Analysis of Simple Sequence Repeats in Genomes of Rhizobia

Simple sequence repeats （SSRs） or microsatellites, as genetic markers, are ubiquitous in genomes of various organisms. The analysis of SSR in rhizobia genome provides useful information for a variety of applications in population genetics of rhizobia. We analyzed the occurrences, relative abundance, and relative density of SSRs, the most common in Bradyrhizobium japonicum, Mesorhizobium loti, and Sinorhizobium meliloti genomes se- quenced in the microorganisms tandem repeats database, and SSRs in the three species genomes were compared with each other. The result showed that there were 1 410, 859, and 638 SSRs in B. japonicum, M. loti, and S. meliloti genomes, respectively. In the genomes of B. japonicum, M. loti, and S. meliloti, tetranucleotide, pentanucleotide, and hexanucleotide repeats were more abundant and indicated higher mutation rates in these species. The least abundance was mononucleotide repeat. The SSRs type and distribution were similar among these species.

The genome of herpes simplex virus type 1 is prone to form short repeat sequences

Herein, we report a very high content of simple sequence repeats (SSRs) covering 66.12% of the herpes simplex virus type 1 (HSV-1) genome when a low threshold is adopted to define SSRs, indicating that repeat sequence is a very important character of the HSV-1 genome. The repeats with two iterations account for 68.33% of the total repeats. In reality, the genome of HSV-1 is prone to form shorter repeat sequences. For mono-, di- and trinucleotide repeats, the repeat numbers decreased with the increase of repeats iterations, implicating that the formation tendency of SSRs might be from low iterations to high iterations. The high iterations SSRs might have subjected to strong selected pressure and survived to perform different functions. The analysis suggested that the repeats formation may be an essential evolutionary driving force for the HSV-1 genome, and the results might be helpful for studying the genome structure, repeats genesis and genome evolution of HSV-1.

Confirmation of Pearl Millet-Napiergrass Hybrids Using EST-Derived Simple Sequence Repeat (SSR) Markers

Prospects for deploying perennial grasses that are currently considered leading candidates for dedicated energy crops over large acreages are debatable because of several limitations, including vegetative propagation or small seed size, low biomass production during the first growing season, and incomplete assessments of crop invasiveness risk. Pearl Millet-Napiergrass hybrids (“PMN”;Pennisetum glaucum [L.] R. Br. × P. purpureum Schumach.), in contrast, are large-seeded, sterile feedstocks capable of high biomass production during establishment year. Novel methods are warranted for confirmation of PMN hybrids, as traditional morphological observations can be inconclusive and chromosome number determination using cytological methods is laborious and time consuming. Six putative PMN lines were produced in this study, and 10 progeny from each line were evaluated using morphological traits, seed fertility, flow cytometry, and expressed sequence tag-simple sequence repeat (EST-SSR) markers. All putative hybrid lines were sterile and failed to produce seed. The PMN hybrids could not be distinguished from either parent using flow cytometry due to highly similar nuclear genome DNA contents. A number of paternal napiergrass-specific EST-SSRs were identified for each PMN line, and four paternal-specific EST-SSRs conserved across all napiergrass accessions were selected to screen the putative PMN hybrids. These EST-SSRs confirmed that all F1 individuals analyzed were PMN hybrids. The use of paternal-specific markers therefore provides a valuable tool in the development of both “Seeded-yet-Sterile” biofuel PMN feedstocks and additional PMN cultivar-and parental species-specific markers.

cpSSR: a New Tool to Analyze Chloroplast Genome of Citrus Somatic Hybrids

Chloroplast simple sequence repeat (cpSSR) markers in Citrus were developed and successfully used to analyze chloroplast genome inheritance of Citrus somatic hybrids. Twenty-two previously reported cpSSR primer pairs from pine (Pinus thunbergii Parl.), rice (Otyza sativa L.) and tobacco (Nicotiana tabacum L.) were tested in Citrus, nine of which could amplify intensive PCR products by agarose gel electrophoresis. Chloroplast genome inheritance of Citrus somatic hybrids from nine fusions was then analyzed, and five of the nine pre-screened primer pairs showed polymorphisms by polyacrylamide gel electrophoresis. The results revealed the random inheritance nature of chloroplast genome in all analyzed Citrus somatic hybrids, which was in agreement with previous reports based on RFLP or CAPS analyses. It was also shown that cpSSR is a more efficient tool in chloroplast genome analyses of somatic hybrids in higher plants, compared with the conventional RFLP or CAPS analyses.

