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Volume 35 Issue 4
Jul.  2022
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Genomic Characteristics and Population Genetic Variation of Dalbergia cultrata Graham ex Benth in China

  • Corresponding author: HUANG Ping, huangping@caf.ac.cn
  • Received Date: 2022-02-22
    Accepted Date: 2022-03-14
  • Objective To analyze the characterization of Dalberiga cultrata genome, and develop a set of high polymorphic SSR molecular markers for assessment of genetic variation of wild population. Method Next Generation Sequencing (NGS) was performed to evaluate the characteristic of genome, and MISA was used to mine candidate genomic SSR loci from the assembled data. The franking primers of candidate SSR loci were designed by Primer Premier v 5.0, and screened by polyacrylamide gel electrophoresis (PAGE). The polymorphic and population genetic analysis was performed by capillary electrophoresis (CE). Result The genome size of D. cultrata was about 706.92 Mb and the heterozygosity was about 1.26%. The rate of repetitive sequence was 55.74%. A total of 27 polymorphic SSR loci were screened and117 alleles were amplified on 27 SSR loci. The value of the polymorphism information content (PIC) varied from 0.149 to 0.803. The population genetic analysis showed the mean expected heterozygosity (He) and the coefficient of genetic differentiation (FST) was 0.504 and 0.034, respectively. AMOVA analysis revealed the genetic variation within the populations (96.54%) was much higher than that among the populations (3.46%). Conclusion The genome of D. cultrata belongs to high heterozygosity and highly repetitive complex genome, which provides important basic data to make a fine assembly strategy. A set of novel genomic SSR markers shows good polymorphism, and stability. Moderate genetic diversity and low genetic differentiation of wild populations of D. cultrata are revealed by SSR markers. This study would facilitate to conserve and assess wild germplasms of D. cultrata.
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Genomic Characteristics and Population Genetic Variation of Dalbergia cultrata Graham ex Benth in China

    Corresponding author: HUANG Ping, huangping@caf.ac.cn
  • 1. State Key Laboratory of Tree Genetics and Breeding; Research Institute of Forestry, Chinese Academy of Forestry; Laboratory of Forest Silviculture and Tree Cultivation, Beijing 100091, China
  • 2. Wenzhou Key Laboratory of Resource Plant Innovation and Utilization, Zhejiang Institute of Subtropical Crops, Zhejiang Academy of Agricultural Sciences, Wenzhou 325005, Zhejiang, China

Abstract:  Objective To analyze the characterization of Dalberiga cultrata genome, and develop a set of high polymorphic SSR molecular markers for assessment of genetic variation of wild population. Method Next Generation Sequencing (NGS) was performed to evaluate the characteristic of genome, and MISA was used to mine candidate genomic SSR loci from the assembled data. The franking primers of candidate SSR loci were designed by Primer Premier v 5.0, and screened by polyacrylamide gel electrophoresis (PAGE). The polymorphic and population genetic analysis was performed by capillary electrophoresis (CE). Result The genome size of D. cultrata was about 706.92 Mb and the heterozygosity was about 1.26%. The rate of repetitive sequence was 55.74%. A total of 27 polymorphic SSR loci were screened and117 alleles were amplified on 27 SSR loci. The value of the polymorphism information content (PIC) varied from 0.149 to 0.803. The population genetic analysis showed the mean expected heterozygosity (He) and the coefficient of genetic differentiation (FST) was 0.504 and 0.034, respectively. AMOVA analysis revealed the genetic variation within the populations (96.54%) was much higher than that among the populations (3.46%). Conclusion The genome of D. cultrata belongs to high heterozygosity and highly repetitive complex genome, which provides important basic data to make a fine assembly strategy. A set of novel genomic SSR markers shows good polymorphism, and stability. Moderate genetic diversity and low genetic differentiation of wild populations of D. cultrata are revealed by SSR markers. This study would facilitate to conserve and assess wild germplasms of D. cultrata.

