃的遺傳多樣性水平；2)唐古特大黃居群的遺傳結(jié)構(gòu)；3)通過多樣性水平調(diào)查,為該物種的遺傳保護(hù)提出合理建議。

1 材料與方法

1.1 實(shí)驗(yàn)材料

本研究采用了唐古特大黃9個(gè)居群87個(gè)樣本,所有樣本均分布在青藏高原地區(qū)(圖1),采樣地點(diǎn)基本覆蓋唐古特大黃現(xiàn)存野生居群的分布地區(qū),樣本信息見表1。采樣時(shí),樣本間隔根據(jù)居群大小確定在3—5m之間,采集的葉片用硅膠干燥后保存?zhèn)溆谩?/p>

1.2 DNA提取,擴(kuò)增及測序

采用改良的CTAB法[15]從干燥的唐古特大黃葉片中提取基因組DNA。利用Hamilton[16]設(shè)計(jì)的‘S’ (5′-GCCGCTTTAGTCCACTCAGC- 3′)和 ‘G’ (5′-GAACGAATCACACTTTTACCAC- 3′)引物進(jìn)行PCR擴(kuò)增,擴(kuò)增體系為：25 μL體系,包括40 ng模板DNA,17.5 μL無菌水,2.5 μL 10×Taq 酶緩沖液,0.5 μL 10 mmol/L dNTPs,5 μmol/L引物各1.25 μL,1 U DNA Taq酶。擴(kuò)增程序?yàn)椋?4 ℃預(yù)變性4.0 min；94 ℃變性1.0 min,51℃退火50 s,72 ℃延伸1.75 min,循環(huán)38次；最后72 ℃延伸8 min。PCR產(chǎn)物用1.0%的瓊脂糖凝膠進(jìn)行電泳檢測,采用純化試劑盒進(jìn)行純化(購自上海生工),然后送至北京六合華大基因公司進(jìn)行核苷酸序列分析。

圖1 唐古特大黃樣本分布圖Fig.1 The samples distributions of R. tanguticum

1.3 數(shù)據(jù)分析

用Clustal X軟件[17]進(jìn)行序列比對并進(jìn)行手工校正。在進(jìn)行單倍型分析時(shí),插入缺失和堿基替換作相同處理。利用DnaSP5.1軟件[18]計(jì)算居群的單倍型多態(tài)性(Hd)、核苷酸多態(tài)性(pi)和物種水平居群間的平均基因流(Nm)。采用PERMUT軟件[19]計(jì)算居群內(nèi)平均遺傳多樣性(Hs)、總遺傳多樣性(Ht)和居群遺傳分化系數(shù)(GST)。應(yīng)用Arlequin3.1軟件進(jìn)行分子變異分析(AMOVA)[20],并進(jìn)行1000次隨機(jī)抽樣的顯著性檢驗(yàn)。采用TFPGA軟件對遺傳距離矩陣和地理距離矩陣進(jìn)行mantel檢測,并進(jìn)行1000次隨機(jī)置換的顯著性檢驗(yàn)。另外,采用MEGA 6.0的鄰接法NJ(Neighbor-Joining)進(jìn)行聚類分析,建立無根系統(tǒng)發(fā)育樹[21]。采用NETWORK 4.6.1軟件[22]中的中點(diǎn)連接法進(jìn)行種內(nèi)譜系的單倍型網(wǎng)絡(luò)分析,并用溯祖理論來推測單倍型間的親緣關(guān)系。

2 結(jié)果

2.1 CpDNA序列變異和單倍型分布

87個(gè)樣本獲得的trnS-G序列長度在944—954 bp之間,校正后總長度為965 bp。共檢測到23個(gè)核苷酸變異位點(diǎn),其中10個(gè)是堿基缺失或插入造成的突變位點(diǎn),13個(gè)為核苷酸置換位點(diǎn)(表2)。

表1 用于實(shí)驗(yàn)的珊瑚菜材料及其來源

基于這些核酸突變,在9個(gè)居群的87個(gè)樣本中共發(fā)現(xiàn)4種單倍型H1—H4(表3,圖2),這些單倍型的代表序列均提交到GenBank數(shù)據(jù)庫(表3)。H1單倍型是Pop4居群特有單倍型,包含7個(gè)樣本；H2單倍型分布在Pop3和Pop8兩個(gè)居群,共16個(gè)樣本；H3是分布最廣泛且最豐富的單倍型,分布在5個(gè)居群(Pop1、Pop2、Pop5、Pop7和Pop9),共48個(gè)樣本,占采樣總數(shù)的55.17%；H4單倍型出現(xiàn)在Pop3和Pop6 這兩個(gè)居群,分別包括7個(gè)和9個(gè)樣本(表3)。

