对除草剂敏感致死水稻bel基因的RAPD和SCAR分子标记

以8077s与抗感的籼稻品种丰35亲本及杂交后自交所得的F2群体为材料，采用群分法（Bulked Segregant Analysis, BSA），从210个10mer随机引物，找到两个水稻苯达松敏感池和抗感池之间表现多态性的特异引物——S20和S316，分别产生的标记片段为S20-440和S316-590。它们与bel基因的连锁距离分别为12.132 cM和7.97 cM。对RAPD扩增标记的片段进行克隆、测序，根据测序结果合成两对特异性的SCAR引物，包含原有的RAPD序列。SC01引物在敏感单株中扩增出一条423 bp带；SC02引物在敏感单株中扩增出一条606 bp带，它们的SCAR标记与bel基因的连锁距离为10.66 cM和7.04 cM。应用SCAR标记对水稻恢复系进行了辅助选育。     

结球甘蓝迟抽薹基因RAPD标记转SCAR标记

本研究以与结球甘蓝迟抽薹基因连锁的N1750为引物,应用RAPD技术进行PCR扩增,检测到迟抽薹基因,对特异片段进行回收、克隆和测序,依据测序结果设计SCAR引物。在166株BC1群体(A21与P02杂交得到F1再与P02回交)中通过与RAPD标记的比较,SCAR扩增结果同RAPD扩增结果完全一致,从而证实了SCAR标记的准确性。实验结果表明,与甘蓝迟抽薹基因连锁的RAPD标记被成功转化为SCAR标记,为甘蓝分子标记辅助选择育种提供了基础。     

红花大金元特异性SCAR引物筛选

建立快速准确的特色烤烟品种红花大金元的分子标记鉴定方法。通过引物设计软件Primer Premier 5设计出SCAR引物,通过SCAR-PCR及凝胶电泳结果筛选出对红花大金元具有特异性的引物。结果表明,设计出的12对SCAR引物分别对红花大金元、云烟85、云烟87和K326基因组DNA进行扩增,其中引物H1、H2和H4只有以红花大金元DNA为模板进行扩增时能获得约220 bp的片段,而非红花大金元则未扩增出该片段。     

甘薯抗茎线虫病基因的遗传分析及SCAR标记

以甘薯抗茎线虫病品种徐781和感茎线虫病品种徐薯18的杂交F1分离群体的174株单株为材料,对甘薯抗茎线虫病基因的遗传进行了分析。结果表明,徐781的抗茎线虫病为单基因控制,推测其基因型为Rrrrrr,徐薯18的基因型为rrrrrr。采用BSA-RAPD相结合的方法,筛选940条随机引物,发现10条引物在抗、感池间表现多态性。用这10条引物检测两亲本及建池单株,发现只有引物OPP03在8株抗池单株中扩增出一条在8株感池单株中所没有的特异条带,认为该标记与甘薯抗茎线虫病基因连锁。根据该标记对F1代174个单株的扩增结果,利用Mapmaker3.0软件计算遗传距离,表明该标记与抗茎线虫病基因间的遗传距离为14.2cM。将该片断回收、克隆、测序,表明其长度为878bp,该标记命名为OPP03878。根据测序结果,设计1对特异引物,进行特异性扩增,成功地将OPP03878标记转化为SCAR标记,用亲本及分离群体验证表明该分子标记稳定性好。     

19个华蕉类栽培品种的SCAR标记鉴别

针对华蕉(Cavendish,AAA)类主栽品种在命名过程中出现的"同物异名"现象较突出的问题,本研究利用SCAR(sequence characterized amplified regions)标记对华蕉品种进行快速鉴别。通过对19个华蕉类品种进行RAPD(random amplified polymorphic DNA)多态性分析,共获得5条具有差异性的DNA片段,并对其进行测序分析,将其转化成相应的5对SCAR引物,再分别以19个华蕉类栽培品种的基因组DNA为模板进行SCAR-PCR扩增。利用这5对SCAR标记在不同华蕉类栽培品种中的差异片段,建立快速鉴别19个华蕉品种的路线图。结果表明:组合使用这5对SCAR引物可以快速、稳定的区分其中的7个华蕉类栽培品种,而其余12个品种则被划分为4个组。本研究将多个SCAR标记进行联合分析,实现快速、稳定、高效的鉴别华蕉类栽培品种,有利于在分子水平上为华蕉类栽培品种的鉴别提供分子依据。     

