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杜鹃花属(Rhododendron)包含近1 000个物种,是杜鹃花科(Ericaceae)最大的属,北温带植物区系中的大属,主产于东亚和东南亚,是重要的园林绿化植物[1-3]。锦绣杜鹃(Rhododendron pulchrum Planch.)是常用的栽培种常绿杜鹃,品种繁多,是杜鹃花园艺分类中毛鹃的代表,优良的田园植物,主要产于广东、广西、湖北、湖南、江西、福建、浙江、江苏等地[4]。目前,锦绣杜鹃中缺乏特异性微卫星标记和遗传连锁图谱,其遗传改良和优良品种选育又是杜鹃花产业的重要任务,因此,筛选可靠的分子标记评价种质资源、遗传多样性分析、分子标记辅助育种等对杜鹃花良种选育和种质资源保护意义重大。
简单重复序列(SSR),又称微卫星,包括基因组SSR和表达序列标签SSR(EST-SSR),是1~6个核苷酸重复基元组成的短串联重复序列,因其具有共显性、多态性强、重复性好、分布广泛等特点而在花卉遗传学中被广泛应用[5-6]。近年来,基于二代测序平台建立的转录组测序技术不仅能够挖掘特定组织在特定时期的表达基因,也具有快速、高效、高通量开发分子标记的优点,已经广泛用于萝卜(Raphanus sativus L.)、大白菜(Brassica rapa L. ssp. pekinensis)、康乃馨(Dianthus caryophyllus L.)、大蒜(Allium sativum L.)、砂梨(Pyrus pyrifolia Nakai.)等经济作物的SSR标记开发等研究[7-11]。
杜鹃属现有的SSR标记非常有限,很难满足杜鹃花遗传多样性和群体结构研究、遗传改良及新品种选育的需要。因此,本研究对锦绣杜鹃花蕾组织进行转录组测序,基于组装的unigenes分析SSR的组成、频率和分布特征,开发SSR引物,同时对SSR标记的多态性和跨物种扩增效率进行评价。本研究基于微卫星标记特征,从批量开发合成的856对引物中筛选多态性强的SSR引物进行有效性和跨物种转移的验证,以期为后续杜鹃花种质资源的遗传多样性研究、遗传图谱构建、基因定位、遗传改良、分子标记辅助新品种选育等研究奠定基础。
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2016年3月初自黄冈师范学院植物资源圃采摘锦绣杜鹃的花蕾组织(表 1),液氮冻存后置于-80 ℃冰箱,供后续总RNA提取和转录组测序。2016年5月至2016年9月分别自黄冈市遗爱湖公园和大别山主峰五脑山采集栽培的锦绣杜鹃群体和野生的映山红群体,摘取顶端健康叶片,用于基因组DNA的提取和遗传多样性检测(表 1)。样品采集按照种群遗传学的原理和方法,遵循代表性、可对照性原则取样(样本间距1~1.96 m)。
表 1 转录组测序和多样性检测材料采集地信息
Table 1. Information of sampling populations used for RNA-seq and genetic diversity analysis
种质
Germplasm样品来源
Sample source经度
Longitude(E)纬度
Latitude(N)海拔
Altitude/m样品个数
Numbers of sample锦绣杜鹃 黄冈师范学院资源圃 114°55′22.62″ 30°26′56.82″ 22 3 锦绣杜鹃 黄冈遗爱湖公园 114°53′28.21″ 30°27′21.67″ 21 30 映山红 大别山主峰五脑山 114°59′57.47″ 31°14′10.95″ 104 30 -
将3份锦绣杜鹃花蕾组织混合,采用高盐Trizol试剂和RNA结合柱的方法提取总RNA,重复3次。采用链特异性纯化试剂盒对总RNA进行mRNA Oligo(dT)磁珠富集、双链cDNA合成、末端修复、接头连接,再用USER酶对cDNA第二条链进行降解,保留真实转录的mRNA第一链,随后进行PCR扩增和2%琼脂糖凝胶电泳检测,回收300~500 bp的片段作为测序文库。采用Agilent 2100 Bioanalyzer和ABI StepOnePlus Real-time PCRSystem对文库进行质检,合格后使用IlluminaHise2500 PE125平台进行测序(2×150 bp),数据过滤采用FASTX (http://hannonlab.cshl.edu/fastx_toolkit/)和CUTADAPT (https://pypi.python.org/pypi/cutadapt/1.4.2)软件,采用Cufflinks软件进行数据组装。本研究的转录组测序数据已经上传到NCBI数据库。
