草菇菌丝细胞的拆分与重建

摘要：分子标记技术筛选亲本单26与单28的遗传差异条带,并成功转化为单26菌株核DNA的特征标记SCAR1和单28菌株核DNA的特征标记SCAR2;将核特征标记SCAR应用于杂交菌株2628的原生质体再生菌株的检测,成功地分离与鉴定出两亲本类型的原生质单核化菌株,表明杂交菌株2628细胞已被拆分;本研究再通过配对杂交、SCAR检测及出菇试验等证明,两亲本类型的原生质单核化菌株又可再重建成异核体的2628菌株;这为丰富食用菌特别是同宗结合真菌的遗传育种理论研究提供了新的思路。     

RAPD-SCAR在鱼类种质鉴定中的应用及前景

种质是利用和改良动植物、微生物的物质基础,更是实施各个育种途径的原材料,因此优良种质鉴定是一个很关键的问题。种质鉴定方法已由形态水平发展到蛋白质和DNA分子水平。RAPD技术具有敏感、快速、简便、产量高、重复性好及检测容易等突出优点,广泛应用于种质鉴定中,但也有不足之处。基于RAPD标记技术建立起来的SCAR（Sequence Characterized Amplified Region）标记克服了RAPD标记的不足,操作简捷,特异性和重复性较高,是一种有效的分子标记。RAPD-SCAR分子标记技术的利用使种质鉴定更为快速准确,提高了育种速度,缩短了育种周期,为相关的研究工作提供分子遗传学依据。     

甘薯抗茎线虫病基因SCAR标记辅助育种初探

用已获得的与抗甘薯茎线虫病基因连锁的RAPD标记OPD01-700引物对高抗亲本徐781和高感亲本徐薯18的F1分离群体的161个品系进行PCR扩增.根据该标记扩增结果,利用Mapmaker3.0软件计算遗传距离,得出此标记与甘薯茎线虫病基因的连锁距离为19.4 cM.对RAPD标记OPD01-700片段进行克隆、测序,得到长度为689 bp的DNA序列,将该序列命名为OPD689.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OPD689标记转化为SCAR标记.将SCAR标记在7个高抗品系和13个高感品系进行初步验证应用,其结果与田间鉴定结果基本一致.初步建立了甘薯抗茎线虫病育种分子标记辅助选择技术.     

Development of reliable PCR markers for the selection of the Vf gene conferring scab resistance in apple

Early selection of scab-resistant apple seedlings can be enhanced by the use of markers tightly linked to the Vf resistance gene. Two sequence characterized amplified regions (SCAR) markers have been obtained from previously described random amplified polymorphic DNA (RAPD) markers. AM19-SCAR is a codominant marker, while AM19-SCAR is dominant, as is the RAPD from which it was derived. A highly detailed map in the vicinity of the Vf gene was built through the cumulative analysis of about 600 seedlings from six different controlled crosses. The usefulness of these and other SCAR markers will be discussed in relation to combining the traditional phenotypic selection with MAS. The availability of two codominant, tightly linked markers flanking both sides of the resistance gene (AL07-SCAR and M18-CAPS) also makes it easy to identify the seedlings homozygous for the resistance gene.     

3个奥利亚罗非鱼群体遗传多样性的RAPD分析和SCAR标记的转化

应用RAPD技术对3个奥利亚罗非鱼(Oreochromis aureus)群体(奥利亚罗非鱼83系、新引进的红色奥利亚罗非鱼与奥利亚罗非鱼02系)进行遗传多样性分析.根据扩增结果统计获得群体内和群体间的遗传距离:奥利亚罗非鱼83系、红色奥利亚罗非鱼、奥利亚罗非鱼02系群体内的遗传距离分别为0.021 0,0.090 3和0.014 9;群体间的遗传距离以奥利亚罗非鱼83系与红色奥利亚罗非鱼群体间的最大,为0.135 1,奥利亚罗非鱼83系与奥利亚罗非鱼02系的遗传距离最小,为0.061 0,红色奥利亚罗非鱼与奥利亚罗非鱼02系群体间的遗传距离为0.121 8.利用MEGA3.1软件进行聚类分析,奥利亚罗非鱼83系与奥利亚罗非鱼02系首先聚为一支,然后再与红色奥利亚罗非鱼聚为一支.3个奥利亚罗非鱼群体总的Shannon多样性指数为:0.234 0,总的Nei基因多样性指数为0.152 7,3个群体的遗传分化指数(Gst)为0.507 1.上述结果说明这3个群体的群体内遗传变异较小,但群体间存在一定程度的遗传变异和遗传分化.对RAPD分析中所获得的6条特异带进行SCAR转化.S97800和S4691400两个特异片段成功转化为SCAR标记.这些标记可作为鉴别3个奥利亚罗非鱼群体的遗传标记,并将为今后的选择育种提供分子标记.     

