白菜低温要求型晚抽薹性的相关序列克隆与分析

为了阐明低温要求型晚抽薹的分子机制,本研究利用易抽薹大白菜BY和晚抽薹芜菁"M.M"杂交获得的81个DH系群体为试材,从中选择了6个极早抽薹和6个极晚抽薹的株系分别组成早抽薹池和晚抽薹池.以两池和双亲为样本,以无低温和3～5℃处理25 d后获得的mRNA为模板,反转录为cDNA,利用256对AFLP引物组合进行cDNA-AFLP分析,筛选与晚抽薹性相关的特异片段.对其中3条差异表达、且丰度较高的cDNA片段进行了克隆测序.序列测定和Blast分析表明,p65m47 cDNA片段与C2结构域蛋白基因有80%的同源性,期望值为7e-17,C2结构域蛋白在细胞的信号转导中起着很重要的作用;p66m55 cDNA I片段与拟南芥AT1G32530 cDNA有85%的同源性,期望值为5e-36,它编码蛋白结合位点/泛素连接酶/锌指结合蛋白;p66m55 cDNA Ⅱ片段与拟南芥AT1G65590 cDNA有87%的同源性,期望值为4e-37,它编码B-N-乙酰已糖胺水解酶.     

大白菜抽薹性状相关SSR分子标记的筛选

耐抽薹大白菜(Brassica rapa L.ssp.pekinensis)新品种选育一直是大白菜育种的一个重要课题。本研究采用耐抽薹性状差异较大的大白菜亲本早抽薹材料‘26’和晚抽薹材料‘502’配制杂交组合,选取62对简单重复序列(simple sequence repeats,SSR)引物,根据集团分离分析法(bulked segregant analysis,BSA),进行大白菜晚抽薹性状基因相连锁的分子标记研究。结果得到了2个在早抽薹基因池中扩增出特征带的SSR标记Na12-E02和BRMS-026,经F2单株验证发现仅BRMS-026与大白菜早抽薹基因紧密连锁,根据Kosambi函数估算其连锁距离为7.2 cM,表明可利用分子标记BRMS-026辅助选育大白菜耐抽薹品种。本研究结果可为最终形成可供育种实践应用的分子标记辅助育种体系和大白菜晚抽薹品种的选育提供理论依据。     

谷子显性雄性不育基因Msch的AFLP标记

利用雄性不育是实现谷子杂种优势利用最经济、有效的途径之一.为了寻找与不育基因Msch紧密连锁的分子标记,提高不育系的选育效率,本研究构建了Msch不育/可育近等基因系(NILs),通过对400对AFLP引物组合进行筛选,找到了与不育基因紧密连锁的两个AFLP标记(P17/M37224和P35/M52208),与不育基因的遗传距离分别是2.1 cM和1.4 cM,而且位于不育基因的同一侧,标记间相距0.7 cM.这两个AFLP标记可有效用于分子标记辅助选择育种.     

AFLP,SSR在黄瓜黑星病抗感材料上的多态性比较

用AFLP和SSR 2种分子标记技术对黄瓜抗感黑星病材料Q6和Q12,及其F2极性集团和F2群体进行了分析,比较了它们的多态性。结果表明,AFLP和SSR 2种分子标记的多态性比率分别为36.5%,9.6%;阳性比率分别为22%,0。在F2群体中找到了1个AFLP标记E20/M64,与目的基因的遗传距离是4.83 cM;1个SSR标记CSWCT02B,与目的基因的遗传距离是28.7 cM。AFLP的多态性比率要比SSR的多态性比率高。分析探讨了2种分子标记技术的优缺点及其在目的基因连锁标记筛选、基因定位等研究中的应用。     

利用抗、染根肿病F_2群体构建大白菜AFLP遗传连锁图谱

以易感染根肿病的大白菜(Brassica campestris ssp.pekinensis)自交系94SK和抗根肿病的CR Shinki DH系为亲本,通过杂交获得F1,并选择一株F1植株进行自交,构建了F2作图群体.利用扩增出与根肿病抗性基因CRb连锁标记的17对AFLP引物组合Pst+GNN/Mse+CNN构建大白菜遗传连锁图谱.这些AFLP引物组合共扩增出322个亲本间表现出多态性的AFLP标记.应用其中6个与CRb基因紧密连锁的AFLP标记转化成的SCAR标记和清晰可见的大于100 bp的211个AFLP标记构建了一张包含179个标记位点、10个连锁群、覆盖长度为576 cM,平均图距3.3 cM的遗传图谱.抗根肿病基因CRb定位在第一条连锁群9 cM的范围内.研究表明,利用Pst/Mse Ⅰ和EcoR Ⅰ/Mse Ⅰ引物组合以及其他分子标记类型构建遗传连锁图谱,并有效减少遗传图谱中聚集和间隙现象的产生提供了一定的科学依据.同时,该图谱的构建对在基因组范围内通过分子标记辅助选择选育大白菜根肿病抗性品种奠定了基础.     

