Development of Starfruit Simple Sequence Repeat (SSR) Using Next Generation Sequencing

Starfruit (Averrhoa carambola L.) is an important fruit for Malaysian export and great attention has been made to improve starfruit fruit quality at Malaysian Agricultural Research and Development Institute (MARDI). The current study used next generation sequencing (NGS) technologies to develop starfruit simple sequence repeat (SSR) from 2 varieties namely B11 and B17 using Illumina HiSeq. The pre-processed reads were de novo assembled to generate approximately 75,000 and 74,000 scaffolds respectively. Total genome size for B11 and B17 were around 345 Mbp and 342 Mbp based on K-mer distribution analysis. In-silico microsatellite mining of each variety has identified more than 17,000 SSR in B11 and B17 respectively. Dinucleotides were the most abundant, accounting for more than 70% of all SSR and repeat motif GA (49%) was most common. A total of 239 SSR primer pairs were designed from contigs larger than 350 nucleotides and tested for amplification. The 30 polymorphic SSRs were used to DNA fingerprint of 12 starfruit hybrids. Polymorphism information content (PIC) ranged from 0.1411 to 0.6838, with an average of 0.3919. The Unweighted Pair-Group Method for Arithmetic Averages (UPGMA) dendrogram clustered 12 starfruit accessions into 2 groups.

Mapping of a Wheat Resistance Gene to Yellow Mosaic Disease by Amplified Fragment Length Polymorphism and Simple Sequence Repeat Markers

Wheat （Triticum aestivum L.） yellow mosaic virus （WYMV） is transmitted by a fungal vector through soil and causes serious wheat yield losses due to yellow mosaic disease, with yellow-streaked leaves and stunted plants. In the present study, the amplified fragment length polymorphisms （AFLP） and simple sequence repeat （SSR） were used to identify the molecular linkages with the resistance gene against WYMV. Bulked segregant analysis was performed with an F2 population derived from the cross of cultivar Ningmai 9 （resistant） × cultivar Yangmai 10 （susceptible）. By screening among the resistant or susceptible parents, the F2 pools and the individuals in the F2 population with 64 combined selective AFLP primers （EcoRI/MseI） or 290 reported SSR primers, a polymorphic DNA segment （approximately 120 bp） was amplified using the primer pair E2/M5, and an SSR marker （approximately 180 bp） was located on wheat chromosome 2A using the primer Xgwm328. Analysis with MAPMAKER/Exp Version 3.0b （Whitehead institute for Biomedical Research, Cambridge, MA, USA） indicated that these two markers were dominantly associated with the resistance gene at distances of 5.4 cM or 17.6 cM, respectively. The resistance gene to WYMV derived from Ningmai 9, is temporarily named YmNM, and was mapped to wheat chromosome 2A.

Identification of necrophagous fly species from 12 different cities in China using ISSR and SCAR markers

Objective:To identify necrophagous fly speies from different regions in China using inter simple sequenc repeat(ISSR) and sequence-characterized amplified region(SCAR) melocular markers and to analyze their gene difference and genetic relationship.Methods:Five carrion fly species were collected from 12 cities and regions in China,including Musca domestica(M.domestica), Lucilia sericata(L.sericata),Chrysomya megacephala(C.megacephala),Helicophagella melanura(H.melanura),Boethcherisca peregrina,and they were studied using ISSR and SCAR markers.Results:Eight ISSR primers were used for amplification of 121 samples.679 clear and stable bands were identified,of which 516 bands were polymorphic.Several species-specific ISSR fragment were cloned and sequenced as an initial effort to derive the SCAR markers.Using M.domestica SCAR specific primers,SCAR-PCR amplification was performed for 8 M.domestca population sample DNA from different regions in China as well as L sericata,C.megacephala, H.melanura and Lucillia cupirina.The result showed only M.domestica produced specificalty 600 bp fragment,but L sericata,C.megacephala,H.melanura and Lucillia cupirina did not produce the same specific fragment.Clustering analysis showed clustering of most flies of M. domestica,C.megacephala and L sericata.M.domestica samples from different regions in China yielded different banding patterns.Conclusions:Application of ISSR-PCR and SCAR markers to identify necrophagous fly species from 12 cities and regions in China is first reported.ISSR-PCR and SCAR markers provide a quick reliable molecular marker technique for the identification of different species of necrophagous fly.