表2 唐古特大黃4個(gè)單倍型變異位點(diǎn)信息

表3 各居群的單倍型分布數(shù)目

圖2 NETWORK單倍型網(wǎng)絡(luò)圖 Fig.2 Haplotype relationship constructed by the software NETWORK 圖中圓圈代表單倍型,H代表檢測到的單倍型,mv代表缺失的單倍型

Network單倍型網(wǎng)絡(luò)圖顯示,4個(gè)單倍型組成了星型遺傳結(jié)構(gòu)(圖2)。單倍型H1、H2和H4在網(wǎng)絡(luò)的邊緣,表明可能是新近發(fā)生的單倍型；H3單倍型和3個(gè)缺失的單倍型在星型網(wǎng)絡(luò)中心,且H3單倍型出現(xiàn)的頻率最高,推測為祖先單倍型。

2.2 遺傳多樣性分析

DnaSP分析顯示,物種水平上,單倍型多樣性Hd為0.6218,核苷酸多樣性指數(shù)pi為0.0049；9個(gè)居群的單倍型多樣性(Hd)范圍在0—0.25之間,核苷酸多樣性(Pi)在0—0.0026之間。其中,Pop3居群含有H2和H4兩種單倍型,其單倍型多樣性和核苷酸多樣性最高(Hd=0.25,Pi=0.0026),而其他8個(gè)居群因只有一個(gè)單倍型(表3),居群單倍型多樣性和核苷酸多樣性均為0。PERMUT分析顯示,居群總的遺傳多樣性指數(shù)(Ht)為0.694,表明了較高的遺傳多樣性水平；居群內(nèi)遺傳多樣性指數(shù)(Hs)為0.028,表明居群內(nèi)遺傳多樣性水平較低。

2.3 遺傳結(jié)構(gòu)分析

由居群總的遺傳多樣性指數(shù)(Ht)和居群內(nèi)遺傳多樣性指數(shù)(Hs)推算,居群間遺傳分化較大,4.03%的變異發(fā)生在居群內(nèi),而其余95.97%的遺傳變異發(fā)生在居群間,居群間分化系數(shù)(GST)為0.960,也驗(yàn)證了上述結(jié)果。AMOVA分析得出居群間遺傳分化系數(shù)(FST)為0.9631,同樣表明遺傳變異主要發(fā)生在居群間。DnaSP分析顯示,居群間基因流(Nm)極小,僅為0.01,表明不同居群間存在有限的基因交流,這也支持了PERMUT和AMOVA的分析結(jié)果。

圖3 基于遺傳距離構(gòu)建的唐古特大黃居群聚類分析圖 Fig.3 NJ dendrogram based on genetic distances of R. tanguticum

基于遺傳距離構(gòu)建的NJ樹顯示了3個(gè)組(圖3)。Pop3和Pop6聚為一組,表明了相近的親緣關(guān)系；Pop7、Pop9、Pop5、Pop1和Pop2聚為一個(gè)小分支,又與Pop8聚為一個(gè)較大的分支,表明相對較近的親緣關(guān)系；Pop4沒有與其他居群聚在一起,表明其與其他居群親緣關(guān)系較遠(yuǎn)。聚類分析表明,地理距離較近的居群并未聚到一起,說明地理距離與遺傳距離沒有直接的相關(guān)關(guān)系。Mantel檢測結(jié)果(r=0.2461,p=0.2060)也驗(yàn)證了這一點(diǎn),表明居群地理距離與遺傳距離存在不顯著的正相關(guān)關(guān)系。

3 討論

3.1 唐古特大黃的遺傳多樣性

遺傳多樣性降低被認(rèn)為是物種瀕危的主要原因[23-26]。但是某些研究發(fā)現(xiàn)瀕危物種也具有較高的遺傳多樣性,例如罌粟科的毛黃堇Corydalistomentella[27](Ht=0.804)和傘形科的珊瑚菜Glehnialittoralis[28](Ht=0.2471)。

本研究基于葉綠體基因trnS-G序列構(gòu)建了9個(gè)野生唐古特大黃居群的遺傳結(jié)構(gòu),結(jié)果顯示該物種具有較高的遺傳多樣性,這一結(jié)果與SSR(He=0.515)、ISSR(He=0.2689)和trnL-F(Ht=0.632)的研究結(jié)果[10-12]一致。物種遺傳多樣性應(yīng)該是長期形成的遺傳分化,唐古特大黃較高的遺傳多樣性可能與該物種的進(jìn)化史和生活史有關(guān)。大黃屬物種分化時(shí)間發(fā)生在360萬—679.6萬年前[29-30],唐古特大黃經(jīng)歷了漫長的進(jìn)化過程,保存了豐富的基因庫；且該物種為多年生草本植物,具有較長的生活史和復(fù)雜的異交繁育系統(tǒng),決定了該物種具有較高的基因擴(kuò)散和交流能力,確保了較高的遺傳多樣性[31]。