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利用MISA (http://pgrc.ipk-gatersleben.de/misa)软件对组装的unigene序列内部的SSR位点进行检测。单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸、六核苷酸最少重复次数分别设置为10、7、5、5、5、5次。对含有SSR位点且侧翼序列大于150 bp的unigenes进行筛选,用于SSR引物的设计。挑选856个富含(AG/CT)n基序的unigenes,采用primer 3.0软件(http://primer3.ut.ee/)设计保守引物,SSR位点距离侧翼序列长度在50~300 bp范围内,退火温度为50~65℃之间,扩增产物大小为80~300 bp,长度18~28 bp,GC含量40%~60%,并且避免产生引物二聚体以及错配。引物在Unigene库中进行BLAST比对验证,去除无效引物。挑选110对引物委托南京金斯瑞生物科技有限公司合成。采用温度梯度PCR确定每个引物的最适合退火温度。随机选取5份锦绣杜鹃材料快速筛选核心引物。
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采用改良的CTAB法提取锦绣杜鹃和映山红2个群体的基因组DNA[12]。采用核心引物对栽培种锦绣杜鹃和野生种映山红2个群体进行PCR扩增,以验证引物的有效性并对2个居群的遗传多样性进行分析。选用10 μL的PCR扩增体系:10×Taq Reaction Buffer 1 μL,2.5 mmol·L-1 dNTPs 0.25 μL,5 U·μL-1 Taq DNA Polymerase 0.15 μL,10 μmol·L-1上下游引物各0.15 μL,1 μL模板DNA(100 ng·μL-1),用灭菌的去离子水补足剩余体积。PCR扩增程序为:94℃预变性10 min,35个扩增循环(94℃变性40 s,最适退火温度下退火30 s,72℃延伸40 s),然后72℃延伸7 min。
PCR扩增产物用6%聚丙烯酰胺凝胶电泳分离,并用硝酸银染色法检测扩增产物。按照PCR扩增条带的电泳位置统计每个个体的基因型,构建基因型数据矩阵。采用POPGENE-pc 2.2软件统计各个群体内的等位基因数(Na)、有效等位基因数(Ne)、Nei氏多样性指数(h)、观察杂合度(HO)、期望杂合度(HE)、Shannon’s信息指数(I)、种群内近交系数(Fis)、总近交系数(Fit)、种群间遗传分化系数(Fst)、基因流(Nm)等遗传参数[13]。
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锦绣杜鹃花蕾组织转录组测序序列总长度为43 766 249 bp,Cufflinks软件组装共得到49 527个unigenes,其中,含有SSR位点的unigene序列有12 113个,共发现16 120个SSR位点。平均每2.7 kb就有1个,SSR位点的出现频率为24.46%。仅含有1个SSR位点的unigene序列有8 943条,占73.83%;含有2个或者2个以上SSR位点的unigene有3 170条,占26.17%。1 695个unigenes中含有复合型SSR位点,占总unigenes的13.99%。
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锦绣杜鹃花蕾转录组内发现的SSR序列共有6种类型,分别是单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸、六核苷酸,且SSR位点的个数大体上随着重复基序长度的增加而减少(表 2)。二核苷酸出现频率最大,有9 624个(59.70%),其次是单核苷酸(3 738个,23.19%)和三核苷酸(2 589个,16.06%)(表 2)。出现频率最低的为五核苷酸,仅有42个,所占比例为0.26%;其次是六核苷酸,有50个,所占比例为0.31%。
表 2 不同SSR重复类型的个数
Table 2. Distribution of SSR repeat types
SSR类型
Types数量
Numbers比例
Percent/%基序类型
Types of motifs单核苷酸/Mono-nucleotides 3 738 23.19 2 二核苷酸/Di-nucleotides 9 624 59.70 4 三核苷酸/Tri-nucleotides 2 589 16.06 10 四核苷酸/Tetra-nucleotides 77 0.48 18 五核苷酸/Penta-nucleotides 42 0.26 17 六核苷酸/Hexa-nucleotides 50 0.31 24 在杜鹃花的花蕾组织转录组序列中,共发现75种SSR重复类型。单核苷酸有A/T和G/C两种,主要类型是A/T,共有3 669个,占总SSR重复类型的22.761%(表 3)。二核苷酸有4种类型,分别是AG/CT(9 047个,56.123%)、AC/GT(343个,2.128%)、AT/AT(226个,1.402%)、CG/CG(8个,0.050%)。三核苷酸有10种类型,出现最多的是AAG/CTT,其次是AGG/CCT、ACC/GGT、AGC/GCT。四核苷酸有18种,主要为AAAG/CTTT(23个,0.143%)、ACAT/ATGT(9个,0.056%)、AGCG/CGCT(6个,0.037%)、ATCC/GGAT(5个,0.031%)。五核苷酸有17种类型,其中,AAGAG/CTCTT最多(7个,0.043%),其次是AAAAG/CTTTT(6个,0.037%)和AGAGG/CCTCT(6个,0.037%)。六核苷酸有24种,主要是AGAGGG/CCCTCT(8个,0.050%),其次是AAGAGG/CCTCTT(5个,0.031%)和AGCAGG/CCTGCT(5个,0.031%)。