  • 刀状黑黄檀(Dalbergia cultrata Graham ex Benth)隶属于豆科(Fabaceae)黄檀属(Dalbergia),高大落叶乔木,其主要分布区位于中国云南省南部以及相毗邻的中南半岛的热带与亚热带地区[1]。刀状黑黄檀是珍贵的红木树种,由于其心材呈黑褐色,新切面具有酸香气味,新切面紫、黑或栗褐,常带紫或黑褐窄条纹,在中国《红木》(GB/T 18107-2017)国家标准中被归类于黑酸枝木系,是制作高档家具、乐器以及工艺品的优质材料;同时,心材和根可入药,具有较好的药用价值[2];刀状黑黄檀根系发达,对土壤要求不高,且具有良好的抗病、抗虫等特性,是困难立地生态修复的优良树种[3]。以往研究主要关注了刀状黑黄檀的野生资源分布以及气候变化对其影响、育苗方法、木材内含物化学成分等方面[3-6]。刀状黑黄檀心材形成周期长导致了突出的供需矛盾,也造成了野生资源的过度开发,而经济社会高速发展过程中的土地利用转变以及气候变化的风险可能进一步挤压其原生栖息地范围,致使野生资源数量进一步减少,野生资源正在遭受遗传侵蚀的风险[4]。尽管刀状黑黄檀早已被列入IUCN(International Union for Conservation of Nature)红色名录(近危级,Near threatened,NT)以及《中国高等植物受威胁物种名录》(易危级,Vulnerable,VU A2c)[7],但是关于该物种种群遗传保护研究尚未见报道,也缺少相关背景基因组信息以及物种特异性的遗传标记。

    分子标记是有效量化和科学评价种群遗传变异程度的分析工具,被广泛应用于遗传图谱构建、数量性状定位、遗传多样性与遗传结构评价等方面[8]。SSR分子标记是群体遗传学研究中最常用的DNA分子标记之一,随着NGS测序技术的发展与成本降低,基于测序数据,通过生物信息学分析方法可快速、高效的发掘出大量的潜在SSR标记位点,进一步结合分子实验筛选验证则可对多态性基因组SSR标记进行鉴定,在林草遗传分析中已有诸多应用,如元宝枫(Acer truncatum Bunge)、象草(Pennisetum purpureum Schum.)和开心果(Pistacia vera L.)等[9-11]

    为了掌握刀状黑黄檀的遗传信息,开发一套可用于遗传分析的适宜分子标记,本研究基于NGS测序技术对刀状黑黄檀基因组的基本特征进行了初步评估,通过实验筛选开发了一套重复性高、多态性好的SSR标记,并在其野生种群中进行了初步验证与评价分析。研究结果不仅可为刀状黑黄檀群体遗传学研究提供可靠分析工具,而且为进一步深入开展种质资源收集保存、分析评价提供科学依据。

    • 基因组测序试验材料:2018年3月在云南省普洱市六顺乡(LSX)采集刀状黑黄檀成熟种子,采种母树凭证标本存放于中国林业科学研究院林业研究所。于当年在中国林科院科研温室播种育苗,育苗基质为草炭土与蛭石(3:1),常规水肥管理,选择长势良好且健康的植株,采集新鲜叶片,液氮保存,用于基因组DNA提取。

      SSR分子标记筛选试验材料:选择云南省普洱市六顺乡(LSX)、江城(JC),西双版纳傣族自治州景洪(JH)等3个地点进行采样,采样地理环境信息见表1。每个采样点随机选择30个个体,共计90个样品,不同个体间距离大于50 m。采集新鲜叶片,经硅胶完全干燥后,放置于−20 ℃冰箱保存,用于DNA提取。

      序号
      No.
      群体
      Populations
      代码
      Code
      经度(E)
      Longitude
      纬度(N)
      Latitude
      海拔
      Altitude/m
      年平均温度
      Annual mean
      temperature/℃
      年平均降水量
      Annual mean
      precipitation/mm
      1六顺乡 LiushunxiangLSX100°47′22°40′800~1 25019.91 460.4
      2江城 JiangchengJC101°43′22°33′800~1 25020.31 783.0
      3景洪 JinghongJH100°31′21°59′600~1 35020.21 450.3

