Analysis of SSR loci and development of SSR primers in <Emphasis Type="Italic">Eucalyptus</Emphasis>

In this study, 28,691 genome sequences and 16,566 expressed sequence tags (ESTs) of Eucalyptus were derived from the GenBank database. A total of 2292 SSR loci were sought out from 1785 effective sequences. Through analyses of SSR loci information, the SSR motif length was negatively correlated with the abundance of the SSRs. In the EST sequences of Eucalyptus, triplet repeat motifs were the most abundant, and dinucleotide repeats motifs had the highest frequencies. Subsequently, 395 pairs of primers were designed based on the SSR loci. Using optimized SSR-PCR conditions, 340 pairs of primers were successfully screened, with a success rate of 86.1%. By construction of a maximum likelihood phylogenetic tree of six eucalypt species, represented by five species of the genus Eucalyptus and one of the genus Corymbia, the genetic relationships of Eucalyptus urophylla and E. camaldulensis suggested by this tree was found to differ from that suggested by traditional morphological taxonomy. The results provide insights for evaluating genetic diversity of Eucalyptus and analysis of Eucalyptus phylogenetics using SSR markers.     

基于桤木属转录组测序的SSR分子标记的开发

[目的]基于转录组数据开发适用于桤木属树种的SSR标记,揭示其在转录组序列中的分布类型及特征,为桤木属树种分子标记辅助育种提供有利工具。[方法]利用Micro SAtellite(MISA)软件对所有转录组序列进行SSRs搜索,并对SSR位点的数量、分布特征进行统计分析。设计100对SSR引物,采用琼脂糖凝胶电泳和毛细管电泳分离检测方法对3种不同倍性桤木属植物(12份材料)进行遗传多样性检测,确定引物多态性及通用性。[结果]85 769条Unigenes序列中发现8 678个SSR位点,分布在8 298条Unigenes中发生频率为9.67%,转录组序列中平均每14.04 kb长度就有一个SSR位点分布。其中,二核苷酸重复类型数量最多,占65.87%。根据转录组Unigenes序列,利用Primer 3软件共设计出4 531对符合要求的引物,挑选出的100对SSR引物中,获得18对多态性高、稳定性好的SSR引物。[结论]本研究可扩增出多态性位点的引物重复单元以二、三核苷酸重复为主。基于桤木属转录组序列的SSR标记开发是可行的,开发的引物为桤木属遗传多样性分析、分子育种、遗传图谱构建和功能基因的挖掘提供了丰富的候选分子标记。     

基于马尾松干旱转录组的抗旱功能SSR位点分析

[目的]了解马尾松干旱胁迫转录组序列的功能分布和SSR位点分布特征,并探索与干旱关联的SSR位点。[方法]对马尾松幼苗进行持续干旱胁迫,选取干旱10、15、25 d及正常供水对照的马尾松针叶样品,通过总RNA提取、Illumina测序、raw reads去冗、Trinity拼接获得unigenes。利用Blast比对,对Unigene序列进行GO、KOG、KEGG功能注释及分类;利用Misa软件进行SSR位点批量搜寻,primer 3. 0软件进行规模化SSR引物设计;利用GOSeq(1. 10. 0)、KOBAS(v2. 0. 12)软件对差异表达的含SSR位点Unigene进行GO、KEGG显著性富集分析。[结果]马尾松转录组194 821个Unigene中,101 806个Unigene获得注释,包含64 943个GO功能注释、35 880个KOG功能注释及30 882个KEGG注释。搜寻到6 728个SSR位点,分布于6 367个Unigene中,SSR出现频率为3. 45%;重复类型以单、三、二核苷酸为主,分别占总SSR的35. 82%、33. 03%和25. 22%;重复基序以A/T、AT/AT、AG/CT、AGC/CTG、AAG/CTT为主;基序长度以10 20 bp的短序列SSR为主;基序重复次数以5～10次重复占优势;批量设计13 338对SSR引物。422个含SSR位点Unigene具有差异表达; KEGG富集分析发现,有11个含SSR位点的差异Unigene参与了光合作用、植物激素信号传导及类胡萝卜素合成等3个与干旱响应相关的代谢途径。[结论]从较高质量的马尾松转录组中获得101 806个具有注释的Unigene;从6 367个Unigene中挖掘出6 728个SSR位点; 422个含SSR位点的差异表达Unigene中筛选出11个与干旱关联的SSR功能位点,为抗旱功能基因定位及马尾松抗旱分子机制研究奠定基础。     