Development and Characterization of Microsatellite Markers in Brassica rapa ssp.chinensis and Transferability Among Related Species

Simple sequence repeat （SSR） or microsatellite marker is a valuable tool for several purposes, such as mapping, fingerprinting, and breeding. In the present study, an intersimple sequence repeat （ISSR）-PCR technique was applied for developing SSR markers in non-heading Chinese cabbage （Brassica rapa）. A total of 190 SSRs were obtained. Among these, AG or CT （54.7%） was the most frequent repeat, followed by AC or GT （31.6%） of the microsatellites. The average number of the SSRs length array was 16 and 10 times, respectively. Based on the determined SSR sequences, 143 SSR primer pairs were designed to evaluate their transferabilities among the related species of Brassica. The number of alleles produced per marker averaged 2.91, and the polymorphism information content （PIC） value ranged from 0 to 0.863 with an average of 0.540. Monomorphism was observed in 16 primer pairs. The transferability percentage in CC genome was higher than in BB genome. More loci occurred in the BBCC genome. This result supported the hypothesis that BB genome was divergent from A and C genomes, and AA and CC genomes were relatively close. The polymorphic primers can be exploited for further evolution, fingerprinting, and variety identification.

Genome-wide development of interspecific microsatellite markers for Saccharum officinarum and Saccharum spontaneum

Sugarcane has a large,complex,polyploid genome that has hindered the progress of genomic research and molecular marker-assisted selection.The user-friendly SSR markers have attracted considerable attention owing to their ideal genetic attributes.However,these markers were not characterized and developed at the genome-wide scale due to the previously lacking high-quality chromosome-level assembled sugarcane genomes.In this present study,744305and 361638 candidate SSRs were identified from the genomes of S.officinarum and S.spontaneum,respectively.We verified the reliability of the predicted SSRs by using 1200 interspecific SSR primer pairs to detect polymorphisms among 11 representative accessions of Saccharum,including S.spontaneum,S.officinarum,S.robustum,and modern sugarcane hybrid.The results showed that 660 SSR markers displayed interspecific polymorphisms among these accessions.Furthermore,100 SSRs were randomly selected to detect the genetic diversity for 39 representative Saccharum accessions.A total of 320 alleles were generated using 100 polymorphic primers,with each marker ranging from two to seven alleles.The genetic diversity analysis revealed that these accessions were distributed in four main groups,including group I(14 S.spontaneum accessions),group II(two S.officinarum accessions),group III(18 modern sugarcane hybrid accessions),and group IV(five S.robustum accessions).Experimental verification supported the reliability of the SSR markers based on genome-wide predictions.The development of a large number of SSR markers based on wet experiments is valuable for genetic studies,including genetic linkage maps,comparative genome analysis,genome-wide association studies,and marker-assisted selection in Saccharum.

水芹SSR分子标记开发与遗传多样性分析

水芹是伞形科水芹属多年生草本植物,是一种重要的药食两用蔬菜作物。在中国,水芹的种植区域十分广泛,然而目前对其种质资源的鉴定、培育及遗传信息的研究较少。本研究利用溧阳白芹基因组开发水芹简单重复序列(SSR)分子标记,分析55份水芹的遗传多样性并用非加权组平均法(UPGMA)构建系统进化树,同时用SSR扩增条带数据构建DNA指纹图谱。结果显示,共鉴定到325699个SSR位点,其中单核苷酸SSR重复单元、二核苷酸SSR重复单元、三核苷酸SSR重复单元、四核苷酸SSR重复单元、五核苷酸SSR重复单元、六核苷酸SSR重复单元的出现频率分别为33.94%、54.62%、9.31%、1.66%、0.17%、0.29%,其中二核苷酸SSR重复单元数最多,有177887个,且A/T(占比为29.98%)和AT/AT(占比为35.70%)是较丰富的重复类型。UPGMA分析结果表明,33对高多态性引物[多态信息含量(PIC)>0.25]可将55份水芹材料分为4组。利用筛选出的4对引物(Oj-084、Oj-110、Oj-112、Oj-156)可以将55份水芹材料完全区分开,并且可构建指纹图谱。研究结果可为水芹种质资源鉴定、保护及分子遗传育种提供有力依据。