Frequency and Distribution of Microsatellites in the Genome of Filamentous Fungus,Neurospora crassa

A total of 38.0 Mb of publicly available DNA sequence in Neurospora crassa was researched for mono- to hexanucleotide simple sequence repeats (SSR or microsatellite) to determine the type, size and frequency. A total of 14 788 SSRs were observed in the whole genomic DNA sequence, about one every 2.57 kb, with the criteria of SSR length >15 bp and 80% matches. The most abundant microsatellite was trinucleotide repeat, the number was 4 729, followed by hexanucleotide and mononucleotide repeats, the numbers were 2 940 and 2 489 respectively, and the least abundance was dinucleotide repeat, only 691 were found. Among the 10 082 ORFs, 4 094 SSRs were harbored in 2 373 ORF (no intron) of the organism. One thousand and fifty six ORFs harbored only one SSR. Similar with other organisms, tri- and hexanucleotide repeats were predominant in ORFs, 54.1 and 48.8% of tri- and hexanucleotide repeats were distributed in ORF region. The density of these two motifs was overpresented in coding regions, because ORF region and coding region constitutes only 46 and 38.3% of genomic sequence, respectively. Upstream and downstream 300 bp of regulatory regions were high density regions of SSRs, particularly density of pentanucleotide SSR in upstream region was as high as five times of average density in genomic DNA, density of di- and tetranucleotide SSR was also more than two times of average density. The density of penta-, tetra-, di- and mononucleotide SSRs was relatively higher than average density. There were 47 SSRs in mitochondria 64 840 bp DNA sequence, their distribution is similar with genomic DNA sequence. These results suggested that SSRs were clustered in regulatory regions of genomic DNA.

SSR Analysis on Diversity of AA Genome Oryza Species in the Southeast and South Asia

To investigate genetic diversities among the AA genome Oryza species in the Southeast and South Asia, a total of 428 accessions of the AA genome Oryza species were genotyped using 36 simple sequence repeats (SSR) markers distributed throughout the rice genome. All of the 36 SSR markers generated polymorphic bands, revealing 100% polymorphism. The number of alleles per locus ranged from 3 to 17 with the mean of 8.6. The Nei's genetic diversity index (He) ranged from 0.337 at RM455 to 0.865 at RM169 with an average value of 0.650. The genetic diversity of the AA genome Oryza species in the Southeast Asia was obviously higher than that in the South Asia. Among the detected Oryza species in the South and Southeast Asia, O. rufipogon showed the highest genetic diversity. Meanwhile, a higher genetic differentiation (Fst) was found among the detected Oryza species in the Southeast Asia than in the South Asia. The Fst value between O. nivara and O. sativa was the highest. The results from the number of specific alleles, specific loci, and allele frequency confirmed the greater genetic variation among the detected species. In addition, the specific allele in RM161 displayed higher frequency (0.193), suggesting its important function in identifying Oryza species of AA genome.

Development of genomic resources for the genus Celtis (Cannabaceae) based on genome skimming data