      Table 1.  Sampling information of D. cultrata

    • 采用CTAB法[12]从叶片样品中提取基因组DNA,分别用1.0%琼脂糖凝胶电泳和ScanDrop 100分光光度计(Molecular Device, California, United States)检测DNA浓度及质量。利用illumina HiSeq TM 2500测序平台(illumina, San Francisco, United States),基于标准规程构建一个插入片段大小为300 bp的文库。基于K-mer分析,估计基因组大小、杂合度以及重复序列比率[13]

      参考Luo等的方法[14],利用SOAPdenovo v 2.04软件进行基因组初步组装。构建Contig序列(K-mer=17)并进一步组装为Scaffold,使用GapCloser v 1.12-r6修补Scaffold内部缺口。以每10 kb作为1个窗口,进行非重叠滑窗统计,计算每个窗口的平均深度与GC含量,获得GC-depth点图,进而估计基因组GC含量。

    • 采用MISA(http://pgrc.ipk-gatersleben.de/misa/misa.html)对初步组装Scaffold进行潜在SSR位点检索,挖掘候选SSR标记。参数设置为:二至六核苷酸基序重复单元最小重复次数值分别设置为6、5、4、4、4。利用Primer Premier v 5.0软件设计引物,引物参数分别为:引物长度18~22 bp,产物大小100~300 bp,退火温度55~62 ℃,最优值为60 ℃。随机挑选300对引物,由睿博兴科生物公司(北京)合成。

    • 从六顺乡(LSX)、江城(JC)和景洪(JH)3个种群90个样品中随机挑选出4个样品对300对引物进行琼脂糖凝胶电泳初筛,选择扩增条带清晰,稳定性良好的引物进行复筛。复筛时在90个样品中随机挑选8个样品进行8.00%非变性聚丙烯酰胺凝胶电泳检测,扩增产物条带明亮、清晰,无背景噪音,即复筛合格引物。然后,利用荧光毛细管电泳检测候选SSR位点多态性,最后利用多态性SSR位点分析3个种群(90个个体)遗传变异程度,初步评估SSR位点适用性以及种群遗传变异程度。

      PCR反应体系为:基因组DNA 50 ng,2 × Taq MasterMix(Dye)(康为世纪,北京)12.5 µL,正向引物(10 μmol·L−1)0.5 µL,反向引物(10 μmol·L−1)0.5 µL,用ddH2O补至25 µL。PCR程序为:96 ℃预变性3 min,然后96 ℃变性30 s,60 ℃退火30 s,72 ℃延伸45 s,共30个循环,最后72 ℃延伸7 min。

      利用GeneMaker v 2.2.0对SSR位点进行基因分型,并进行二次人工复核。

    • 采用GenAlEx v 6.502[15]计算下列群体遗传参数,包括观测等位基因数(Na),有效等位基因数(Ne),观测杂合度(Ho),期望杂合度(He),Shannon's信息指数(I),种群间遗传分化系数(FST),哈温平衡偏离显著性(HWE(p))等。采用GeneALEx v 6.502[15]进行分子方差分析(AMOVA)以检测遗传变异来源。采用CERVUS v 3.0.4[16]计算位点多态性信息含量(PIC)。

    2.   结果与分析
    • 基于illumina HiSeq TM 2500平台测序共生成94.52 Gb高质量数据,覆盖度约为基因组的137倍。测序质量值Q20和Q30分别为96.24%和90.23%,表明测序质量良好,可用于后续分析。使用SOAPdenovo软件进行组装和拼接,选择K-mer为17,初步组装基因组,共获得463 258个Scaffold,Scaffold N50长度约为2 381 bp(表2)。将所有高质量数据用于K-mer分析,K-mer分布曲线结果显示:K-mer期望深度(主峰)对应的测序深度为126 × ,GenomeScope估计基因组约为706.92 Mb,基因组杂合度为1.26%,重复序列比率为55.74%,平均GC含量为34.11%。