20份燕麦种质遗传多样性分析

【目的】了解20份燕麦种质间的相对亲缘关系。【方法】利用8对SSR多态性引物对20份燕麦种质进行遗传多样性分析。【结果】8对引物共扩增出44个位点,其中多态性位点有40个,平均每对引物扩增出5.5个位点。SSR标记位点平均多态性信息含量、平均有效等位基因数、平均香农指数、平均期望杂合度和平均观测杂合度分别为0.6308、3.0524、1.2710、0.6731和0.5521。聚类结果表明:20份燕麦种质的遗传相似系数变幅为0.14~1.00,在相似系数0.59处,可将20份材料分为7组。主成分分析结果显示前3个主成分占总变异的83.44%,根据第一及第二主成分可将20份种质分为4个大组,且与聚类结果基本一致。【结论】所选用引物多态性较好,遗传多样性较高,不同燕麦种质间遗传关系与其地理来源具有一定相关性,研究结果可为燕麦遗传改良及分子育种提供参考。

SSR分子标记在玉米研究中的应用

SSR简单重复序列也称微卫星DNA,是由特异性的引物进行PCR扩增分析的一种分子标记技术。简要介绍了SSR分子标记的原理,分析了SSR分子标记在玉米的种质资源、品种纯度鉴定、真伪鉴定、遗传多样性、种质性状等方面的应用。通过研究表明:能够利用SSR分子标记技术对玉米的品种纯度鉴定、真伪性鉴定、遗传结构、亲缘关系、优劣群体的划分、种质性状等方面进行分析,同时也能为以后研究玉米的遗传连锁图谱构建、分子标记辅助育种、基因定位和种质资源等方面提供参考。

Simple Sequence Repeat Genetic Linkage Maps of A-genome Diploid Cotton (Gossypium arboreum)

This study introduces the construction of the first intraspecific genetic linkage map of the A-genome diploid cotton with newly developed simple sequence repeat （SSR） markers using 189 F2 plants derived from the cross of two Asiatic cotton cultivars （Gossypium arboreum L.） Jianglingzhongmlan x Zhejiangxiaoshanl（ishu. Polymorphisms between the two parents were detected using 6 092 pairs of SSR primers. Two-hundred and sixty-eight pairs of SSR primers with better polymorphisms were picked out to analyze the F2 population. In total, 320 polymorphic bands were generated and used to construct a linkage map with JoinMap3.0. Two-hundred and sixty-seven loci, including three phenotypic traits were mapped at a logarithms of odds ratio （LOD）≥ 3.0 on 13 linkage groups. The total length of the map was 2 508.71 cM, and the average distance between adjacent markers was 9.40 cM. Chromosome assignments were according to the association of linkages with our backbone tetraploid specific map using the 89 similar SSR loci. Comparisons among the 13 suites of orthologous linkage groups revealed that the A-genome chromosomes are largely collinear with the At and Dt sub- genome chromosomes. Chromosomes associated with inversions suggested that allopolyploidization was accompanied by homologous chromosomal rearrangement. The inter-chromosomal duplicated loci supply molecular evidence that the A-genome diploid Asiatic cotton is paleopolyploid.

SSR分子标记在作物遗传育种中的应用

SSR(simple sequence repeat)是建立在PCR技术上的一种广泛应用的分子标记,具有含量丰富、多态性高、共显性等优点。本文简要介绍了SSR分子标记技术的原理和特点,重点介绍了SSR分子标记技术在作物遗传育种中的应用,主要在作物遗传多样性、基因定位、分子辅助标记、遗传图谱构建、品种鉴定和纯度鉴定等方面进行阐述。