Celtis is a Cannabaceae genus of 60e70 species of trees,or rarely shrubs,commonly known as hackberries.This woody genus consists of very valuable forest plants that provide important wildlife habitat for birds and mammals.Although previous studies have identified its phylogenetic position,interspecific relationships within Celtis remain unclear.In this study,we generated genome skimming data from five Celtis species to analyze phylogenetic relationships within the genus and develop genome resources.The plastomes of Celtis ranged in length from 158,989 bp to 159,082 bp,with a typical angiosperm quadripartite structure,and encoded a total of 132 genes with 20 duplicated in the IRs.Comparative analyses showed that plastome content and structure were relatively conserved.Whole plastomes showed no signs of gene loss,translocations,inversions,or genome rearrangement.Six plastid hotspot regions(trnH-psbA,psbA-trnK,trnG-trnR,psbC-trnS,cemA-petA and rps8-rpl14),4097 polymorphic nuclear SSRs,as well as 62 low or single-copy gene fragments were identified within Celtis.Moreover,the phylogenetic relationships based on the complete plastome sequences strongly endorse the placement of C.biondii as sister to the((((C.koraiensis,C.sinensis),C.tetrandra),C.julianae),C.cerasifera)clade.These findings and the genetic resources developed here will be conducive to further studies on the genus Celtis involving phylogeny,population genetics,and conservation biology.

EFFICIENT ALGORITHMS FOR IDENTIFYING ORTHOLOGOUS SIMPLE SEQUENCE REPEATS OF DISEASE GENES

Dynamic mutations of simple sequence repeats （SSRs） have been demonstrated to affect normal gene function and cause different genetic disorders. Several conserved and even partial functional SSR patterns are discovered in inherited orthologous disease genes. To explore a wide range of SSRs in genetic diseases, a comprehensive system focusing on identifying orthologous SSRs of disease genes through a comparative genomics mechanism is constructed and accomplished by adopting online Mendelian inheritance in man （OMIM） and NCBI HomoloGene databases as the fundamental resources of human genetic diseases and homologous gene information. In addition, an efficient and effective algorithm for searching SSR patterns is also developed for providing annotated SSR information among various model species. By integrating these data resources and mining technologies, biologists and doctors can systematically retrieve novel and important conserved SSR information among orthologous disease genes. The proposed system, Orthologous SSR for Disease Genes （OSDG）, is the first comprehensive framework for identifying orthologous SSRs as potential causative factors of genetic disorders and is freely available at http：//osdg.cs.ntou.edu.tw/.

了哥王叶绿体基因组分析

【目的】阐明药用植物了哥王Wikstroemia indica的叶绿体基因组结构特点及系统进化地位,为了哥王的资源保护和可持续利用提供科学依据。【方法】采用Illumina测序平台进行了哥王叶绿体基因组测序,并通过生物信息技术和软件进行序列拼接、注释以及比对和系统进化分析。【结果】了哥王叶绿体基因组全长为149864 bp,由86347 bp的大单拷贝区(LSC)、10601 bp的小单拷贝区(SSC)以及穿插在它们之间均为26458 bp的一对反向重复区(IR)构成,具有环状双链四分体结构,包含124个基因。在了哥王叶绿体基因组中共找到64种24180个密码子,其中30种为高频使用密码子,高频使用密码子中又有29种是以A/T结尾;搜索到93个简单重复序列(SSR),其中单核苷酸重复居多(72个),且以A或T及两者组合形成的基序为优势基序。了哥王与近缘植物的叶绿体基因组IR边界存在较为明显的变异。序列比较和系统进化树显示了哥王与同属细轴荛花W.nutans具有最高的序列同源性。【结论】了哥王叶绿体基因组具有植物叶绿体基因组的典型结构,有密码子使用偏好性,含多态性较为丰富的SSR,且与细轴荛花的亲缘关系最近。

五指毛桃叶绿体基因组结构与序列特征分析

【目的】阐明药食两用植物五指毛桃的叶绿体基因组结构及其系统进化关系,为其资源利用和产品开发等研究提供基因组信息。【方法】采用高通量测序技术对五指毛桃叶绿体基因组测序,并借助生物信息工具和软件进行序列拼接、注释、比对和系统进化分析。【结果】五指毛桃叶绿体基因组为环状双链四分体分子,长度160 340 bp,GC含量为35.9%,包含130个基因。该基因组含有26 679个密码子,偏好使用以A/T结尾的密码子;共有93个简单重复序列,其中以A/T形成的单、二核苷酸重复为优势重复基序。五指毛桃与其他榕属植物相比,叶绿体基因组序列同源性较高,碱基变异位点数量较少,主要分布在非编码区域如rpoB-trnC、trnT-trnL、rpl32-trnL等,与薜荔具有最高的序列相似度。【结论】五指毛桃叶绿体基因组具有典型的高等植物叶绿体基因组结构和基因组成,存在密码子偏好性,包含类型丰富的简单重复序列,且与同属植物薜荔的亲缘关系最近。