      组装信息
      Information of assembly
      长序列片段
      Scaffold
      重叠群
      Contig
      拼接序列数量 Number of sequences 463 258 720 549
      基因组大小 Total length/bp 777 543 915 776 970 961
      平均长度 Average Length/bp 1 678.43 1 078.30
      最大长度 Maximum Length/bp 96 195 96 195
      N50长度 N50 Length/bp 2 381 1 576
      N90长度 N90 Length/bp 692 514

      Table 2.  Information of the assembled genome sequences of D. cultrata

    • 利用MISA进行SSR候选位点检索,从463 258个Scaffold中总共鉴定出142 891个SSR标记(表3),其中,二核苷酸(Di-)位点数量最多,占总数的61.21%,主要重复类型为AT/AT,占54.75%;三核苷酸(Tri-)次之(24.88%),主要类型为AAT/ATT,占54.23%;四核苷酸(Tetra-)占比为7.45%,主要类型为AAAT/ATTT,占比为68.11%;五核苷酸(Penta-)占比为1.11%,主要类型为AAAAT/ATTTT。

      SSR重复基序
      SSR repeat motif
      SSR数量
      Number of SSR
      频率
      Frequency/%
      主要类型
      Main type
      二核苷酸 Dinucleotide 87 464 61.21 AT/AT
      三核苷酸 Trinucleotide 35 558 24.88 AAT/ATT
      四核苷酸 Tetranucleotide 10 651 7.45 AAAT/ATTT
      五核苷酸 Pentanucleotide 1 593 1.11 AAAAT/ATTTT
      其它 Others 7 625 5.34
      总计 Total 142 891

      Table 3.  Characteristic and distribution of SSR in D. cultrata genome

    • 琼脂糖凝胶电泳初筛结果显示:128对引物具有清晰稳定的扩增条带,占检测引物比例为42.67%;经过8.00%非变性聚丙烯酰胺凝胶复筛检测,最终共筛选了27对扩增稳定,条带清晰明亮,多态性良好的引物,约占检测引物总数的9.00%,引物详细信息见表4