SSR标记遗传距离与粳稻杂种优势的相关性分析

利用SSR分子标记对30个粳稻品种进行遗传多样性分析,继而研究分子标记遗传距离与按照NCⅡ设计获得的200个杂交组合主要产量性状杂种优势的相关性,探讨分子标记遗传距离预测杂种优势的可行性。结果表明,64对SSR引物共检测到185条多态性片段,平均每对引物2.9条,每个SSR位点的多态性信息含量指数(PIC值)变化范围为0.064～0.844,平均为0.380。以SSR标记遗传相似系数为原始数据,按UPGMA聚类方法将30个亲本材料划分为7大类群,分类结果与系谱关系基本相符。分子标记遗传距离与杂种性状平均值的相关除每穗总粒数外均达到显著或极显著水平,与杂种优势的相关均达到极显著水平,相关系数介于-0.361～0.359,说明分子标记可用于水稻杂种优势群的划分和遗传多样性分析,但相关程度还不足以预测产量杂种优势。

ISSR-PCR在石斛种间鉴别中的应用

目的 采用ISSR-PCR方法对石斛属9种植物进行鉴别,探讨不同种石斛在DNA分子水平上的差异。方法 选取10条由SSR组成的引物,对9种石斛进行PCR扩增及琼脂糖凝胶电泳分析。结果 10条引物中有7条扩增出多态性条带。每条引物可检测的多态性位点最少7个,最多14个,扩增片段大小为220-1260 bp。其中,引物UBC-807和UBC-864具有较高的多态性条带比率,均可以独立将所有被测种区分开来。结论ISSR-PCR作为一种简便、可靠的分子标记鉴定技术,可以作为石斛属种间鉴别的方法之一。

水稻亚种间单片段代换系的建立

利用微卫星标记辅助选择 ,建立了 2 9个以台中 6 5为受体、低脚乌尖或窄叶青为供体的单片段代换系 ,该代换系群体的代换片段分布于 9条染色体上 ,总长度为 6 43.6 5cM ,平均长度为 2 2 .19cM ,覆盖全基因组 42 7.5 5cM ,覆盖率为2 3.47%。

应用SSR分子标记鉴定超级杂交水稻组合及其纯度

应用SSR分子标记技术对超级杂交稻5 个组合(HYS 1/R105、培矮64S/E32、两优培九、88S/0293、J23A/Q611)及其9个亲本进行了鉴定。用144对SSR引物进行筛选,有47对能够在实验材料中显示较好的多态性,其中,RM337与RM154呈现丰富多态性,可鉴别供试组合并分别与其亲本区分开。对于水稻的每一条染色体,各筛选出两条产生多态性的引物,共24对,并提供一组作为鉴定参考的图谱;通过杂种表现为父母本互补带型的特点,找到在杂交稻组合及其亲本间具有多态性的引物,筛选出5对引物分别作为鉴定上述5个超级杂交稻组合的特异引物,进而针对杂交稻不同的纯度问题设计鉴定方法。

基于转录组数据的马氏珠母贝EST-SSR位点的信息分析及其多态性检测

为获得稳定可靠的马氏珠母贝SSR分子标记,本文利用MISA软件对转录组测序数据进行了大规模的EST-SSR标记发掘,并分析了位点信息及其多态性。结果表明,马氏珠母贝转录组测序所获得的74007条Unigenes中检测出9872个EST-SSR位点,位点出现频率为13.34%,平均每5102 bp含有1个SSR位点。在转录组SSR中共有132种重复基元类型,其中单核苷酸重复基元为主要类型,占SSR总数的81.46%;单核苷酸重复以A/T基序为主,占SSR总数的71.27%。基于筛选的SSR序列,应用Primer3软件进行引物的批量设计,共为5922条EST-SSR序列成功设计出17766对引物。随机选择80对引物对EST-SSR多态性进行检测,共有62对引物成功扩增出稳定条带,占引物总数的77.5%;其中,17对EST-SSR引物表现出个体多态性,多态性比率为27.42%。以上研究为马氏珠母贝遗传多样性、遗传图谱构建及分子辅助育种研究提供了有效工具,对于马氏珠母贝种质资源保护、优良品种培育和珍珠养殖业的健康发展均具有重要意义。

SSR标记技术及其在植物遗传学中的应用

SSR是建立在PCR技术上的新型分子标记 ,与RFLP、RAPD、AFLP相比 ,SSR具有多态性高 ,结果稳定可靠 ,重复性好 ,操作简便等特点 .阐述了SSR的原理和方法 。