3.2 唐古特大黃遺傳結(jié)構(gòu)

唐古特大黃居群間存在強(qiáng)烈的遺傳分化。Buso等[32]認(rèn)為,當(dāng)GST>0.25時(shí),表明居群間存在極強(qiáng)的遺傳變異。本研究中,唐古特大黃居群GST為0.960,表明唐古特大黃居群間存在強(qiáng)烈的遺傳分化,其中95.97%的遺傳分化來源于居群間,4.03%的遺傳分化來自居群內(nèi)。AMOVA分析與DnaSP、PERMUT分析均取得了一致的結(jié)果,均表明遺傳分化主要來自于居群間。產(chǎn)生這種遺傳結(jié)構(gòu)的原因可能與其特殊的高山生態(tài)環(huán)境有關(guān)。唐古特大黃主要生長在青藏高原地區(qū),青藏高原的高山和深谷可能阻礙了花粉和種子的傳播,從而降低了居群間的基因流,導(dǎo)致居群間遺傳分化加大。唐古特大黃居群間較低的基因流(Nm=0.01)也證實(shí)了這一推論。另外,在經(jīng)濟(jì)利益的驅(qū)使下,人類對唐古特大黃野生資源過度采挖,也可能導(dǎo)致部分居群消失,造成某些等位基因的缺失,打破了居群間連續(xù)的基因交流,最終導(dǎo)致居群遺傳分化加劇。單倍型網(wǎng)絡(luò)圖分析結(jié)果驗(yàn)證了這一點(diǎn),4個(gè)單倍型的簡約網(wǎng)絡(luò)圖并未確定所有祖源單倍型,而是表明存在3個(gè)缺失的古老單倍型。

聚類分析結(jié)果表明,9個(gè)居群分為3組,居群間的遺傳距離與地理距離無顯著的正相關(guān)關(guān)系,地理相距較近的居群并沒有聚到一起,如西藏地區(qū)的居群4和居群9以及青海地區(qū)的6個(gè)居群分別聚到不同的分支(圖3),推測現(xiàn)存居群可能起源于不同的冰期避難所,支持唐古特大黃經(jīng)歷了冰期的退縮和間冰期的擴(kuò)張,在間冰期擴(kuò)張過程中發(fā)生了遺傳漂變,部分單倍型丟失,居群保留下來的單倍型由于來源不同,造成居群間的遺傳分化較大。遺憾的是根據(jù)本研究結(jié)果,尚無法得知唐古特大黃的避難所所在,增加數(shù)據(jù)量對該植物開展分子親緣地理學(xué)研究,可能會更好地揭示青藏高原地區(qū)第四紀(jì)冰期以來該植物居群變化的歷史過程。

3.3 唐古特大黃遺傳多樣性保護(hù)

遺傳變異是物種進(jìn)化和發(fā)展的基礎(chǔ),因此居群遺傳多樣性的保護(hù)是瀕危物種保護(hù)的基本要求。Hamrick和Bhagwat等指出,若某一居群遺傳多樣性占總遺傳多樣性比例達(dá)到99%,則建議該居群單獨(dú)保護(hù)以維持其較高的多樣性[24,33],否則應(yīng)當(dāng)對所有居群加以保護(hù)。基于以上調(diào)查結(jié)果,發(fā)現(xiàn)9個(gè)居群中遺傳多樣性最高的為Pop3(Hd=0.25,Pi=0.0026),占總遺傳多樣性比例為53.06%,未達(dá)到單獨(dú)保護(hù)的標(biāo)準(zhǔn)。另外,唐古特大黃遺傳多樣性主要存在于居群間(GST=0.960,FST=0.9631),而居群內(nèi)的遺傳多樣性較低(Hs=0.028),居群呈片段化存在,居群間的基因交流受阻(Nm=0.01),這種格局極易造成居群的退化甚至滅絕。綜合以上因素,提出如下建議：1)對唐古特大黃所有野生居群進(jìn)行就地保護(hù),包括一些遺傳多樣性程度不高,但擁有特殊等位基因的種群,維持居群數(shù)量,確保居群間的基因交流,維持物種的進(jìn)化潛力；為了避免遺傳多樣性高的小居群走向滅絕,如果就地保護(hù)不利的情況下建議遷地保護(hù)；2)采集和保存種子建立種質(zhì)資源庫,開展異地繁育等保護(hù)措施。以上這些保護(hù)策略將有利于增加唐古特大黃居群數(shù)量,并逐步擴(kuò)大其分布范圍。