      位点
      Locus
      重复单元
      Repeat Motif
      正向引物序列
      Forward Primer
      反向引物序列
      Reverse Primer
      产物大小
      Product Size/bp
      退火温度
      Tm/(℃)
      HHT5(TTAT)4GCAGCCAACCTATGTTCCATTAAGTGCTGAGTGATTGCGG18060
      HHT10(CCTGG)4GGCCAGTTTGCATAAACCATAGTCAGGGCACTGAAGGAGA19560
      HHT14(CAC)7GACCTCGACAAGCTCAAAGCCAGCCCAATAAGTTGTCGGT25460
      HHT23(AAAAT)4GATCGGGCTCGTGGTTAATAATCTTCGGGCCATTTTCTTT16160
      HHT45(AGAAAA)4TCCATCATCCGTCACAAAGAAATGCTGTGCTTTCCAGGTC27660
      HHT49(AAT)12CAGAAGCCAGAAACGTCGTAGGGGCCGAAAGAAAAGATAG18660
      HHT60(CTAT)5CCACTACTGCTCCTTCGCATACAACGTACGTAGGCAACCC16060
      HHT62(AAC)7CGCCCTTCTCCATATTCAAAGGAAATTGGTTGGCTTCGTA27760
      HHT85(TC)10ACACGGCACACATTCTCGTACGCTCCATGAAGGAAGAAAG23460
      HHT97(GCA)11CCACAACCAGCAACAACAACAAGTCCTTCATCATCCCGTG22360
      HHT117(ATT)7ATAAGTATGTGCCGGCATGGGAGATGGATGCATCGGTAAAA25360
      HHT119(AC)10TCATGGTACTGTCATCCCCACTCAAGGCTGACCAAAGGAG25560
      HHT121(AT)10GCTTGAGGAGATGGTGAAGGCCAAATTTCGGTAATGACAAGA27959
      HHT124(GA)10GGAAATGCCCACACAAGAGTTGACACATGTGGGATATGGA18758
      HHT126(TG)10CAGATGATCCAACCCAATCATACCAAACCCAAACCCAATC24260
      HHT160(CT)11TCAAATCGCAAATGGCAATAGGAATACGACGTCATGCAAA18860
      HHT161(AC)10GACACAGGAAAAGGAAGATGGGTGGAGCCGACAAGAAATGT18560
      HHT211(TTTTA)4ATATCATTTCCCCACAGCCACCAAAAACAACAAGCTGGAA22159
      HHT216(AAAAC)4AGGGCTGCAAAAAGTGAAGAGGTTGTAGCCGAGGAAAGAA26359
      HHT219(AATAAA)4TGGTGAAACTGGTCCATGAGTTTGACCGCATGTTACCTGA23660
      HHT223(ATTAG)4CCGCAAAATATATGACGGCTGAGAGAGGCACATTTGACCG20361
      HHT224(AAAAT)4AGCCCAATCCTTTTCTTCGTATTGCAATACATGGACCCGT26560
      HHT226(TTTTA)4TGTCTCGCTCTCGCAGATAACAACCGCCAAAATTACATGA27159
      HHT229(ATTTT)4TGGAGGGTGTTTTGTTGCTTGCTGGAGAAGAGGTCATTGC27960
      HHT230(TTTGCA)4CTTACGAGTGGGAATGGGAAAAAAGAGATGGGGCGAAGAT21460
      HHT247(TTGAG)4GAAGTAGGCAGGAAGCATCGATTAGCCGGTTGTGGTTTTG19960
      HHT255(CAAACA)5TATGATGGCTTTTGGGGAGACCGGTGTTGTTGTTGCTATG27360

      Table 4.  Characteristics of flanking primers of 27 SSR markers developed for D. cultrata

      利用筛选后具有多态性的SSR位点对3个种群的90个刀状黑黄檀个体进行SSR基因分型(图1)。检测结果显示:在27个SSR位点上共检测到117个等位基因,观测等位基因数(Na)最少为2个,最多为10个,均值为4.333;有效等位基因数(Ne)最少为1.193,最多为5.757,均值为2.397。位点水平的观测杂合度(Ho)最小值为0.111(HHT226),最大值为0.833(HHT119),均值为0.451;期望杂合度(He)最小值为0.162(HHT226),最大值为0.826(HHT121),均值为0.523。多态性信息含量(PIC)最小值为0.149(HHT226),最大值为0.803(HHT121),均值为0.463,其中,11个位点具有高多态性(PIC>0.5),13个位点具有中等多态性(0.25<PIC≤0.5),仅有HHT23、HHT224和HHT226等3个位点多态性低于0.25。Shannon's信息指数(I)的平均值为0.957,其中,最小值为0.300,最大值为1.894。哈温平衡(HWE)检验结果显示:HHT5、HHT10等8个位点符合哈温平衡(表5)。

      Figure 1.  Fluorescence capillary electrophoresis test results of part of SSR loci

