小麦抗叶锈病基因Lr45的AFLP分子标记

 利用AFLP技术对小麦抗叶锈基因Lr45进行了标记，从60对AFLP引物中筛选出2对在亲本及TcLr45×Thatcher F2抗感群体间揭示多态性的引物P-AGG/M-GAG和P-ACA/M-GGT。其扩增片段为261 bp和105 bp，2个标记与Lr45的遗传距离分别为0.6 cM和1.3 cM。测序比较261 bp片段与大麦属Vulgare HotrI基因部分序列同源性达86%，105 bp片段与一粒小麦磷脂酰丝氨酸脱羧酶基因部分序列同源性高达96%。2个测序片段均包含开放阅读框（ORF）序列。     

Identification of <Emphasis Type="Italic">Lr24</Emphasis> with targeted region amplified polymorphism (TRAP) analysis in wheat

This research is aimed at developing TRAP markers, as a probe for library screening, closely linked to or co-segregated with Lr24. Ninety TRAP primer pairs were used to test the resistant and susceptible parents, as well as the resistant bulk and the susceptible bulk in our study. The polymorphic TRAP primers of TcLr24 were employed to genotype the F₂ population from TcLr24×Thatcher subsequently. Ten of 90 TRAP primer pairs displayed polymorphism between TcLr24 and Thatcher, accounting for 11.11%. A further study found that primer ARBI1/RGA-2F generated a 161 bp fragment presented only in the resistance plants of F₂ population. Forty-five other wheat leaf rust resistant NILs and 30 diploid materials of wheat were also tested to detect the specificity of the primer. This specific band was amplified in TcLr19, TcLr29, TcLr38, TcLr42 and TcLr44, but absented in all the 30 diploid materials. It was concluded that this marker ARBI1/RGA-2F was closely linked to Lr24, which could be used to detect Lr24 in the F₂ population of TcLr24×Thatcher, and be further used as a probe for cDNA and BAC library screening of TcLr24.     

小麦抗叶锈病基因Lr19的AFLP标记

 以Thatcher和23个以Thatcher为遗传背景的小麦抗叶锈病近等基因系及TcLr19与Thatcher杂交F2代植株为材料，利用AFLP技术开展了小麦抗叶锈病基因Lr19的分子标记研究。共获得7个与小麦抗叶锈病基因连锁的分子标记：P-AGT/M-GAG289bp（3.3cM）、P-ACA/M-GGT102bp（4.1cM）、P-ACA/M-GGT106bp（4.1cM）、P-AAC/M-CAG123bp（4.9cM）、P-AAC/M-GGT203bp（5.0cM）、P-ACA/M-GGT290bp（5.7cM）和P-ATC/M-GAG293bp（9.6cM）。这些特异性片段经回收、克隆、测序得出了特异带的序列。该研究可促进遗传图谱、物理图谱的构建和小麦抗叶锈病基因Lr19的克隆。     

小麦抗叶锈病基因Lr24的TRAP分析

 【目的】获得与Lr24基因紧密连锁并可能作为探针的TRAP分子标记。【方法】应用TRAP技术,选用90对引物组合对小麦抗叶锈病基因Lr24、感病亲本Thatcher及其F2代抗感各10株组成的抗、感基因池（Br、Bs）的扩增带型差异进行分析,用筛选获得的多态性引物对TcLr24×Thatcher F2群体进一步筛选,进而用获得的特异引物对45个小麦抗叶锈病近等基因系和30个小麦二倍体材料进行分析。【结果】获得10对能够在TcLr24、Br与Thatcher、Bs间产生多态性的引物,多态性引物检出率为11.11%。其中1对在F2抗感群体中有差异且稳定扩增的TRAP引物ARBI1/RGA-2F,其161bp 扩增产物仅在F2抗病单株中出现,感病单株中缺失。用该引物对45个近等基因系和30个二倍体材料检测发现,近等基因系TcLr19、TcLr29、TcLr38、Lr42和TcLr44中有相同大小片段的扩增产物,30个二倍体材料中未出现相应扩增产物。【结论】本研究获得的一个与Lr24紧密连锁的TRAP标记,该标记可筛选含有Lr24基因的育种后代群体,可作为探针用于文库的筛选。     