五爪金龙叶绿体基因组特征分析

为了阐明药用植物五爪金龙Ipomoea cairica的叶绿体基因组特征,选用五爪金龙叶片为样品,采用试剂盒法提取DNA并建立测序文库,随后使用新一代细胞器基因组测序技术进行高通量测序,并利用生物信息学手段进行序列组装和特征分析。结果显示,五爪金龙叶绿体基因组为环状双链四分体分子,全长161780 bp,由88205 bp的大单拷贝区(Large Single Copy,LSC)、30747 bp的反向重复区(Inverted Repeat,IR)A、12081 bp的小单拷贝区(Small Single Copy,SSC)和30747 bp的反向重复区B(IRB)排列而成,含有131个基因;共有28757个密码子,偏好使用以A或T结尾的密码子;存在67个简单重复序列(Simple Sequence Repeat,SSR),并以A/T单核苷酸重复居多。五爪金龙与近缘植物在IRB-SSC边界之间存在较为明显的变异,与同属三裂叶薯I.triloba、番薯I.batatas等的亲缘关系更近。本研究报道了五爪金龙叶绿体基因组的结构和序列组成,并分析其特征,为五爪金龙的物种鉴定、遗传进化及开发利用提供了基因组信息。

毛茛铁线莲叶绿体全基因组解析及系统发育分析

为了解析药用植物毛茛铁线莲的叶绿体全基因组特征及系统发育位置,利用高通量测序技术对毛茛铁线莲叶片样品进行测序,并对毛茛铁线莲叶绿体基因组进行组装、注释和特征分析,采用最大简约法(MP)、最大似然法(ML)及贝叶斯法(BI)构建分子系统发育树.结果表明,毛茛铁线莲的叶绿体基因组为典型的四分体结构,全长159 741 bp,共编码112个基因,包括79个蛋白编码基因、29个tRNA基因和4个rRNA基因;毛茛铁线莲叶绿体基因组共含有84个简单重复序列,以单核苷酸重复基序居多,共57个;毛茛铁线莲叶绿体基因组使用频次最高的氨基酸是亮氨酸;基于叶绿体全基因组构建的MP,ML和BI树,拓扑结构基本一致;铁线莲属是明显的单系类群,与银莲花属关系最近,毛茛铁线莲与绣球藤亲缘关系最近.

Exploration of presence/absence variation and corresponding polymorphic markers in soybean genome

This study was designed to reveal the genome‐wide distribution of presence/absence variation（PAV） and to establish a database of polymorphic PAV markers in soybean. The 33 soybean whole‐genome sequences were compared to each other with that of Williams 82 as a reference genome. A total of 33,127 PAVs were detected and 28,912 PAV markers with their primer sequences were designed as the database NJAUSoyPAV_1.0. The PAVs scattered on whole genome while only 518（1.8%） overlapped with simple sequence repeats（SSRs） in BARCSOYSSR_1.0database. In a random sample of 800 PAVs, 713（89.13%） showed polymorphism among the 12 differential genotypes. Using 126 PAVs and 108 SSRs to test a Chinese soybean germplasm collection composed of 828 Glycine soja Sieb. et Zucc. and Glycine max（L.） Merr. accessions, the per locus allele number and its variation appeared less in PAVs than in SSRs. The distinctness among alleles/bands of PCR（polymerase chain reaction） products showed better in PAVs than in SSRs, potential in accurate marker‐assisted allele selection. The association mapping results showed SSR t PAV was more powerful than any single marker systems.The NJAUSoyPAV_1.0 database has enriched the source of PCR markers, and may fit the materials with a range of per locus allele numbers, if jointly used with SSR markers.