      位点
      Locus
      观测等位基因
      Na
      有效等位基因
      Ne
      观测杂合度
      Ho
      期望杂合度
      He
      多态性信息含量
      PIC
      群体间遗传分化系数
      FST
      Shannon’s信息指数
      I
      哈温平衡
      HWE(p
      HHT53.0002.0320.3670.5080.3900.1150.7430.058
      HHT103.0001.9700.4380.4920.3870.0080.7420.539
      HHT144.0002.6270.4770.6190.5590.0561.1160.000**
      HHT233.0001.2820.1700.2200.1980.0310.4000.012*
      HHT453.0002.2720.5330.5600.4890.0040.9360.290
      HHT496.0002.8550.4120.6500.5900.0601.2450.000**
      HHT604.0002.2030.4440.5460.4670.0220.9290.151
      HHT622.0001.9790.6020.4950.3720.0210.6880.042*
      HHT859.0004.0140.7190.7510.7160.0271.6970.000**
      HHT976.0004.4420.7220.7750.7420.0251.6180.000**
      HHT1173.0001.7950.2330.4430.3970.0330.7800.000**
      HHT1196.0003.3110.8330.6980.6510.0451.3820.000**
      HHT12110.0005.7570.5730.8260.8030.0821.8940.000**
      HHT1246.0002.5580.3440.6090.5620.0301.1660.000**
      HHT1268.0001.9330.2020.4830.4670.0341.1240.000**
      HHT1608.0001.9130.4670.4770.4380.0241.0010.999
      HHR1615.0002.9140.6670.6570.5970.0261.2380.000**
      HHT2113.0002.3430.5840.5730.5040.0230.9600.089
      HHT2163.0002.0470.5340.5110.4110.0220.7960.222
      HHT2192.0001.6360.5060.3890.3130.0080.5770.005**
      HHT2234.0002.0480.5170.5120.4430.0140.9010.066
      HHT2242.0001.2900.1460.2250.2000.0030.3850.001**
      HHT2262.0001.1930.1110.1620.1490.0510.3000.003**
      HHT2292.0001.6000.2950.3750.3050.1030.5620.047*
      HHT2304.0002.6840.4090.6270.5500.0231.0680.000**
      HHT2472.0001.4680.3980.3190.2680.0020.4990.020*
      HHT2554.0002.5560.4770.6090.5420.0111.0890.005**
      均值Mean4.3332.3970.4510.5230.4630.0340.957--
      注:“*”表明显著性水平(*p < 0.05,**p < 0.01)。
        Notes:“*”Indicates significant levels (*p < 0.05, **p < 0.01).

      Table 5.  Characterization of locus-level polymorphic information at 27 SSR loci

    • 在3个野生种群中检验了27个SSR位点在群体遗传分析中有效性与适用性,结果(表6)显示:在六顺乡(LSX)、江城(JC)以及景洪(JH)种群中,观测等位基因(Na)数量分别为3.407、3.444、3.741;有效等位基因(Ne)数量分别为2.194、2.225、2.453。在3个种群中,具有中高度多态性的SSR位点分别占比85.19%(LSX)、88.89%(JC)、85.19%(JH)。种群水平的期望杂合度(He)分别为0.491(LSX)、0.493(JC)、0.528(JH),均值为0.504;观测杂合度(Ho)分别为0.472(LSX)、0.416(JC)、0.461(JH),均值为0.450,结果表明3个刀状黑黄檀野生种群具有中等的遗传多样性。

      群体
      Population
      观测等位基因
      Na
      有效等位基因
      Ne
      Shannon's信息指数
      I
      观测杂合度
      Ho
      期望杂合度
      He
      六顺乡 LSX3.4072.1940.8590.4720.491
      江城 JC3.4442.2250.8620.4160.493
      景洪 JH3.7412.4530.9490.4610.528
      均值 Mean3.5312.2910.8900.4500.504

      Table 6.  Genetic diversity of 3 wild populations of D.cultrata in China

      FST值可用于评价种群间遗传分化程度,3个种群之间的遗传分化系数(FST)为0.002~0.115,平均值为0.034(表5)。AMOVA分析结果显示:刀状黑黄檀种群内遗传变异和种群间遗传变异分别为96.54%和3.46%,这表明刀状黑黄檀遗传变异主要来源于种群内(表7)。

      变异来源
      Source of variation
      自由度
      Degree of freedom
      方差和
      Sum of squares
      变异组分
      Variance component
      占变异的比例
      Proportion in total variation/%
      种群间 Among population
      247.52223.7613.46
      种群内 Within population
      1771 263.41714.31796.54
      总计 Total
      179100.00