基于松属不同树种EST序列的马尾松SSR引物开发

利用松属5个树种的EST序列,分别树种查找SSR位点,采用Primer3．0软件进行引物设计。设计的5个树种的EST-SSR引物中：所占比例最高的均为三核苷酸重复,其次是六核苷酸重复,两者之和都达到了64％以上;四核苷酸重复所占比例均为最低。从设计的引物中分别不同树种各选取20对进行引物合成和通用性检测以及马尾松群体检测,结果表明：松属5个树种的EST序列开发的马尾松SSR引物,其在马尾松中的扩增成功率为60％-80％;最高的是辐射松,最低的是海岸松,平均为72％。通用性最好的为辐射松,属内的通用性为94％;属间和科间通用性也分别达到75％和44％。不同树种序列来源的EST-SSR引物,其扩增成功率在10％-30％;最高的是北美短叶松,其次是辐射松,最低的是扭叶松和火炬松;平均为17％。     

基于芭蕉属EST序列的地涌金莲SSR引物开发

对31 308条来自NCBI的芭蕉属(Musa)EST序列进行拼接得到全长为13.51 Mb的21 129条无冗余EST序列(含3 818条contigs及17 311条singletons),其中,4 944条(23.40%)EST序列含有5 416条SSRs,SSR的出现频率为25.63%,平均分布距离2.49 Kb,有234条(1.11%)含有1个以上的SSR。在所检测的SSR中,二、三、四核苷酸重复是主导重复类型,分别占EST-SSR总数的21.80%(1 181条)、52.55%(2 846条)和14.55% (788条)。AG/CT、AAG/CTT与AGG/CCT和AAAG/CTTT与AAAT/ATTT分别是二、三、四核苷酸的优势重复基元。随机设计了238对EST-SSR引物在24个地涌金莲个体中进行筛选,116对有扩增产物,其中,78对EST-SSR引物扩增出清晰稳定的目的片段,49对引物表现出多态性。本研究检测的15对引物扩增等位基因数范围是2~7个,平均3.067个;表观杂合度(Ho)范围是0.042 0.750,平均0.250;期望杂合度(He)范围是0.232~0.823,平均0.522。     

杜仲基因组微卫星特征及SSR标记开发

为全面解读杜仲的遗传背景,本研究在基因组测序(数据未公布)的基础上,通过MISA软件搜索杜仲基因组(26 947/854 758 160 bp)中的完整型(1～7核苷酸重复)及复合型SSR序列,共查找出25 694个Scaffolds含有488592个SSR位点,占总Scaffolds的95.3％.从SSR位点分布密度上讲,平均每1 749 bp出现1个微卫星,其中单核苷酸重复单元的SSR含量最多,约占总数的54.34％,其次为二核苷酸(20.47％),而复合模式、三核苷酸、四核苷酸、五核苷酸、六核苷酸和七核苷酸重复分别占20.29％、23.89％、0.77％、0.13％、0.10％和0.01％,并发现SSR中均以含A、T的重复类型占主导地位.根据SSR位点设计并合成引物290对,162对SSR引物扩增出清晰的目的条带,其中16对引物多态性高、稳定性好,在8份杜仲资源中共检测到84个等位基因,平均每个SSR位点检测到5.25个等位基因.本研究对于进一步利用SSR分子标记分析杜仲遗传多样性、遗传图谱构建、杜仲雌雄株早期鉴定等具有较高的应用价值.     

杨树抗逆转录因子基因内SSR标记的开发

利用直接测序法开发小叶杨抗逆转录因子基因内一套新的核基因组SSR标记。通过对3个抗逆转录因子共21个成员在36个小叶杨基因型个体中的序列比较分析后共检测到31个SSR多态性位点,SSR出现的频率为1/1916bp。在小叶杨自然群体中,SSR多态性位点的碱基重复呈现出2～5碱基形式,基元重复次数变异范围为3～20次,其中以二碱基重复的位点较多,占总数的51.6%。在此基础上,依据SSR位点两侧的保守序列,设计31对SSR位点PCR扩增引物对。利用设计的引物检测所开发的SSR位点在杨属内22个基因型个体中PCR扩增的有效性及SSR位点的保守性。PCR扩增结果显示,93.5%的SSR位点能够在杨属内至少4个派内有效扩增,每对引物组合可检测到SSR多样性位点数3～12个,平均6个。基于抗逆转录因子基因内开发的SSR标记位点为分子标记辅助小叶杨抗逆性状育种提供工具。     