表 3 主要SSR重复类型的百分比含量
Table 3. Percent of the main SSR repeat types
重复类型Repeats 数量Numbers 比例Percent/% A/T 3 669 22.761 AG/CT 9 047 56.123 AAG/CTT 758 4.702 AGG/CCT 440 2.730 ACC/GGT 398 2.469 AGC/GCT 223 1.383 AAAG/CTTT 23 0.143 AAGAG/CTCTT 7 0.043 AGAGGG/CCCTCT 8 0.050 6种不同长度类型的SSR位点个数均随重复次数的增加而减少(表 4)。单核苷酸重复次数最多的为9~12次(1 996个,53.4%),其次是13~16次(864个,23.11%),大于24次重复的个数仅仅含有179个(4.79%)。二核苷酸重复次数最多的为5~8次(3 425个,35.59%),其次是9~12次(2 737个,28.44%),仅210个重复次数大于24(2.18%)。三核苷酸的重复次数主要集中在5~8次(2 484个,95.94%),重复次数在9~12次的有89个,在13~16次的有14个,而重复次数在17~20和21~24次的各有1个(0.04%),没有重复次数大于24的三核苷酸重复基序。四核苷酸和六核苷酸的重复次数均集中在5~8次。五核苷酸的重复次数主要在5~8次(95.24%),重复次数在9~12和13~16次的各有1个。
表 4 不同SSR重复类型的分布频率
Table 4. Frequency distribution of different SSR repeat types
SSR类型Types 5~8 9~12 13~16 17~20 21~24 >24 单核苷酸/Mono-nucleotides 0 1 996 864 353 346 179 二核苷酸/Di-nucleotides 3 425 2 737 1 548 1 250 454 210 三核苷酸/Tri-nucleotides 2 484 89 14 1 1 0 四核苷酸/Tetra-nucleotides 77 0 0 0 0 0 五核苷酸/Penta-nucleotides 40 1 1 0 0 0 六核苷酸/Hexa-nucleotides 50 0 0 0 0 0 -
选取856个SSR位点长度大于20 bp且两端侧翼序列长度均大于150 bp的unigenes设计引物,占含SSR位点unigenes总序列的8.62%。经BLAST比对后,去除无效引物115对。在有效引物中随机挑选110对进行合成,选用转录组测序用的锦绣杜鹃嫩叶基因组DNA进行PCR扩增验证,产物经3%的琼脂糖凝胶电泳检测。共筛选出68对SSR引物能够扩增出与预期大小一致,且条带清晰的目的DNA产物,占总挑选引物数的61.82%。设置温度梯度PCR程序,对68对SSR引物的最适合退火温度进行筛选。选取5份锦绣杜鹃种质基因组DNA对此68对引物进行核心引物的筛选,获得多态性强的引物40对,选用13对多态性最强、无明显影子带的引物进行后续的群体遗传多样性的检测(表 5)。
表 5 多态性SSR引物序列及基序信息
Table 5. Characteristic of Polymorphic SSR primer pairs
引物
Locus上游引物序列(5′-3′)
Forward primer squence下游引物序列(5′-3′)
Reverse primer sequence基序
motif片段大小
Size range/bp退火温度/℃
Annealing TemperatureRp01 CTTGCCACTTTGAGTTTGAG AGAGTAATTTGGAGGAAGCG (CT)28 209~221 58 Rp02 TGAACCCTTCTTCTTCTTCC TTTGATTGAAGGGTGGAGTG (TC)23 250~258 60 Rp03 CTCTCTCTCTCCTTCCTTCA GGATTCTTACTCGTGTCTGG (CT)27 217~229 58 Rp04 GCAGCACACGGATATTTAAG CTCAATACCACACTACACCC (CT)29 201~209 60 Rp05 TAGCTGCTTACTGTTGAAGG AGTAAAAGGGCTGAAACTGT (AG)22 150~160 56 Rp06 TCTTCTTCCTTCTTTGCTCC GGGGAAAGGAGAAGAGAAAG (CT)25 179~195 58 Rp07 GCCCTATCCCTCAACTTTAC GAGGAGCGTGGTTAGTAATT (TC)21 230~240 58 Rp08 GTATGGGACCTGTGATTTCC CTCCAACTAGCTACTCCAAC (GA)24 229~239 58 Rp09 GAAATCTCGAATCACCTCCA AAGGTGTTGGTGGACTAATC (TC)21 150~166 58 Rp10 TTGAAGAACACTCAAGTTGC ACGTAGAACATTGCTTTCCT (GA)21 187~203 60 Rp11 CCCTTCCTCTTCTCAAATCC CGTCATTTTCACACACAGAG (CT)20 174~190 62 Rp12 CTCTCCCAAAATTAGCCGAT GAATTGGCTGTTGGATGATG (CT)21 234~250 60 Rp13 AGAAAACTGGGAGATGTGTC AGGTGATCATCTTTGCATGT (CT)21 245~258 56 -
选用的13个SSR标记在2个群体内扩增的DNA片段范围为150~258 bp,在栽培种锦绣杜鹃和野生映山红群体中分别扩增出71个和74个等位基因,平均每个SSR位点扩增出的等位基因数为5.462和5.692个(表 6)。2个种群内近交系数Fis变化范围为-0.515~1.000,平均值为0.014。总近交系数Fit变化范围为-0.315~1.000,平均值为0.080。遗传分化系数Fst变化范围为0.010~0.159,平均值为0.066,即6.6%的遗传变异发生在群体间,发生在群体内的遗传变异高达93.4%。基因流Nm变化范围为1.324~24.762,平均值为3.514 (表 6)。
表 6 13个微卫星位点的遗传分化和基因流信息
Table 6. Summary of genetic variation and gene flow
位点
Locus种群内近交系数
Fis总近交系数
Fit分化系数
Fst基因流
NmRp01 1.000 1.000 0.141 1.519 Rp02 -0.515 -0.297 0.144 1.491 Rp03 1.000 1.000 0.067 3.457 Rp04 -0.285 -0.265 0.015 16.032 Rp05 -0.292 -0.226 0.050 4.708 Rp06 -0.232 -0.212 0.016 15.044 Rp07 -0.382 -0.289 0.067 3.478 Rp08 -0.216 -0.180 0.030 8.128 Rp09 0.286 0.362 0.107 2.098 Rp10 0.877 0.881 0.032 7.512 Rp11 -0.328 -0.315 0.010 24.762 Rp12 0.016 0.172 0.159 1.324 Rp13 -0.223 -0.191 0.027 9.169 平均Mean 0.014 0.080 0.066 3.514 锦绣杜鹃群体中,等位基因数Na在各SSR位点之间变化范围为3~9,有效等位基因数变化范围为1.684~5.930,Rp03标记扩增的等位基因最少(3个),Rp08扩增的等位基因最多(9个)。观察杂合度HO和期望杂合度HE的变化范围分别为0.000~1.000和0.433~0.848,平均值分别为0.696±0.426和0.705±0.129。Shannon’s信息指数I和Nei氏多样性指数h的变化范围分别为0.736~1.961和0.406~0.831,平均值分别为1.376±0.339和0.683±0.131。Shannon’s信息指数I、期望杂合度HE、Nei氏多样性指数h呈现相似的变化趋势,最大值均出现在Rp08处,最小值在位点Rp03处(表 7)。
表 7 SSR引物在2个群体中的遗传多样性
Table 7. Genetic diversity of two populations revealed by SSR markers
位点Locus 锦绣杜鹃(R. pulchrum) 映山红(R.simsii) Na Ne HO HE I h Na Ne HO HE I h Rp01 4 2.909 0.000 0.700 1.213 0.656 4 3.922 0.000 0.764 1.376 0.745 Rp02 6 2.586 1.000 0.633 1.157 0.613 5 3.412 1.000 0.719 1.361 0.707 Rp03 3 1.684 0.000 0.433 0.736 0.406 5 3.600 0.000 0.754 1.424 0.722 Rp04 4 3.982 1.000 0.775 1.384 0.749 7 5.200 1.000 0.824 1.777 0.808 Rp05 5 3.193 0.882 0.708 1.273 0.687 5 4.360 1.000 0.789 1.533 0.771 Rp06 8 4.308 1.000 0.782 1.718 0.768 9 6.922 1.000 0.871 2.049 0.856 Rp07 5 3.767 1.000 0.752 1.429 0.735 4 3.485 1.000 0.742 1.308 0.713 Rp08 9 5.930 1.000 0.848 1.961 0.831 7 5.365 1.000 0.830 