一种可用于PCR分析的水稻DNA简易提取法

以水稻黄化苗为材料,用NaOH溶液抽提DNA,对其用于基于PCR技术的DNA标记中的效果进行了分析。结果发现,用0.5mol/LNaOH对黄化苗进行直接处理,并用等量1mol/LTrisHCl进行稀释、中和,离心所得的上清液即可直接用于各种PCR扩增和随后的DNA标记鉴别,包括转基因PCR片段检测、微卫星标记多态性分析(琼脂糖电泳法或毛细管电泳法),用这种方法提取的DNA可以在短期内保存,在PCR分析中其效果与用常规CTAB法提取的DNA相当。

林木基因组中的微卫星(SSR)及其应用

微卫星ＤＮＡ 是一种简单重复序列（Ｓｉｍｐｌｅ Ｓｅｑｕｅｎｃｅ Ｒｅｐｅａｔ，简称ＳＳＲ） ，其核心单位由２～４ 个核苷酸组成，两侧一般是保守序列。由于ＳＳＲ 具有共显性，多态性高，可进行ＰＣＲ 扩增分析，因此是一种很有价值的分子标记。微卫星在林木基因组中广泛存在，其核心单位主要是（ＡＧ）ｎ ，（ＡＣ）ｎ 。现已在许多林木中发现有微卫星，如：松树、杨树、桉树、苹果树。林木的微卫星标记可扩充现有的ＲＦＬＰ、ＲＡＰＤ、ＡＦＬＰ 遗传图谱，以及ＱＴＬ 分析，并应用于基因型鉴别，分子标记辅助选择育种。

灰盖鬼伞基因组中微卫星序列的组成

本研究利用已公布的灰盖鬼伞基因组测序结果,对该真菌基因组中的微卫星(microsatellite)或简单重复序列(simplesequence repeats,SSRs)进行了系统分析。结果表明,在已公布的36.2 Mb的基因组序列中,共有7 859个SSR序列(长度大于15bp,匹配值大于80%)。SSR的碱基总数达143 kb,约占整个基因组碱基数的0.40%,平均4.61 kb中就有1个大于15 bp的SSR序列。其中数量最多的是3碱基SSR,数量达到3 033个,其次为6碱基重复序列(2 121个)、5碱基重复序列(1 820个),这3种SSR总数达6 974个,占SSR总数的84.9%,单碱基重复序列数量最少,仅有285个。与子囊菌中的稻瘟病菌和粗糙脉孢菌相比,灰盖鬼伞菌基因组中每百万碱基中的SSR数量和密度都较小。这些研究结果可为该担子菌基因组的特征描述、注释及分子标记的筛选提供基础信息。

花椰菜转录组SSR位点分析及其分子标记开发

【目的】开发适合花椰菜的SSR分子标记,为花椰菜品种遗传关系鉴定、遗传图谱绘制等提供候选标记。【方法】以24份花椰菜材料为研究对象,通过MISA软件对其转录组SSR位点信息进行分析,设计SSR引物,对这些引物进行PCR扩增,开发高多态性花椰菜的SSR分子标记;利用差异条带对不同花椰菜材料的亲缘关系进行分析。【结果】从转录组数据中得到66 450条Unigene基因,包含10 715个SSR位点,发生频率为12.09%,平均分布距离为5.9kb。SSR位点中优势类型为二核苷酸重复基序,出现频率占总SSR的51.16%;其次是三核苷酸,占总SSR的47.37%。重复序列基序共49种,其中出现频率较高的重复基序主要为AG/CT、GA/TC和AAG/CTT。有1 164个长度≥20bp的SSR位点,获得具有潜在高多态性的SSR引物6 119对。随机设计的40对引物中有31对引物能进行有效扩增,其中有17对引物在24份花椰菜品种中表现出多态性。UPGMA分析显示,在遗传距离为0.625时,17对多态性引物可将24份花椰菜分为3类。【结论】开发的花椰菜SSR标记类型丰富,出现频率高,具有较高的可用性。