      Table 7.  AMOVA analysis among and within D.cultrata populations

    3.   讨论
    • 自Galbraith等[17]利用流式细胞术测定植物细胞核DNA含量以来,流式细胞术成为了测定植物基因组大小的标准方法。随着高通量测序技术的发展,基于K-mer分析的生物信息学估计基因组特征方法的出现,对于探索未知基因组物种提供了极大帮助,已经广泛应用于植物基因组大小评估和特征分析[18],并且K-mer分析相较于流式细胞术分析法的预测结果可信度更高、信息更丰富。以往普遍认为黄檀属植物的基因组可能比较大,在DNA-C值数据库中黄檀属植物基因组(1C)大小范围为1 430~1 928 Mb,平均值为1 729 Mb[19]。然而,在本研究中,基于K-mer分析结果估计刀状黑黄檀的基因组大小约为706.92 Mb,基因组杂合度为1.26%,重复序列比率为55.74%。这一结果表明,其基因组属于高杂合、高重复的复杂基因组,基因组大小略高于降香黄檀(Dalbergia odorifera T. Chen)的653.45 Mb[20],这可能是由于物种差异以及较高的基因组杂合度等因素共同导致的[21]。刀状黑黄檀的基因组高杂合性可能与该物种复杂的分化过程有关,Vatanparast等[22]基于分子证据认为,黄檀属起源于南美洲,但其只用了ITS片段,结果缺乏有力支撑;崔菲等[23]利用核基因和叶绿体片段构建了系统发育树,但基部类群也并未明确,暂未确定黄檀属的明确起源地区与分化趋势。然而,从基因组水平来解读物种系统演化的过程将是未来植物分类与系统进化研究发展的趋势。因此,对于刀状黑黄檀基因组特征初步分析和评价有利于制定合适的基因组精细组装策略,选用最新的三代Pacbio测序平台HiFi模式测序可能对于解决高杂合度在基因组组装时所产生的不利影响具有较大帮助,结合Hi-C技术和三代测序平台的全长转录组分析,将有助于基因组染色体水平的组装并获得更加精确的基因组注释信息。

    • SSR分子标记是保护遗传学中最常用的DNA标记之一,与RFLP、AFLP和RAPD相比,SSR标记具有信息丰富、易于判定、稳定和共显性的特点[24]。以往研究也发现,在遗传分析方面,基因组SSR相比于EST-SSR以及SNP具有更高的多态性[25]。刀状黑黄檀基因组包含了大量的SSR分子标记位点,平均5 442 bp存在1个,且重复类型丰富,变异基序多样,以双、三核苷酸重复基序的占比较高。以往研究认为,重复单元长度变化与选择压力有关,重复单元长度越长,所受的选择压力越大,拷贝数就越少,因此,基因组中长度较短的微卫星变异速率较快,而较长的重复单元变异速率较慢,所以,基因组中低级重复单元较多则表示该物种进化水平较高,而高级重复单元比例高的物种其进化时间短或变异频率较低[26-27]

    • 本研究基于NGS测序和初步组装数据发掘了刀状黑黄檀基因组SSR候选位点,并结合实验室筛选,验证了27个多态性较好,扩增稳定的SSR位点。位点水平的观测等位基因数量(Na)变异范围为2~10个,均值为4.333,这也表明筛选验证的位点具有较好的等位基因变异,其中,超过80%以上的SSR位点具有中高度的多态性信息含量。研究发现,27个SSR位点中仅有8个位点符合哈温平衡,类似的状况在生态位较为接近的降香黄檀种群中也有所体现,同样具有一定比率的SSR位点不符合哈温平衡[28]。符合哈温平衡需要满足随机交配且足够大的种群数量,而在资源调查中发现,刀状黑黄檀的野生种群均为零星分布,且遭遇了严重的生境片段化,几乎没有连续分布的刀状黑黄檀纯林[4],因此,种群有效个体数量偏低,非随机交配等因素可能是造成SSR标记位点偏离哈温平衡的主要原因。