1.775 0.814 Rp09 6 4.624 0.765 0.808 1.633 0.784 4 1.667 0.080 0.408 0.794 0.400 Rp10 4 2.579 0.071 0.623 1.091 0.612 4 2.344 0.074 0.584 1.008 0.573 Rp11 5 4.000 1.000 0.767 1.485 0.750 6 4.093 1.000 0.769 1.576 0.756 Rp12 6 1.862 0.333 0.490 1.038 0.463 7 5.521 0.929 0.834 1.825 0.819 Rp13 6 5.729 1.000 0.842 1.767 0.825 7 5.244 1.000 0.824 1.800 0.809 平均值Mean 5.462 3.627 0.696 0.705 1.376 0.683 5.692 4.241 0.699 0.747 1.508 0.730 标准差 1.664 1.330 0.426 0.129 0.339 0.131 1.600 1.416 0.459 0.125 0.350 0.122 在映山红群体内,各SSR位点有效等位基因数Ne变化范围为1.667~6.922,平均值为4.241。观察杂合度HO和期望杂合度HE的变化范围分别为0.000~1.000和0.408~0.871,平均值分别为0.699和0.747。Shannon’s信息指数I和Nei氏多样性指数h的变化范围分别为0.794~2.049和0.400~0.856,平均值分别为1.508和0.730。与锦绣杜鹃类似,Shannon’s信息指数I、期望杂合度、Nei氏多样性指数也呈现相似的变化趋势,然而其最大值出现在Rp06处,最小值在Rp09处(表 7)。
基于锦绣杜鹃花蕾转录组的SSR标记开发及应用
Development and Application of SSR Markers Based on Buds Transcriptomic Data of Rhododendron pulchrum Planch
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摘要:
目的 探究锦绣杜鹃(Rhododendron pulchrum Planch.)EST-SSR的类型、分布频率和分布特征,开发有效的SSR标记,并验证其在遗传多样性研究和跨物种转移中的应用潜力。 方法 采用RNA-seq技术对锦绣杜鹃‘紫鹤’品种的花蕾进行转录组测序,MISA软件对组装的unigenes内部的SSR位点进行检索,分析其类型、分布频率。利用Primer 3.0软件设计引物,并对锦绣杜鹃群体和其近缘种映山红群体进行多样性检测,利用POPGENE-PC 2.2软件计算等位基因数、有效等位基因数、多态性位点百分率、Shannon's信息指数、Nei氏多样性指数、观察杂合度、期望杂合度等参数。 结果 锦绣杜鹃‘紫鹤’花蕾转录组共有49 527个unigenes(43 766 249 bp),从中筛选出16 120个SSR位点(24.46%),发生频率为1·(2.7 kb)-1。微卫星的重复次数主要集中在5~24次之间,二核苷酸发生频率最高(9 624个,59.70%),其次是单核苷酸(3 738个,23.19%),频率最低的为五核苷酸(42个,0.26%)。频率最高的重复基序有A/T、AG/CT、AAG/CTT、AGG/CCT、ACC/GGT、AGC/GCT、AAAG/CTTT、AAGAG/CTCTT、AGAGGG/CCCTCT等。选用13个多态性SSR标记对锦绣杜鹃群体进行PCR扩增,共得到71个等位基因,平均每个位点3~9个;有效等位基因数在各SSR位点之间变化范围为1.684~5.930;观察杂合度HO和期望杂合度HE的变化范围分别为0.000~1.000和0.433~0.848,平均值分别为0.696±0.426和0.705±0.129;Shannon's信息指数I和Nei氏多样性指数h的变化范围分别为0.736~1.961和0.406~0.831,平均值分别为1.376±0.339和0.683±0.131。此13个标记在映山红群体中的跨物种扩增成功率为100%,并反映出丰富的遗传多样性。2个群体间的遗传差异93.4%存在于群体内部。 结论 基于AG/CT重复基序开发的13个SSR标记具有高度的多态性,为后续杜鹃花属植物的遗传多样性研究、遗传图谱构建、基因定位、分子标记辅助育种等研究奠定了基础。大别山野生映山红资源具有较高的遗传多样性,且杂合度过剩,遗传变异主要发生在群体内部。 