Identification of AFLP Markers Linked to Lr19 Resistance to Wheat Leaf Rust

AFLP analyses were carried out on Thatcher, 23 near-isogenic lines and F2 generation of TcLrl9 × Thatcher, to develop molecular markers for gene Lr19 resistance to wheat leaf rust. Seven markers linked to Lr19 resistance trait were obtained,which were P-AGT/M-GAG289 bp (3.3 cM), P-ACA/M-GGT102 bp (4.1 cM), P-ACA/M-GGT106 bp (4.1 cM), P-AAC/M-CAG123 bp(4.9 cM), P-AAC/M-GGT203 bp (5.0 cM), P-ACA/M-GGT290 bp (5.7 cM), and P-ATC/M-GAG293 bp (9.6 cM). All of these specific fragments were isolated from the polyacrylamide gels, reamplified, cloned, and sequenced. The research may facilitate genetic mapping, physical mapping, and the eventual cloning of Lr19.     

小麦抗叶锈基因Lr45的SCAR标记

 【目的】建立小麦抗叶锈基因Lr45 SCAR（sequenced characterized amplified region序列特征扩增区域）标记。【方法】以小麦抗叶锈病基因材料TcLr45和感病材料Thatcher为亲本,利用黑麦基因组特异的RAPD标记引物OPH20进行PCR扩增,对获得的与预期大小相同的1.5kb的特异片段进行克隆测序,并根据该序列设计一对特异PCR引物LRYR和LRYF,对TcLr45×Thatcher F2代单株构建的分离群体进行扩增,验证该标记与Lr45的连锁关系,Mapmaker 3.0软件绘制遗传连锁图。【结果】该标记在TcLr45中扩增出单一条带,片段大小为1 272 bp（命名为Ypsc20H1272）,而在感病亲本中则无扩增条带。F2代分离群体进行连锁分析,遗传距离为8.2 cM,该标记为与Lr45连锁的SCAR标记。【结论】将Lr45的RAPD标记转化成SCAR标记。     

小麦抗叶锈病基因Lr19 的SSR标记

 选取小麦感叶锈病亲本Thatcher、6个以Thatcher为遗传背景的小麦抗叶锈近等基因系及TcLr19与Thatcher杂交F2代为材料，开展了小麦抗叶锈基因Lr19的微卫星分子标记研究；从13对微卫星引物中筛选出了1对在亲本及TcLr19×Thatcher F2抗感群体间揭示多态性的引物Xgwm44，并获得了1个与小麦抗叶锈基因Lr19紧密连锁的SSR标记Xgwm44139bp，此标记位点与Lr19基因之间的遗传距离为0.9cM。该研究可为分子标记辅助育种及构建遗传图谱、物理图谱和基因克隆奠定了基础。     

A SSR Marker for Leaf Rust Resistance Gene Lr19 in Wheat

Microsatellite was carded out in Thatcher, six near-isogenic lines and F2 progeny of TcLr19xThatcher to develop molecular markers for leaf rust resistance gene Lr19. Thirteen primer pairs were screened, of which one primer pair Xgwm44 displayed polymorphsim in the population of TcLr 19, Thatcher, and their F2 generations. One marker closed linked to Lr19 resistance trait was obtained, and was named Xgwm44139bp with the genetic distance 0.9 cM. The research shows that Lr19 has more potential in marker-assisted breeding programs in wheat and provides a step stone for mapping genetic map, physical map and the eventual cloning.     