    • 以3个野生种群为对象,对刀状黑黄檀遗传多样性进行了初步分析与评价,结果显示3个种群的期望杂合度(He)均值为0.504,明显低于异交植物的平均值0.65[29],通过与其他黄檀属内植物遗传多样性比较发现(表8),3个刀状黑黄檀野生种群的期望杂合度明显高于海南分布的降香黄檀(He = 0.370)[28],但低于泰国、老挝等地分布的交趾黄檀(Dalbergia cochinchinensis Pierre)(He = 0.550 & uHe = 0.570)以及柬埔寨和越南等地分布的奥氏黄檀(Dalbergia oliveri Gamble ex Prain)(He = 0.730)[30-31]和南美洲巴西热带地区分布的巴西黑黄檀(Dalbergia nigra (Vell.) Benth.)(He = 0.735 & He = 0.682)[32-33]。比较结果表明:中国野生分布的刀状黑黄檀种群遗传多样性处于较低的水平,这也暗示中国分布的刀状黑黄檀野生资源遭受了明显的遗传侵蚀,这可能是由于过去的过度砍伐与土地利用转变等因素造成的。在IUCN红色名录中,交趾黄檀(VU, A1cd)和降香黄檀(VU, A1d)濒危等级都高于刀状黑黄檀(NT级别),在中国受威胁高等植物名录中,刀状黑黄檀被列为VU级,从野生种群遗传多样性水平角度而言,本研究结果也支持将刀状黑黄檀的濒危等级提高到与交趾黄檀和降香黄檀相同的级别。尽管刀状黑黄檀已经被列入国家二级保护植物,但在野外调查研究中发现,土地利用转变导致的生境片段化也可能影响其野生种群的稳定性及限制种群间的基因交流与种群内的繁殖更新[3]。因此,今后的工作应将全面深入开展遗传多样性评估、繁殖生物学及景观遗传学等方面的研究作为重点方向,用于制定科学合理的资源保护与种群恢复策略。

      物种名
      Species name
      拉丁名
      Scientific
      name
      分布区域
      Distribution
      种群数量
      No. of
      populations
      样本数量
      No. of
      individuals
      标记类型
      Type of
      marker
      群体遗传参数
      Genetic
      Parameters
      参考文献
      Reference
      刀状黑黄檀Dalbergia cultrata中国云南等,热带390SSRHe = 0.504本研究
      降香黄檀Dalbergia odorifera中国海南,热带742SSRHe = 0.370[28]
      交趾黄檀Dalbergia. cochinchinensis泰国、老挝、
      柬埔寨等,热带
      26677SSRHe = 0.550[30]
      uHe = 0.570[31]
      奥氏黄檀Dalbergia oliveri老挝、柬埔寨和
      越南等,热带
      23616SSRHe = 0.730[30]
      巴西黑黄檀Dalbergia nigra巴西,热带3235SSRHe = 0.735[32]
      1107He = 0.682[33]
      黄檀Dalbergia hupeana Hance中国四川和陕西,
      亚热带
      390SSRHe =0.659~0.740,
      uHe = 0.670~0.752
      [34]
      多体蕊黄檀Dalbergia polyadelpha Prain中国云南,热带129SSRHe = 0.512, uHe = 0.522[34]
      南岭黄檀Dalbergia balansae Prain中国云南,热带129SSRHe = 0.635, uHe = 0.648[34]

      Table 8.  Comparative analysis of genetic diversity in Dalbergia genus

    4.   结论
    • 本研究利用NGS技术对刀状黑黄檀基因组特征进行了分析,结果表明,其基因组大小约为706.92 Mb,杂合度为1.26%,属于高杂合的复杂基因组,提出了基于PacBio三代测序和Hi-C辅助组装的精细测序组装策略。通过实验发掘并验证了27个扩增条带清晰、多态性良好的SSR位点,并初步评估了3个野生刀状黑黄檀种群的遗传多样性,结果表明其具有中等水平的遗传多样性和较低的遗传分化程度。

Reference (34)

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