天山樱桃野生居群遗传多样性SSR分析

为给天山樱桃种质资源的保护和开发利用提供参考,应用SSR标记技术对新疆天山樱桃4个居群44份种质的遗传多样性进行分析。结果表明:筛选的14对SSR引物共检测到191个位点,包含148个多态性位点。Nei’s基因多样性指数和Shannon信息指数分别为0.239 8和0.367 6,各居群遗传分化系数为0.193 5,基因流为2.084 3。天山樱桃遗传分化水平较低,各居群间基因交流频繁,遗传变异主要存在于居群内。由基于遗传距离的聚类结果可知,裕民县与大西沟居群遗传关系最近,与特克斯居群遗传距离较远,4个天山樱桃居群中大西沟居群遗传多样性最高。     

日本落叶松EST-SSR标记挖掘及特征分析

基于日本落叶松转录组测序(RNA-seq)结果,利用SSRIT工具查找SSR位点,检测到1 788个EST-SSR位点,平均长度是17.5 bp。进一步统计分析发现在落叶松中重复类型SSR位点出现次数最多的是六、三核苷酸,其余依次是五、二、四核苷酸重复类型;同时检测到656种重复基元类型,种类丰富。用Primer Premier 5.0设计500对引物,随机合成102对,以红豆杉科、柏科、松科材料为模板进行检测,结果显示在红豆杉科和柏科里的扩增率分别为95%、50%;在松科不同属的通用性为落叶松属99.01%、黄杉属67.4%、雪松属63.8%、冷杉属67.4%、云杉属70.2%和松属75.7%。利用101对SSR引物对7种不同落叶松品种亲缘关系进行了分析,获得79个多态性位点,在遗传相似系数0.69处将其区分为4个类群。     

欧洲黑杨育种基因资源SSR多态性比较研究

本研究利用13对SSR引物对120份(105份国外引进、15份我国新疆)欧洲黑杨基因资源进行了遗传多态性分析,共检测出171个等位基因,每个多态性位点检测到7～19个等位基因,平均为13.2,多态信息指数为0.396～0.909,平均为0.808;遗传距离为0～0.456 4.欧洲黑杨基因种源具有丰富的遗传多样性.通过UPGMA等类分析,将120份材料划分为8类,地理分布较近的材料基本聚在一起,表明遗传距离与地理距离相关性强.     

锥栗基因组SSR开发及农家品种的遗传多样性分析

本文对锥栗SSR富集文库进行Illumina MiSeq高通量测序,利用生物信息学对得到的序列进行SSR特征分析,开发锥栗基因组SSR并对农家品种进行了遗传多样性分析。在2051475条序列中,总共搜索到2117345个SSR,以复合形式存在的SSR数量为640155个。二核苷酸为重复单元的SSR数量最多,占总数的73.22%,之后依次为三核苷酸(12.61%)、单核苷酸(12.56%)、四核苷酸(1.33%),单碱基重复、二碱基重复和三碱基重复的优势重复单元分别为:A/T、AC/GT、AAG/CTT。对SSR的长度多态性进行了评价。以8个农家品种为材料,在100对基因组SSR引物中筛选出多态性和特异性较好的引物10对。对25个锥栗农家品种进行了遗传多样性分析观测等位基因和期望杂合度分别为6.3和0.705,有效等位基因数为3.628,Shannon信息指数为1.441,表明锥栗农家品种具有较高的遗传多样性水平。     