桃果实非酸/酸性状分子标记的筛选

以桃品种‘京玉’、‘美味’的正反交F1代群体69株为试材,采用RAPD、AFLP技术与BSA相结合的方法筛选与桃果实非酸/酸性状连锁的分子标记。在筛选的301个RAPD引物中,只有引物S332在两基因池间产生的多态性片段与非酸性状连锁,但重组率高达28.9%。通过128对AFLP引物组合在两基因池间的分析结果表明,18对引物组合产生多态性。单株验证结果表明,只有3对引物组合E-ACT/M-CAT、E-TA/M-CTC和E-AT/M-CTA产生的多态性片段与果实非酸/酸性状连锁,连锁距离分别为16.2、1.47和2.99 cM。利用获得的连锁标记,构建D/d基因连锁群,连锁距离最近的2个标记AFTA-CTC、AFAT-CTA分别位于D/d基因位点两侧。     

Molecular Characterization of Atlas 66-Derived Wheat Near-lsogenic Lines Contrasting in Aluminum （Al） Tolerance

Aluminum （Al） toxicity is the major limiting factor for wheat growth in acidic soils. Genetic improvement of Al tolerance is one of the most cost-effective solutions to improve wheat productivity. The objective of this study was to characterize near isogenic lines （NILs） contrasting in Al tolerance derived from Atlas 66 in the backgrounds of Al-sensitive cultivars Chisholm and Century using amplified fragment length polymorphism （AFLP） and simple sequence repeat （SSR）. A total of 200 AFLP and 88 SSR primer pairs were screened and 12 markers （11 AFLPs and one SSR） were associated with Al-tolerance in NILs of at least one recurrent parental background. Among them, nine were linked to A1 tolerance in the Chisholm-derived NILs, seven were associated with Al-tolerance in the Century-derived NILs, and three AFLPs derived from the primer combinations of pAG/mGCAG, pCAG/mAGC and pGTG/mGCG, and one SSR, Xwmc331 on chromosome 4D, associated with A1 tolerance in NILs of both recurrent parental backgrounds. Those common markers across two backgrounds may be the major marker loci associated with Al-tolerance in Atlas 66 and could be useful for marker-assisted breeding to improve Al tolerance in wheat. In addition, evaluation of Al tolerance among different genotypes using hematoxylin stain and relative root growth revealed that Atlas 66 was more tolerant to Al stress than the NILs, therefore suggested that the Al-tolerant NILs might not carry all Al-tolerance loci from Arias 66 and inheritance of Al tolerance in Arias 66 is more likely multigenic.     

番茄抗黄化曲叶病毒病基因的AFLP分子标记

用番茄抗黄化曲叶病毒病品系‘T0727’与高感黄化曲叶病毒病品系‘T9179’配制杂交组合,接种鉴定其F₁代及F₂代分离群体的黄化曲叶病毒病发生情况。用64对EcoRⅠ/MseⅠ引物组合对‘T0727’、‘T9179’两个亲本及其F₁代和F₂代抗病和感病基因池进行AFLP分析,共扩增出4 023条可分辨的条带,其中3条为稳定的差异。用‘T0727’和 ‘T9179’杂交产生的F2代分离群体对3个特异条带与目的基因的遗传连锁性进行分析,发现特异条带E-ACC/M-CAG与抗黄化曲叶病毒病基因紧密连锁,二者之间的遗传距离为9.5 cM。将E-ACC/M-CAG片段回收、克隆和测序,成功地将其转化为SCAR标记,暂定名为Afty-196,可以用于对番茄黄化曲叶病毒病基因的标记辅助选择。     

柿(Diospyros kaki L.) AFLP标记连锁图谱的构建

以甜柿禅寺丸和涩柿磨盘柿杂交,用获得的F1代分离群体的96个单株为实验材料,以AFLP标记尝试构建柿属植物的AFLP分子标记连锁图谱。先用2个亲本筛选引物,从256对AFLP引物组合中筛选得到了27对多态性好的引物。用筛选得到的27对引物对2个亲本及其F1群体进行AFLP分析,共得到多态性标记447个,其中符合孟德尔定律的标记有400个,占总多态性标记的89.5%。符合1∶1分离比率的多态性标记共有300个,在禅寺丸中得到147个,在磨盘柿中得到153个。符合3∶1比率的多态性标记有100个。用Mapmaker 3.0软件和拟测交策略,分别构建了禅寺丸和磨盘柿的分子标记AFLP连锁图谱。禅寺丸的连锁图谱包括24个连锁群,由149个AFLP标记组成,总图距为2 105.5cM,标记间平均距离为14.1cM。磨盘柿的连锁图谱包括22个连锁群,由181个AFLP标记组成,总图距为2 253.8cM,标记间平均距离为12.5cM。     