北陵鸢尾叶绿体基因组及其特征分析

本研究为探究北陵鸢尾(Iris typhifolia)叶绿体基因组的基本特征,明确其在鸢尾属的系统发育位置,采用Illumina二代测序技术对北陵鸢尾叶绿体基因组进行测序,对其叶绿体基因组基本特征进行分析,结果表明:北陵鸢尾叶绿体基因组大小为152405 bp,具有典型的环状四分体结构,包含82335 bp的大单拷贝区(LSC)、18018 bp的小单拷贝区(SSC)和一对26026 bp的反向重复区(IRs),总GC含量为38.04%;共注释得到133个基因,包含87个蛋白编码基因、8个rRNA基因和38个tRNA基因;共检测到57个SSR位点,其中单核苷酸重复数量最多;系统发育分析表明北陵鸢尾和溪荪(I.sanguinea)关系较近,支持率为100%。本研究通过对北陵鸢尾叶绿体基因组重复序列、IR边界、系统发育等进行分析,为北陵鸢尾的分子标记开发、系统发育分析、物种鉴定和种质资源开发及利用提供依据。

大银鱼全基因组简单重复序列标记开发及在不同生态型群体中的检验

利用Krait软件对2020年公布的拼接程度更高的大银鱼全基因组中完美型微卫星的分布特征进行分析,并据此开发具有多态性的微卫星DNA(又称简单重复序列)标记。结果显示:在大银鱼全基因组内共获得587 554个完美型微卫星位点,序列总长度为11 803 017 bp,占全基因组长度的2.53%;在6种重复类型的微卫星中,二核苷酸数量最多(401 585个,占比68.35%)。针对微卫星位点设计的99对引物中,有39对具有多态性,选择其中多态性较好的14个微卫星标记分别对大银鱼洄游型群体、陆封型群体及移植型群体中选择的1个具有代表性的群体进行检验,结果表明,多态性较好的14个微卫星标记在3个具有代表性的群体中均能进行有效扩增。对3个群体的遗传多样性和遗传结构进行分析发现,洄游型群体(崇明岛群体)遗传变异丰富(平均期望杂合度为0.614、平均多态信息含量为0.576),与淡水群体[太湖群体(陆封型)和连环湖群体(移植型)]分属于2个不同的遗传群组,两者间具有较大的遗传距离和极高的遗传分化水平(遗传分化指数高于0.25,P<0.05);淡水群体(太湖和连环湖群体)的遗传变异相对匮乏,2个群体间的遗传距离较小,虽存在显著的遗传分化,但遗传分化水平较低(遗传分化指数为0.102,P<0.05)。本研究结果表明,洄游型群体具有潜在的种质资源保护价值,可为后续大银鱼全基因组微卫星标记开发和遗传图谱构建等提供依据,并为后续更大范围的群体种质资源评估与管理提供参考。

长叶竹柏叶绿体基因组特征及系统发育分析

本研究以长叶竹柏叶片为材料,对其进行叶绿体基因组测序及构建系统发育树,分析长叶竹柏叶绿体基因组的结构特征。结果表明,长叶竹柏的叶绿体基因组结构(GenBank No.OL435123)缺乏反向重复(IR)区,叶绿体基因组大小为133870 bp,总GC含量为37%,共注释基因数为119个,包括80个蛋白编码基因,35个tRNA基因和4个rRNA基因。叶绿体基因组注释显示,大多数的基因与光合作用及基因转录翻译相关,其中43个光合作用相关基因、64个自我复制基因、8个其他蛋白编码基因和4个未知功能基因。密码子偏好性分析表明,共检测出44623个密码子,亮氨酸(Leu)是长叶竹柏叶绿体基组中占比最大的密码子,占10.54%,密码子偏向使用A/U两种碱基。长叶竹柏叶绿体基因组有49个SSRs,其中包含26个单核苷酸、22个二核苷酸和1个三核苷酸3种类型的SSRs。系统进化树显示,长叶竹柏(Nageia fleuryi,OL435123.1)与竹柏(Nageia nagi,AB830885.1和LC572146.1)聚在同一支,具有较近的亲缘关系。