木荷1代育种群体遗传多样性分析

木荷为我国亚热带地区主要的珍贵优质阔叶用材树种和生态防护树种.利用筛选的14对多态性强的SSR引物,对木荷1代育种群体中来自15个产地133个亲本进行遗传多样性分析,为其优异种质资源保存、杂交亲本选配及新种质创制提供科学依据.结果表明:14对引物共扩增86个位点,每对引物检测到的等位基因数(Na)变异范围为2～11个,平均等位基因数(Na)为6.14个,平均有效等位基因数(Ne)为3.23个,平均观察杂合度(Ho)为0.572 0,平均Shannon信息指数(I)和平均Nei's基因多样性指数(Nei)分别为1.224 7和0.599 0,说明木荷1代育种群体具有丰富的遗传多样性,其中,福建建瓯产地遗传多样性最高,浙江遂昌产地遗传多样性最低.木荷1代育种群体中成对亲本间遗传距离为0.023 3～1.633 8,平均为0.6067.不同产地遗传多样性与纬度呈显著的负相关关系(r=-0.5162,p=0.048 9).通过UPGMA聚类,可将133个育种亲本分成3个类群,其中,类群3又分为4个亚类群.木荷亲本选配时,应充分考虑优树的产地来源.     

杨树基因组SSR标记在康定柳中的通用性分析

SSR分子标记因具有共显性遗传、高度多态性、重复性和稳定性好、易于实现自动化等优点,在遗传育种等方面有着广泛的应用前景。本研究在杨树基因组中随机选取622对SSR引物对来自5个不同群体的康定柳雌雄各20个个体,共计200个进行了通用性分析。结果表明：杨树基因组SSR标记在康定柳中的通用性达到7.88%。     

锥栗主栽农家品种遗传多样性的SSR分析

为给锥栗杂交育种和遗传作图的亲本选配提供参考,应用SSR分子标记技术,分析了福建省建瓯市17个锥栗主栽农家品种的遗传多样性。结果表明:不同农家品种遗传多样性丰富,利用筛选出的12对引物组合扩增的片段大小主要在50~2 000 bp之间,共扩增出180个位点,多态位点163个,占90.56%;17个锥栗主栽农家品种的遗传相似性系数在0.509 2~0822 1之间,乌壳长芒和牛角仔(晚熟)的相似性系数最大,中尖嘴与白露仔的相似性系数最小,且在相似性系数为0.60处,大尖嘴和中尖嘴单独聚为I类,在相似性系数为0.77处,第II类又被分为A~H共8个组;SSR引物组合Cs CAT33和Cm TCR4共同构建了17个锥栗主栽农家品种的DNA指纹图谱。     

竹类植物SSR引物开发策略及应用

微卫星(SSR)指的是以核苷酸为单位,串联重复的DNA序列,分布于真核生物的整个基因组中。SSR标记是基于DNA长度多态性的分子标记技术,广泛应用于群体遗传结构分析、构建遗传图谱、基因定位、亲缘关系等领域。本文综述了竹类植物中的SSR引物开发方法及应用,旨在为SSR分子标记在竹类植物中的应用提供参考。     

'辽宁2号'核桃品种矮化机制初探

'Liaoning 2' is a dwarf walnut variety bred by Chinese scientists through artificial hybridization, but little is known about its dwarfing mechanism. In this study, the dwarfing mechanism of 'Liaoning 2' is discussed through the comparison of biological characteristics, genomics and the expression of growth-related gene JrGAI,with a growth normally, closely related(half-sib families) variety of 'Liaoning 1' as control. By observing the phenology and tree phenotypes, it was found that there was no significant difference in phenological phase between 'Liaoning 1' and 'Liaoning 2', but 'Liaoning 2' was obviously dwarf, the height, crown and trunk diameter were smaller, the average length of internode reduced significantly, but there was no significant difference in the number of development branch internode. The dwarf phenotype of 'Liaoning 2' is mainly caused by inter-shortening. The qRT-PCR technique was performed to analyze the expression of GA negative regulation gene JrGAI and the results showed that during the shoot development, the expression of JrGAI gene exhibited decreasing expression in the shoot rapid growing period in both varieties and the decline in 'Liaoning 1' was remarkably reduced. The SSR analysis with 25 pairs of primers showed that SSR loci of two varieties were different, especially the homozygosity of 'Liaoning 2' SSR loci was higher. It is concluded that different expressions of JrGAI gene and homozygosity of SSR loci could be important internal reasons of dwarf phenotype of 'Liaoning 2'.     