一个与西瓜核不育育性基因连锁的AFLP分子标记的获得

以西瓜G17AB核雄性不育两用系为材料,采用BAS法构建了不育池和可育池。利用AFLP分子标记技术,通过筛选256对AFLP引物组合,获得了一个与育性位点紧密连锁的AFLP标记,片段长度在310~350 bp之间,该标记与育性基因的遗传距离为5.0 cM。     

AFLP and RAPD Analysis of the Boer and Indigenous Breeds of the Goat in Jiangsu

Blood and tissue samples were collected from 105 goats including 60 Boer goats (30 for eachsex), 30 Xuhuai goats (15 for each sex) and 15 Haimen goats (7 stud and 8 does). DNA was extracted andDNA pools were constructed on the basis of goat breeds. In 36 selective primer combinations, 29 combinationsamplified totally 3 253 markers including 92 polymorphic markers by amplified fragment length polymor-phism (AFLP). On average, 3.17 polymorphic markers were amplified per combination, with a polymorphicfrequency of 2.8%. The primer combinations amplifying more polymorphic markers (showed in brackets)were involved in E00+ACG/M00+CAA (13), E00+ACG/M00+CAG (10), E00+AAC/M00+CAC (8)and E00+AAC/M00+ACT (7). A total of 183 markers including 60 polymorphic markers were amplified byRAPD from the pooled DNA of three breeds using 22 primers with strong polymorphism and high reproduc-ibility selected from 93 RAPD primers. On average, 2.73 polymorphic markers were amplified per primer,with a polymorphic frequency of 32.8%. The results of AFLP and RAPD coincidently suggested that the ge-netic distance is the closest between Xuhuai and Haimen goat, next between Xuhuai and Boer goat, and the far-thest between Haimen and Boer goat. According to the UPGMA method, Haimen and Xuhuai goats can begathered together as a cluster, then Boer goat. Both methods can be used to implicate the genetic difference ofthese three breeds, in particular AFLP has more polymorphic markers.     

小麦抗叶锈近等基因系TcLr38的cDNA-AFLP分析

 【目的】分析经叶锈菌诱导小麦抗叶锈近等基因系TcLr38的基因表达情况,寻找与抗病基因表达相关的片段。【方法】应用cDNA-AFLP技术从mRNA表达水平研究TcLr38和感病对照Thatcher与小麦叶锈菌05-22-65（THTS）互作时基因表达的差异。【结果】76对引物组合检测到约3 800条条带,平均每一对选择性引物可以获得50条条带,其中28对能够在小麦近等基因系TcLr38和感病对照Thatcher之间扩增出特异性条带。多态性条带划分为8种类型,其中3种类型可能与抗病基因相关。最终得到95条小麦抗叶锈近等基因系TcLr38的特异性表达的转录衍生片段（TDF）,其中19条差异TDFs只在接种叶锈菌的TcLr38中出现,为上调表达,而8条差异TDFs为下调表达。对可能与抗病相关的特异TDFs中的21条片段进行序列测定与分析,经BLASTx比较,17个TDFs所推导的蛋白质序列在数据库中找到其所对应的同源序列,15个TDFs为已知功能的基因。【结论】通过对功能已知的基因分析,推测决定蛋白激酶C、ATP结合蛋白、类受体激酶、信号传导组氨酸激酶（HPK）、肽酶家族M23、Dnak抑制蛋白DksA、甲硫氨酸tRNA合成酶、RanGTP酶激活蛋白1、烯酰辅酶A水合酶的TDFs可能与抗病或防御过程相关。     