Abstract:Objective The types, distribution frequencies and characteristics of expressed sequence tag-simple sequence repeats (EST-SSRs) were clarified through transcriptome analysis of buds of Rhododendron pulchrum Planch ("Zihe" variety). Moreover, the development of polymorphic SSR markers were performed, as well as their usage in genetic diversity analysis and potential for cross-amplification in related species. Method RNA-seq data from buds of R. pulchrum were searched with MISA software. The characteristics of SSR loci were analyzed and SSR primers were designed with Primer 3.0. The effective primer pairs were used in genetic diversity analysis of R. pulchrum and R. simsii populations. The genetic parameters of population were calculated with POPGENE-pc 2.2 software. Result Totally, 49 527 unigenes (43 766 249 bp) were obtained, and 16 120 SSR loci 1.(2.7 kb)-1 were searched, which accounted for 24.46% of the total unigenes. Repeat numbers of most SSR loci ranged between 5-24. Dinuclotide repeat was the most abundant type with a frequency of 59.70% (9 624), followed by mono-nucleotide repeat (3 738, 23.19%), and the least type was penta-nucleotide repeat (42, 0.26%). Moreover, the typical motifs were A/T, AG/CT, AAG/CTT, AGG/CCT, ACC/GGT, AGC/GCT、AAAG/CTTT, AAGAG/CTCTT, and AGAGGG/CCCTCT. The availability and polymorphism of the thirteen SSR markers selected were clarified in R. pulchrum population. A total of 71 alleles was scored, and the amount of allele (Na) and effective number of allele (Ne) per locus ranged from 3-9 and 1.684-5.930, respectively. The observed heterozygosity (HO) and expected heterozygosity (HE) varied from 0.000 to 1.000 and from 0.433 to 0.848, with the mean values of 0.696±0.426 and 0.705±0.129, respectively. Shannon's information index (I) and Nei's gene diversity (h) ranged from 0.736 to 1.961 and from 0.406 to 0.831, with the average values of 1.376±0.339 and 0.683±0.131, respectively. All these SSR markers could be successfully cross-amplified in the related species R. simsii, which also showed high genetic diversity. The genetic variation existed mainly among populations. Conclusion The 13 polymorphic SSR markers based on unigenes containing (AG/CT)n loci will benefit for following genetic diversity analysis, genetic map construction, gene mapping, and molecular marker assisted breeding of Rhododendron species. Natural R. simsii germplasm resources possess high genetic diversity. Moreover, excess heterozygosity is observed, and genetic variation is mainly maintained among populations. -