结球甘蓝(Brassica oleracea var. capitata)迟抽薹基因SCAR标记转CAPS标记

为了更加精确结球甘蓝迟抽薹基因标记。本研究以结球甘蓝冬性强的迟抽薹材料‘P02’和冬性弱的易抽薹材料‘A21’杂交得F1代,F1代自交至F3代为试验材料,并以课题组所设计的特定序列扩增(sequence characterized amplified regions, SCAR)标记的SCN1/248为引物。在F3代的785株结球甘蓝中有711株扩增出目的条带,有74株未能扩增出目的条带。经测序,该序列长度为248 bp,运用该序列共设计3对酶切扩增多态性序列(cleaved amplified polymorphic sequence, CAPS)引物,其中1对引物均扩增出目的条带,多态性消失,另外两对分别命名为SC01和SC02。结合田间127株结球甘蓝抽薹性状调查,有65株表现为迟抽薹,59株表现为易抽薹,3株表现为中间型,与SCAR扩增检测结果基本一致,将SCAR标记转换成CAPS标记,建立CAPS标记体系。本研究进一步精化了分子标记辅助选择,为做精确定位功能基因,选育优良的迟抽薹结球甘蓝品种提供了理论依据。     

小麦抗病种质贵农775中抗白粉病基因的RAPD标记

运用RAPD技术,采用分离群体分组分析法（BSA）进行了小麦种质贵农775抗白粉病基因连锁的分子标记研究,其中有一个引物S2018在抗病亲本贵农775和抗病材料中扩增出了特异的DNA片段,而在感病材料和感病亲本丰产3号中没有扩增出同样的DNA片段。此片段长度约为880 bp。用F2分离群体（106株植株）进行遗传连锁性分析,引物S20188     

甜椒胞质雄性不育恢复系SRAP及SCAR标记

以甜椒胞质雄性不育恢复系5-2R1和相应的保持系5-2为试材,利用SRAP分子标记技术筛选与甜椒恢复基因相关的分子标记。128对SRAP引物共扩增获得了3796条从100bp至800bp大小不同的条带,平均每对引物组合可扩增出30条清晰的条带,检测到4个多态性位点,其中恢复系材料仅有1个。对该特异片段进行回收、克隆和测序,结果表明该片段全长为259bp。经数据库比对分析表明,该片段与线粒体中的NADH脱氢酶第5亚基(GenBank:EF151914.1)具有81%的同源性;同时,该片段与辣椒BAC克隆PEPBAC 158K24(GenBank:FJ597540.1)的部分序列高度同源。根据序列特点设计特异SCAR引物,对恢复系和保持系进行扩增验证,仅在恢复系中扩增出目的条带,表明已成功地将此SRAP标记转化为简单、稳定的SCAR标记。我们推测本研究获得的SCAR标记可能与胞质雄性不育恢复性状连锁。     

小麦抗条锈基因Yr6分子标记初步研究

小麦抗条锈基因Yr6在我国小麦育种中应用很少,寻找该基因的分子标记将有助于促进该基因的合理利用。将Yr6与其他有效的抗条锈基因相结合可以拓宽品种的抗病谱,延长育成品种的使用年限。本研究中,共用653条RAPD引物和小麦7B染色体上的36对SSR引物对Yr6的近等基因系进行了DNA多态性分析,对PCR产物具有多态性的SSR引物进一步检测了Yr6基因的2个载体品种。结果共有93条RAPD引物(占总数的14.2%)和5对SSR引物(占总数的13.9%)在抗病近等基因系Yr6/6×Avocet S和感病材料Avocet S间稳定扩增出了差异条带,其中SSR标记Xwmc76和Xwmc276在Yr6基因的载体品种Heines Kolben和Heines Peko中检测出了与Yr6/6×Avocet S中相同、而与感病亲本Avocet S中不同的多态性扩增条带,说明这2个SSR标记可能与Yr6基因连锁。     

小麦抗叶锈病基因Lr24的一个新STS标记

来源于长穗偃麦草的基因Lr24对小麦叶锈病具有很高的抗性,本研究旨在开发用于Lr24基因分子标记辅助育种的新的分子标记。从定位于小麦3D染色体的22对SSR、EST-SSR引物中筛选出4对揭示TcLr24多态性的引物,用468株F2抗感群体对这4对引物进一步检测,得到1个与Lr24共分离的EST-SSR标记Xcwem17。对该标记进行测序,并设计了STS引物。用该STS引物及已知的Lr24SCAR引物对试验群体进行验证,两对引物在该F2群体中均表现共分离,且Xcwem17可在TcLr24单基因系和已知含Lr24的农家品种泰山1号中可扩增出180bp单一条带,感病对照及其余7个近等基因系无扩增。该EST-SSR标记可直接用于分子标记辅助选择。     

Development and application of SCAR markers for sex identification in the dioecious species <Emphasis Type="Italic">Ginkgo biloba</Emphasis> L.