秀珍菇全基因组SSR位点分析及其在遗传多样性评估中的应用

利用GenBank数据库中秀珍菇(Pleurotus pulmonarius)的全基因组序列进行简单重复序列(SSR)位点挖掘。在秀珍菇PM_ss5基因组中共检测出2348个SSR位点,相对丰度为平均1 Mb中含有59个SSR位点;所有SSR位点中,以二核苷酸SSR为主(51.8%),三核苷酸SSR次之(27.7%);经鉴定的秀珍菇SSR位点包含141种碱基基序,优势碱基基序为GA/TC、CT/AG;SSR长度变化范围为10~156 bp,其中,10~15 bp的SSR位点占比为77.2%;在秀珍菇和其他4种侧耳属真菌基因组中,都是以短核苷酸SSR为主,秀珍菇二核苷酸SSR占比高于其他侧耳属菌株;利用筛选的53对多态性引物对18个秀珍菇菌株进行遗传多样性分析,结果发现参试菌株表现出中度遗传多样性,平均Shannon信息指数为0.38,平均Nei’s基因多样性为0.23,平均有效等位基因数为1.35。

直立型半野生大豆叶绿体基因组分析

采用Illumina高通量测序技术对直立型半野生大豆(Glycine gracilis)叶绿体基因组(Chloroplast DNA,cpDNA)进行测序和组装,获得完整基因组,以探究其结构特征,为后续基于cpDNA的相关分析以及大豆资源多样性保护和系统发育等研究提供基础或依据。结果表明,直立型半野生大豆cpDNA全长148320 bp,为典型四分体结构,注释基因108个,包含66个蛋白编码基因、29个tRNA基因和4个rRNA基因。基因组序列上共检测到87个SSR位点,其中与单核苷酸、双核苷酸、三核苷酸、四核苷酸、五核苷酸对应的位点分别为55,19,4,7,2个。在系统发育上,直立型半野生大豆与已公布的普通型一年生半野生大豆(Glycine gracilis)、栽培大豆(Glycine max)及野大豆(Glycine soja)亲缘关系较近。

4种金花茶叶绿体基因组比较分析

利用全基因组重测序数据组装得到了夏石金花茶等4种金花茶的叶绿体全基因组序列,并进行了注释和比较分析.结果表明,4种金花茶植物叶绿体基因组序列高度相似,约为156657~157046 bp,均预测注释134个基因,包含89个蛋白编码基因、8个rRNA和37个tRNA;金花茶植物的叶绿体基因组在结构和进化上具有保守性,它们具有相似的密码子偏好性且均未发生大面积的倒位和基因重排;通过叶绿体基因组的比较分析和核苷酸多态性分析发掘了rps16和ycf1等高变异片段,这些片段可作为金花茶植物的分子标记;基于金花茶叶绿体基因组数据构建的系统进化树具有较高的支持度,说明金花茶叶绿体基因组序列的公布对其系统发育的研究具有重要意义.

露兜树叶绿体基因组结构与序列特征分析

目的 明确露兜树Pandanus tectorius Soland叶绿体基因组的结构、序列特征和系统进化关系。方法 以露兜树叶片总DNA为材料,采用illumina NovaSeq高通量测序列平台进行叶绿体基因组测序,利用生物信息学工具对其进行组装、注释和特征分析,并基于最大似然法构建系统进化树。结果 露兜树叶绿体基因组呈典型的环状四分体结构,全长为157 747 bp,其中大单拷贝区的长度为85 929 bp,小单拷贝区的长度为17 992 bp,两个反向互补区的长度则均为26 913 bp。共含有131个基因,其中蛋白编码基因85个,核糖体RNA基因8个,转运RNA基因38个。露兜树叶绿体基因组密码子偏向使用A或T碱基结尾;共检测到144个简单重复序列,其中以A或T碱基的单核苷酸重复为主。基于叶绿体基因组序列的系统进化分析显示露兜树与同目植物巴拿马草同源性较高。结论 获得了露兜树叶绿体基因组序列及其特征信息,为该药用植物的分子标记开发和遗传多样性研究奠定了基础。