白桦EST-SSR信息分析与标记的开发

对NCBI(美国国立生物技术信息中心)中2 5 48条欧洲白桦ESTs的序列进行分析,结果表明:其中260条ESTs中含有306个SSRs,为全部 ESTs序列的10.2%,SSR频率为12.01%.其中,二核苷酸重复比例为81.37%,三核苷酸重复比例为16.67%,四核苷酸重复比例为1.96%.不同软件设计引物的效率不同,Pri mer3设计出176对SSR引物,在白桦中扩增成功的引物比例为59.09%,而具有多态性的引物比例仅为25.96%;SSR Primer设计出100对引物,在白桦中扩增成功的引物比例为37%,而具有多态性的引物比例为48.65%.     

Analysis of genetic relationship among Arbutus unedo L. genotypes using RAPD and SSR markers

The strawberry tree (Arbutus unedo L.) is an underutilized, drought tolerant, fire resistant species with a south western distribution in Europe, and with ecological and putative socio-economical impact in Portugal and Mediterranean countries. Our aim was to develop an appropriate set of molecular markers to enable genetic diversity to be assessed and to fingerprint Arbutus unedo genotypes for breeding and conservation purposes in Portugal. Twenty-seven trees from a broad geographic range were screened with 20 random amplified polymorphic DNA (RAPD primers) and 11 microsatellite markers (SSR). The RAPDs generated 124 bands, 57.3% of which were polymorphic, with an expected heterozygosity of 27%. We cross-amplified 11 SSR primers developed for Vaccinium spp., and 5 were found to be polymorphic in A. unedo, with 75% of expected heterozygosity, a number of alleles of 11.6, a null allele frequency of 7.6% and a polymorphic information content of 71%. Although the SSRs were more polymorphic and informative than the RAPDs, both markers displayed high genetic variability with the gathered data. No geographic pattern was observed in the genetic variation distribution based on both marker systems, and the lack of correlation between genetic and geographical matrices was confirmed by Mantel tests. Likely, no correlation was found between pairwise SSR and RAPD band-sharing matrices. These results and their implications on A. unedo breeding and conservation programs are discussed.     

Analysis of EST-SSRs in silver birch(Betula pendula Roth.)

Simple sequence repeats(SSRs) defined as sequence repeat units between 1 and 6 bp occur abundantly in both coding and non-coding regions in eukaryotic genomes and these repeats can affect gene expression. In this study, ESTs(expressed sequence tags) of Betula pendula(silver birch) were analyzed for in silico mining of ESTSSRs, protein annotation, open reading frames(ORFs),designing primers, and identifying codon repetitions. In B.pendula, the frequency of ESTs containing SSRs was 7.8 %with an average of 1SSR/4. 78 kb of EST sequences. A total of 188 SSRs was identified by using MISA software and dinucleotide SSR motifs(65.9 %) were found to be the most abundant type of repeat motif followed by tri-(27.1 %),tetra-(4.8 %), and penta-(2.2 %) motifs. Based on ORF analysis, 175 of 178 sequences were predicted as ORFs and the most frequent SSRs were detected in 50 UTR(58.43 %),followed by in ORF(31.46 %) and in 30UTR(8.43 %). 102 of 178 ESTs were annotated as ribosomal protein, transport protein, membrane protein, carrier protein, binding protein,and transferase protein. For a total of 102 SSRs(57.3 %)with significant matches, a set of 102 primers(100 %) with forward and reverse strands was designed by using Primer 3 software. Serine(Ser, 19.6 %) was predominant in putative encoded amino acids and most of amino acids showed nonpolar(35.3 %) nature. Our data provide resources for B.pendula and can be useful for in silico comparative analyses of Betulaceae species, including SSR mining.     

紫胶虫主要生产种的RAPD分子标记分析

用RAPD技术研究了紫胶虫7种主要生产种12个群体之间的亲缘关系,研究中每个群体使用了24个标本,共有288个个体用于研究.在试用的45种随机引物中,筛选出9种引物用于随机引物扩增,共得到121个清晰稳定的位点,其中118个为多态性位点,多态百分率为97.52%,每条引物检测到的位点数为11～16个,长度为150～3 000 bp.根据扩增结果,用UPGMA法对Nei's遗传距离进行聚类分析,构建分子系统树.7种紫胶虫聚类结果显示:紫胶虫各群体间的遗传距离为0.044 3～0.791 7,其中,种间的遗传平均距离为0.443 0,种内的遗传平均距离为0.170 7.根据聚类分析,讨论了紫胶虫主要生产种的亲缘关系.