Development of molecular markers linked to the <Emphasis Type="Italic">Fom-1</Emphasis> locus for resistance to Fusarium race 2 in melon

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis (F.o.m), is a worldwide soil-borne disease of melon (Cucumis melo L.). The most effective control measure available is the use of resistant varieties. Resistance to races 0 and 2 of this fungal pathogen is conditioned by the dominant gene Fom-1. An F₂ population derived from the ‘Charentais-Fom1’ × ‘TRG-1551’ cross was used in combination with bulked segregant analysis utilizing the random amplified polymorphic DNA (RAPD) markers, in order to develop molecular markers linked to the locus Fom-1. Four hundred decamer primers were screened to identify three RAPD markers (B17₆₄₉, V01₅₇₈, and V06₁₀₉₂) linked to Fom-1 locus. Fragments amplified by primers B17₆₄₉ and V01₅₇₈ were linked in coupling phase to Fom1, at 3.5 and 4 cM respectively, whereas V06₁₀₉₂ marker was linked in repulsion to the same dominant resistant allele at 15.1 cM from the Fom-1 locus. These RAPDs were cloned and sequenced in order to design primers that would amplify only the target fragment. The derived sequence characterized amplified region (SCAR) markers SB17₆₄₅ and SV01₅₇₄ (645 and 574 bp, respectively) were present only in the resistant parent. The SV06₁₀₉₂ marker amplified a band of 1092 bp only in the susceptible parent. These markers are more universal than the CAPS markers developed by Brotman et al. (Theor Appl Genet 10:337–345, 2005). The analysis of 24 melon accessions, representing several melon types, with these markers revealed that different melon types behaved differently with the developed markers supporting the theory of multiple, independent origins of resistance to races 0 and 2 of F.o.m.     

玉米免DNA制备单株SSR和SCAR基因分型方法研究

探讨采用免DNA制备法分析玉米单株SSR与SCAR分子标记的基因型.分别刮取各单株少许叶肉细胞到1×PCR缓冲液中,置于PCR仪上95℃10 min,冷却,添加PCR其它组份,完成SSR与SCAR的PCR扩增与显色成像.免除模板DNA制备过程,对掖478与丹340两个亲本及57个(共58个)F2分离群体单株分别实现SSR标记-phi402893基因型快速鉴定,对掖478与丹340两个亲本及5个F2分离群体单株分别实现SCAR标记-MITE185基因型快速鉴定.     

与黄瓜矮生基因连锁的ISSR标记及其SCAR转换

以黄瓜矮生品系D8与蔓生品系JIN5杂交并自交得到的124株F₂代为试材,应用ISSR分子标记技术和BSA（混合群体分组分析法）法寻找与黄瓜矮生基因连锁的分子标记。从80条ISSR引物中筛选到一个黄瓜矮生性状特异的多态性引物UBC818,经F₂代单株验证,UBC818的扩增片段在蔓生黄瓜JIN5植株中稳定出现,而在矮生黄瓜D8植株中没有。连锁关系分析表明,标记与黄瓜矮生基因间的遗传距离为11.1 cM。根据对该片段的序列分析结果重新设计了1对引物,将ISSR标记成功转化成了SCAR标记, 并命名为UBC818-S。     

空间诱变绿豆长荚型突变系的分子生物学分析

中绿1号绿豆种子于1994年经返回式卫星搭载后,经过3年的地面种植和筛选得到了基本稳定的长英型突变系,该突变系荚长16cm左右,每荚种子粒数为15～19粒。通过对3个形态和荚长都基本稳定的突变系和原始对照品系之间的RAPD分析,从100个10-mer Operon引物中筛选出3个能产生稳定的遗传多态性的引物,其特异扩增产物分子量依次为1.4kb、1kb、2kb,因此分别命名为OPZ-13#-（1400）、OPY-07#-（1000）、OPY-04#-（2000）。OPZ-13#-（1400）和OPY-07#-（1000）的克隆和鉴定工作已经完成,其中OPY-07#-（1000）转换成了稳定的SCAR标记。3个突变系进行RAPD分析,彼此之间没有发现扩增产物的差异,它们与原始对照之间却有着共同的差异,表明长荚型突变品系在DNA分子水平上已接近同源。     

烟草PVY抗性的遗传分析与分子标记筛选

本文以抗马铃薯Y病毒(简称PVY)的烟草品种RY5,感病品种Coker176为亲本,构建F1、正反交F2和正反交BC1群体,苗期摩擦接种PVY的抗性遗传分析结果表明,接种后第21d群体PVY抗性数据符合孟德尔单基因隐性质量性状的遗传模型。接种后第28d群体PVY抗性数据偏离孟德尔单基因隐性质量性状的遗传模型。提取F2代群体中抗病和感病单株DNA,从多条RAPD引物和一对SCAR引物中,筛选出两个紧密连锁的分子标记。RAPD标记O12V3695与RY5的抗病基因对应的显性等位基因位点(Va)间的遗传距离为2.10cM,而SCAR标记与Va间的遗传距离为2.52cM,这两个分子标记可用于抗PVY抗性育种。