亚麻抗枯萎病基因FuJ7(t)的分子标记

 用高抗枯萎病亚麻品种“晋亚 7号”与高感枯萎病品种“晋亚 1号”配制杂交组合 ,接种鉴定其正反交F1代以及F2 代分离群体的枯萎病发生情况 ,结果表明 ,晋亚 7号对枯萎病的抗性属于细胞核遗传 ,受 2个显性基因控制。用 4 8个EcoRI/MseI引物组合对“晋亚 7号”、“晋亚 1号”两个亲本及其F2 代抗病和感病基因池进行AFLP分析 ,共扩增出约 330 0条可分辨的带 ,其中 3条为稳定的差异。用“晋亚 7号”和“晋亚 1号”杂交产生的F2 代分离群体对 3个特异条带与目的基因的遗传连锁性进行分析 ,发现特异条带AG/CAG与暂定名为FuJ7(t)的抗枯萎病基因紧密连锁 ,二者之间的遗传距离为 5 .2cM。将AG/CAG片段回收、克隆和测序 ,成功地将其转化为SCAR标记 ,可以更加方便地用于对FuJ7(t)基因的分子检测和标记辅助选择。     

Molecular Mapping QTLs for Gel Consistency in Rice

A population of 152 recombinant inbred lines (RIIs) derived from a cross between CT9993, a japonica rice with hard consistency (GC) and Khoa Dawk Mali105 (KDML 105 ), a famous Thai rice(Oryza sativa L. ) with medium GC, was used for GC quantitative trait loci (QTLs) analysis. Three linkage maps were constructed with RFLP, AFLP and microsatellite (SSLP) markers. The first one consists of 83 RFLP markers with 17.53 cM of an average distance between markers. The second one consists of 83 RFLP and 69 AFLP markers with 13.22 cM average distance between markers and the third one consists of 83 RFLP, 69 AFLP and 15 SSLP markers, and has an average distance of 12.98 cM between markers. Based on these three maps, QTLs conferring GC were analyzed. The results show that GC is mainly controlled by two linked loci,which are located at the two flanks of RFLP marker R2170 on chromosome 3. The distance between the two linked QTLs is 25 cM and the two QTLs were determined by a LOD scores larger than 16.0. The number of minor QTIs controlling GC varies from 4- 9 in the three different linkage maps.     

甘蓝型油菜细胞质雄性不育恢复基因的分子标记及定位

以甘蓝型油菜恢复系CP015和不育系77A构建的F2群体为供试材料,运用RAPD,SSR和AFLP 3种标记技术,对甘蓝型油菜恢复基因进行分子标记和图谱定位。在260条RAPD引物、185对SSR引物、58对AFLP引物中,在两亲本间筛选多态性高的RAPD引物121条,SSR引物128对,AFLP引物组合26对,进一步通过BSA法筛选,获得了与甘蓝型油菜恢复基因Rf连锁的1个RAPD标记S1009-750和1个AFLP标记E5M11-150,标记与基因Rf之间的遗传距离分别为4.9cM和8.6cM。     

小麦抗叶锈基因Lr44的AFLP分子标记

利用AFLP技术借助于小麦抗叶锈近等基因系和TcLr44×ThatcherF2代分离群体材料,获得4个Lr44的分子标记,分别为Paag:Mcta300,Paac:Mcgt85,Paag:Mcta395和Paac:Mcgt90,与目的基因的遗传距离分别为0 01cM、1 1cM、3cM、2 2cM和2 8cM,为分子辅助育种、构建密集的遗传图谱、克隆Lr44以及研究基因编码特性等奠定基础。     

5种蔷薇遗传多态性的AFLP分析

[目的]研究AFLP标记在蔷薇遗传多态性方面的应用和5种不同蔷薇品种的遗传背景。[方法]应用10条人工设计的与接头序列相识别的AFLP选择性引物,用PstⅠ酶切,对5种蔷薇基因组DNA进行AFLP反应,分析不同蔷薇间的遗传相似系数和遗传距离。[结果]获得了108个AFLP标记,单引物获得的标记数在4～16,5种蔷薇的遗传相似系数为0.824(0.732～0.947),遗传距离为0.053～0.268。[结论]该研究为评价蔷薇的遗传稳定性提供了相关的参数。