There is an urgent need for early sex identification to support field planting in Ginkgo biloba L., due to the different economic and medicinal values between male and female trees. An easy, rapid and reliable molecular method for sex type determination of G. biloba was reported in the paper. Random amplification of polymorphic DNA (RAPD) and sequence-characterized amplified region (SCAR) were used to search for specific molecular markers linked to the sex locus. A total of 48 primers were used for screening of specific RAPD markers in six male and three female samples. Only one primer, S10, showed different amplification band patterns associated with sex types. Then the sex-specific bands, S10-BandA and S10-BandB, were cloned and sequenced. Based on the sequences two pairs of SCAR primers, GBA and GBB, were designed. The GBA primers amplify a single 571 bp band in male samples but not in female samples, and DNA amplification using GBB primers could generate a 688 bp band only in the female individuals. Finally, the SCAR primers were used to test 16 sex-unknown samples. SCAR primers developed in this paper can be used as effective, convenient and reliable molecular markers for sex identification in G. biloba.     

Identification and characterization of RAPD and SCAR markers linked to anthracnose resistance gene in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, one of the destructive foliar diseases of sorghum growing in warm humid regions, is incited by the fungus Colletotrichum graminicola.The inheritance of anthracnose resistance was studied using the parental cultivars of Sorghum bicolor (L.) Moench, HC 136 (susceptible to anthracnose) and G 73 (anthracnose resistant). The F₁ and F₂ plants were inoculated with the local isolates of C. graminicola cultures. The F₂ plants showed a segregation ratio of 3 (susceptible): 1(resistant) indicating that the locus for resistance to anthracnose in sorghum accession G 73 segregates as a recessive trait in a cross to susceptible cultivar HC 136. RAPD (random amplified polymorphic DNA) marker OPJ 01₁₄₃₇ was identified as marker closely linked to anthracnose resistance gene in sorghum by bulked segregant analysis of HC 136 × G73 derived recombinant inbred lines (RILs) of sorghum. A total of 84 random decamer primers were used to screen polymorphism among the parental genotypes. Among these, only 24 primers were polymorphic. On bulked segregant analysis, primer OPJ 01 amplified a 1437 bp fragment only in resistant parent G 73 and resistant bulk. The marker OPJ 01₁₄₃₇ was cloned and sequenced. The sequence of RAPD marker OPJ 01₁₄₃₇ was used to generate specific markers called sequence characterized amplified regions (SCARs). A pair of SCAR markers SCJ 01-1 and SCJ 01-2 was developed using Mac Vector program. SCAR amplification of resistant and susceptible parents along with their respective bulks and RILs confirmed that SCAR marker SCJ 01 is at the same loci as that of RAPD marker OPJ 01₁₄₃₇ and hence, is linked to anthracnose resistance gene. Resistant parent G 73 and resistant bulk amplified single specific band on PCR amplification using SCAR primer pairs. The RAPD marker OPJ 01₁₄₃₇ was mapped at a distance of 3.26 cM apart from the locus governing anthracnose resistance on the sorghum genetic map by the segregation analysis of the RILs. Using BLAST program, it was found that the marker showed 100 per cent alignment with the contig{_}3966 located on the longer arm of chromosome 8 of sorghum genome. Therefore, these identified RAPD and SCAR markers can be used in the resistance-breeding program of sorghum anthracnose by marker-assisted selection.An erratum to this article can be found at     

大豆抗胞囊线虫病S11700特异性抗性基因标记

大豆胞囊线虫病是世界大豆产区危害最重的病害之一.本文以高抗大豆胞囊线虫3号生理小种的黑豆品种小粒黑豆为父本,以高感大豆胞囊线虫3号生理小种的品种辽豆10号为母本配制杂交组合.利用分离群体分组分析法(BSA)对辽豆10号×小粒黑豆杂交组合的F2代大豆材料基因组DNA进行了RAPD分析,供试的143个随机引物有92个产生了RAPD扩增产物,其中产生RAPD多态性的随机引物有25个.筛选到一个与抗大豆胞囊线虫基因相关的特异性DNA片段S 11700,此RAPD标记具有较高的重复性和稳定性,可用于优良的抗大豆胞囊线虫新品种的辅助选育.     