4 結(jié)論

本研究基于葉綠體基因片段trnS-G 的序列變異對9個(gè)唐古特大黃野生居群的遺傳結(jié)構(gòu)進(jìn)行了分析,結(jié)果表明該物種具有較高的遺傳多樣性,遺傳分化主要來自于居群間,居群間的基因流較低。產(chǎn)生這種遺傳結(jié)構(gòu)的原因可能與唐古特大黃特殊的高山生境和人類活動的干擾有關(guān)。為保護(hù)唐古特大黃的遺傳多樣性,建議對其所有的野生居群進(jìn)行就地保護(hù),并采集和保存種子建立異地種質(zhì)資源庫,開展異地繁育等保護(hù)工作。

致謝：感謝劉建全教授在野外考察和材料收集過程中給予的指導(dǎo)和幫助。

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Geneticdiversityofanendangeredspecies,Rheumtanguticum(Polygonaceae),ontheQinghai-TibetanPlateau

WANG Ailan1, LI Weiwei2,*

1SchoolofLifeandSciences,LudongUniversity,Yantai264025,China2EditorialOfficeofLudongUniversityJournal,LudongUniversity,Yantai264025,China

Rheumtanguticum(Polygonaceae), an endangered traditional Chinese and Tibetan medicines, is distributed in the Tibetan Plateau. To investigate endangered reasons and protect the wild resources, we collected 87 individuals fromnine populations ofR.tanguticum, and estimated the genetic diversity of this speciesbased on the cpDNAtrnS-G region.The results showed that there was high genetic diversity within this species. The length of the totaltrnS-G region of the 87 individuals ranged from 944 to 954bp, and covered 965 nucleotide positions after being aligned. Variations were detected in 23 nucleotides, including 13 nucleotide substitutions and 10 indel sites. On the basis of these nucleotide mutations, a total of four nucleotide haplotypes (H1—H4) were identified. The development of a parsimony network showed that the four haplotypes made up a shallow star genetic tree. Haplotypes H1, H2, and H4 were located at the tips of the network, representing recent haplotypes.Haplotype H3 and three missing haplotypes were in the center of the star network.Moreover,since haplotype H3 had the highest frequency, it was identified as an ancestral haplotype.The total diversity (Ht) among the individuals collected was 0.694, indicating a high genetic diversity. In addition, a high level of population differentiation was observed (GST=0.960), as determined by PERMUT analysis, showing 95.97% genetic differentiation between populations, whereas the remainder of the total genetic variation (4.03%) was attributed to within-population diversity (Hs=0.028). AMOVA analysis (FST=0.9631) also indicated that the genetic diversity among populations was greater than that within populations. The value for gene flow was fairly low (Nm=0.01), indicating a limited gene flow between populations, which is consistent with the findings of PERMUT and AMOVA analysis.The NJ tree of populations based on genetic distance revealed three supported groups. However, populations with close geographic distance did not cluster together, indicating that there was no direct relationship between the geographic distances and genetic distances of the nine populations, which was confirmed by the Mantel test(r=0.2461,p=0.2060). The high genetic diversity of the examined populations is probably associated with the long evolutionary history and life history ofR.tanguticum.The large genetic differentiation between populations might be related tothe alpine geographical environment and human activity. Therefore, we suggest that more wild populations with high genetic diversity should be protected by an in-situ conservation program at sites distant from human activity. We also recommend the collection and conservation of seeds from populations with rich genetic diversity for the ex-situ conservation of genetic stocks and their propagation. These conservation measures will certainly provide the basic resources required to increase the number of populations of this species and extend its range of distribution.

Rheumtanguticum;trnS-G region; genetic diversity; gene flow

國家自然科學(xué)基金項(xiàng)目(31000104)

2016- 08- 09; < class="emphasis_bold">網(wǎng)絡(luò)出版日期

日期:2017- 07- 11

*通訊作者Corresponding author.E-mail: lwwal@163.com

10.5846/stxb201608091635

王愛蘭,李維衛(wèi).青藏高原瀕危植物唐古特大黃的遺傳多樣性.生態(tài)學(xué)報(bào),2017,37(21):7251- 7257.

Wang A L, Li W W.Genetic diversity of an endangered species,Rheumtanguticum(Polygonaceae), on the Qinghai-Tibetan Plateau.Acta Ecologica Sinica,2017,37(21):7251- 7257.