Key words:
- Rhododendron pulchrum Planch
- / RNA-seq
- / microsatellite
- / genetic diversity
- / cross-amplification
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表 1 转录组测序和多样性检测材料采集地信息
Table 1. Information of sampling populations used for RNA-seq and genetic diversity analysis
种质
Germplasm样品来源
Sample source经度
Longitude(E)纬度
Latitude(N)海拔
Altitude/m样品个数
Numbers of sample锦绣杜鹃 黄冈师范学院资源圃 114°55′22.62″ 30°26′56.82″ 22 3 锦绣杜鹃 黄冈遗爱湖公园 114°53′28.21″ 30°27′21.67″ 21 30 映山红 大别山主峰五脑山 114°59′57.47″ 31°14′10.95″ 104 30 表 2 不同SSR重复类型的个数
Table 2. Distribution of SSR repeat types
SSR类型
Types数量
Numbers比例
Percent/%基序类型
Types of motifs单核苷酸/Mono-nucleotides 3 738 23.19 2 二核苷酸/Di-nucleotides 9 624 59.70 4 三核苷酸/Tri-nucleotides 2 589 16.06 10 四核苷酸/Tetra-nucleotides 77 0.48 18 五核苷酸/Penta-nucleotides 42 0.26 17 六核苷酸/Hexa-nucleotides 50 0.31 24 表 3 主要SSR重复类型的百分比含量
Table 3. Percent of the main SSR repeat types
重复类型Repeats 数量Numbers 比例Percent/% A/T 3 669 22.761 AG/CT 9 047 56.123 AAG/CTT 758 4.702 AGG/CCT 440 2.730 ACC/GGT 398 2.469 AGC/GCT 223 1.383 AAAG/CTTT 23 0.143 AAGAG/CTCTT 7 0.043 AGAGGG/CCCTCT 8 0.050 表 4 不同SSR重复类型的分布频率
Table 4. Frequency distribution of different SSR repeat types
SSR类型Types 5~8 9~12 13~16 17~20 21~24 >24 单核苷酸/Mono-nucleotides 0 1 996 864 353 346 179 二核苷酸/Di-nucleotides 3 425 2 737 1 548 1 250 454 210 三核苷酸/Tri-nucleotides 2 484 89 14 1 1 0 四核苷酸/Tetra-nucleotides 77 0 0 0 0 0 五核苷酸/Penta-nucleotides 40 1 1 0 0 0 六核苷酸/Hexa-nucleotides 50 0 0 0 0 0 表 5 多态性SSR引物序列及基序信息
Table 5. Characteristic of Polymorphic SSR primer pairs
引物
Locus上游引物序列(5′-3′)
Forward primer squence下游引物序列(5′-3′)
Reverse primer sequence基序
motif片段大小
Size range/bp退火温度/℃
Annealing TemperatureRp01 CTTGCCACTTTGAGTTTGAG AGAGTAATTTGGAGGAAGCG (CT)28 209~221 58 Rp02 TGAACCCTTCTTCTTCTTCC TTTGATTGAAGGGTGGAGTG (TC)23 250~258 60 Rp03 CTCTCTCTCTCCTTCCTTCA GGATTCTTACTCGTGTCTGG (CT)27 217~229 58 Rp04 GCAGCACACGGATATTTAAG CTCAATACCACACTACACCC (CT)29 201~209 60 Rp05 TAGCTGCTTACTGTTGAAGG AGTAAAAGGGCTGAAACTGT (AG)22 150~160 56 Rp06 TCTTCTTCCTTCTTTGCTCC GGGGAAAGGAGAAGAGAAAG (CT)25 179~195 58 Rp07 GCCCTATCCCTCAACTTTAC GAGGAGCGTGGTTAGTAATT (TC)21 230~240 58 Rp08 GTATGGGACCTGTGATTTCC CTCCAACTAGCTACTCCAAC (GA)24 229~239 58 Rp09 GAAATCTCGAATCACCTCCA AAGGTGTTGGTGGACTAATC (TC)21 150~166 58 Rp10 TTGAAGAACACTCAAGTTGC ACGTAGAACATTGCTTTCCT (GA)21 187~203 60 Rp11 CCCTTCCTCTTCTCAAATCC CGTCATTTTCACACACAGAG (CT)20 174~190 62 Rp12 CTCTCCCAAAATTAGCCGAT GAATTGGCTGTTGGATGATG (CT)21 234~250 60 Rp13 AGAAAACTGGGAGATGTGTC AGGTGATCATCTTTGCATGT (CT)21 245~258 56 表 6 13个微卫星位点的遗传分化和基因流信息
Table 6. Summary of genetic variation and gene flow
位点
Locus种群内近交系数
Fis总近交系数
Fit分化系数
Fst基因流
NmRp01 1.000 1.000 0.141 1.519 Rp02 -0.515 -0.297 0.144 1.491 Rp03 1.000 1.000 0.067 3.457 Rp04 -0.285 -0.265 0.015 16.032 Rp05 -0.292 -0.226 0.050 4.708 Rp06 -0.232 -0.212 0.016 15.044 Rp07 -0.382 -0.289 0.067 3.478 Rp08 -0.216 -0.180 0.030 8.128 Rp09 0.286 0.362 0.107 2.098 Rp10 0.877 0.881 0.032 7.512 Rp11 -0.328 -0.315 0.010 24.762 Rp12 0.016 0.172 0.159 1.324 Rp13 -0.223 -0.191 0.027 9.169 平均Mean 0.014 0.080 0.066 3.514 表 7 SSR引物在2个群体中的遗传多样性
Table 7. Genetic diversity of two populations revealed by SSR markers
位点Locus 锦绣杜鹃(R. pulchrum) 映山红(R.simsii) Na Ne HO HE I h Na Ne HO HE I h Rp01 4 2.909 0.000 0.700 1.213 0.656 4 3.922 0.000 0.764 1.376 0.745 Rp02 6 2.586 1.000 0.633 1.157 0.613 5 3.412 1.000 0.719 1.361 0.707 Rp03 3 1.684 0.000 0.433 0.736 0.406 5 3.600 0.000 0.754 1.424 0.722 Rp04 4 3.982 1.000 0.775 1.384 0.749 7 5.200 1.000 0.824 1.777 0.808 Rp05 5 3.193 0.882 0.708 1.273 0.687 5 4.360 1.000 0.789 1.533 0.771 Rp06 8 4.308 1.000 0.782 1.718 0.768 9 6.922 1.000 0.871 2.049 0.856 Rp07 5 3.767 1.000 0.752 1.429 0.735 4 3.485 1.000 0.742 1.308 0.713 Rp08 9 5.930 1.000 0.848 1.961 0.831 7 5.365 1.000 0.830 1.775 0.814 Rp09 6 4.624 0.765 0.808 1.633 0.784 4 1.667 0.080 0.408 0.794 0.400 Rp10 4 2.579 0.071 0.623 1.091 0.612 4 2.344 0.074 0.584 1.008 0.573 Rp11 5 4.000 1.000 0.767 1.485 0.750 6 4.093 1.000 0.769 1.576 0.756 Rp12 6 1.862 0.333 0.490 1.038 0.463 7 5.521 0.929 0.834 1.825 0.819 Rp13 6 5.729 1.000 0.842 1.767 0.825 7 5.244 1.000 0.824 1.800 0.809 平均值Mean 5.462 3.627 0.696 0.705 1.376 0.683 5.692 4.241 0.699 0.747 1.508 0.730 标准差 1.664 1.330 0.426 0.129 0.339 0.131 1.600 1.416 0.459 0.125 0.350 0.122 -
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