Conversion of the RAPD OPC021150 marker of the Rfo restorer gene into a SCAR marker for rapid selection of oilseed rape

The Rfo fertility restorer gene for the Ogura cytoplasmic male sterility (CMS) applied for oilseed rape hybrid seed production can be monitored with the use of the RAPD OPC02₁₁₅₀ marker while molecular breeding. The aim of this work was to convert the RAPD marker into a more suitable SCAR marker. Total DNA was isolated from a doubled haploid line derived from the line BO20 (INRA, France). A fragment of 1150‐bp linked to the Rfo gene was PCR amplified with the use of the RAPD OPC02 primer, cloned and sequenced. A pair of primers was designed and PCR amplification was performed to develop a SCAR marker for the Rfo gene. The new marker was applied for analysis of 220 oilseed rape lines comprising doubled haploid and inbred restorer lines, restored hybrids as well as F₁ and F₂ recombinant generations involving restorer lines. Simultaneously, the RAPD OPC02 marker was used and it revealed that the markers are equivalent to each other. However, the developed new SCAR marker has made the analysis more practical, rapid and efficient.     

An extended random primer amplified region (ERPAR) marker linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata)

Similar to SCAR, an extended random primer amplified region (ERPAR) marker is a PCR amplified genomic DNA fragment at a single genetically defined locus. However, ERPAR uses specific primer pairs derived from RAPD primers by adding bases sequentially to their 3′-ends. As an example, an ERPAR marker was derived from a RAPD marker (OT11₉₀₀) linked to a dominant male sterility gene in cabbage (Brassica oleracea var. capitata). After two cycles of base adding and primer pair screening, a primer pair (5′-TTCCCCGCGACT-3′and 5′-TTCCCCGCGAGA-3′) amplified a single intense band with the same size as OT11₉₀₀. The identity of the new marker and OT11₉₀₀ was verified by segregation analysis. The new marker amplified by this extended primer pair was named as EPT11₉₀₀. The development of ERPAR exploits the importance of 3′-end bases of primers in PCR ERPAR shares advantages of SCAR, but eliminates the need for cloning and sequencing. It is a fast and universal way of converting RAPD markers into stable markers. This revised version was published online in July 2006 with corrections to the Cover Date.     

南瓜抗CMV基因的RAPD分子标记筛选

以抗病自交系K01和感病自交系K02杂交后自交所得的F2群体为材料,采用分离群体分析法筛选与南瓜抗CMV基因连锁的RAPD分子标记。通过520个随机引物和310组双引物的RAPD扩增分析,共找到了2个与南瓜抗CMV亲本K01中的抗病基因相连锁的分子标记S4391400和S19 S345600。这2个标记与K01的抗病基因的重组率分别为7.5%和11.8%,遗传距离分别为7.1和11.7 cM。     

EST辅助的甘蓝型油菜显性核不育AFLP标记转化

甘蓝型油菜显性核不育广泛应用于轮回选择和杂种优势利用,不育基因标记的开发与应用对于基因克隆和育种实践具有重要意义。基于AFLP标记SA12MG14的序列信息,从拟南芥整合数据库中,检索与标记序列同源的甘蓝型油菜EST,结合标记和EST序列设计特异引物,转化成新的SCAR标记。获得的SCAR标记S6B3,具有很高的检测稳定性,在回交群体Popu2上分析验证,结果与AFLP标记完全一致。该标记与不育基因相距0.3 cM,将其用于临保系同源的纯合型不育系选育,可有效提高育种工作效率。     

N基因标记基因PCR检测方法的建立及其在遗传育种中的应用

烟草的N基因是一个较为有效的抗烟草花叶病毒(tobacco mosaic virus,TMV)基因,在抗病育种中发挥着重要的作用。为建立其标记基因PCR检测体系,评估已知目的基因cDNA序列作为分子标记的目的基因辅助育种的可行性,本文依据N基因cDNA序列设计4对扩增范围在150～950bp的不同片段长度引物,并对含有N基因的coker176基因组DNA进行PCR扩增。扩增结果表明,有3对可扩增出特异产物。测序结果表明,有2对引物的扩增产物含有内含子。根据所获得的DNA序列设计出2对用于分子标记辅助育种的引物,并分别对C151、NC89、coker176等11个品种的基因组DNA进行扩增,结果表明在coker176、龙江912、吉烟9号和龙江911品种中有扩增产物,除龙江911外,这一结果与已知的N基因在11个品种的分布相一致,成功建立了N基因PCR检测体系,并证明以cDNA序列设计引物扩增DNA序列的目的基因辅助育种技术完全可行。并对实验中出现的问题进行了研究。