甘蓝型油菜白花性状的遗传及AFLP标记

甘蓝型油菜的白花性状是一种有用的指示性状,本研究以甘蓝型白花油菜及其分离群体为材料,对甘蓝型油菜的白花性状的遗传规律进行了研究。结果表明,甘蓝型油菜的白花性状受1对不完全显性基因控制。利用集团分离法(BSA)对白花基因进行AFLP分析,在256对AFLP引物中共获得6个与白花基因紧密连锁的分子标记。EA11MC04与EA12MC01为基因两侧最近的分子标记,其遗传距离分别为0.8,1.0 cM。     

谷子抗除草剂“拿捕净”基因的AFLP标记

对由一对显性核基因(Srf)控制的谷子抗除草剂拿捕净(Sethoxydim)种质抗性进行了分子标记的研究.通过采用F2代群分法,在谷子F2代抗感池间随机筛选了330对AFLP引物,找到了与谷子抗除草剂基因连锁的两个AFLP标记AP1284(M55/15)和AP2350(M55/14).它们位于抗除草剂基因的一侧,与该基因的遗传图距分别为6.3 cM和2.9 cM.二者间的遗传     

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。     

与茄子青枯病抗性相关基因连锁的AFLP标记研究

本文以茄子高感青枯病亲本自交系5810、高抗亲本自交系5556单1、两自交系杂交得到F1,F1单株自交得到F2群体为试材,采用苗期接种鉴定及BSA法结合AFLP技术,探讨了茄子青枯病抗性的遗传规律,获得与茄子青枯病抗性相关基因连锁的AFLP分子标记。结果表明：抗病亲本5556单1青枯病抗性受一对单隐性基因控制,感病亲本控制的感病基因是不完全显性的。在抗、感池中未发现标记,而通过抗、感池单株分析得到了相引相AFLP分子标记E13M10,如及相斥相AFLP标记E16M5240,估算它们与目标基因间的遗传距离分别为10．14cM和7．56cM。     

玉米S组CMS育性恢复基因的分子标记定位

以[Mo17(rf_3rf_3)CMS-唐徐×HZ_1(Rf_3Rf_3)N]×Mo17(rf_3rf_3)N回交群体作为基因定位群体。采用RFLP和RAPD分子标记技术,定位了Rf_3/rf_3基因。RFLP分析表明,Rf_3/rf_3基因位于第二染色体长臂上的UMC36A和UMC49分子标记之间,其遗传距离分别为12.7cM和4.8cM。采用BSA法,筛选了340个10mer随机引物,找到与Rf_3基因紧密连锁的分子标记RAPD Eo8-1.2,将Rf_3/rf_3基因的标记距离缩小到2.7cM,为Rf_3/rf_3基因辅助选择提供了有价值的分子标记。     

水稻苯达松敏感致死基因(ben)的电子杂交定位和基因预测

来自水稻突变体的苯达松敏感致死基因（ben）能够作为一种除草剂筛选标志用于杂交水稻种子的安全生产。本研究利用我们已得到的与Ben/ben基因紧密连锁的两个RAPD标记（OPG18/972,OPG18/943）以及与该标记高度同源的跨叠克隆Contig10968（来自中国水稻基因组框架序列,全长8 273 bp）的序列信息,沿标记的两端设计新的PCR引物     

花生黄曲霉侵染抗性的AFLP标记

本研究利用抗、感黄曲霉菌侵染的花生品种为亲本配制杂交组合“J11×中花5号”,以其F₂分离群体为研究材料，采用AFLP技术和BSA分析方法，获得了与花生黄曲霉菌侵染抗性连锁的2个分子标记，标记与抗性间的遗传距离分别为8.8 cM和6.6 cM;利用获得的分子标记对抗、感黄曲霉的花生种质资源进行了分子鉴定，实验结果表明分子标记与抗性鉴定结果具有较高的一致性，证实了两标记应用于研究群体之外的育种潜力。该抗侵染分子标记的建立为开展花生抗黄曲霉辅助选择育种提供了有效的筛选技术。     

Development of molecular markers linked to the wheat powdery mildew resistance gene Pm4b and marker validation for molecular breeding

Powdery mildew, caused by Blumeria graminis (DC.) E.O. Speer f. sp. tritici, is an important disease in wheat (Triticum aestivum L.). Bulk segregant analysis (BSA) was employed to identify SRAP (sequence‐related amplified polymorphism), sequence tagged site (STS) and simple sequence repeat (SSR) markers linked to the Pm4b gene, which confers good resistance to powdery mildew in wheat. Out of 240 SRAP primer combinations tested, primer combinations Me8/Em7 and Me12/Em7 yielded 220‐bp and 205‐bp band, respectively, each of them associated with Pm4b. STS‐₂₄₁ also linked to Pm4b with a genetic distance of 4.9 cM. Among the eight SSR markers located on wheat chromosome 2AL, Xgwm382 was found to be polymorphic and linked to Pm4b with a genetic distance of 11.8 cM. Further analysis was carried out using the four markers to investigate marker validation for marker‐assisted selection (MAS). The results showed that a combination of the linked markers STS_?241, Me8/Em7_?220 and Xgwm382 could be used for marker‐assisted selection of the resistance gene Pm4b in wheat breeding programmes.     

A codominant SCAR marker linked to the genic male sterility gene (ms1) in chili pepper (Capsicum annuum)

As one of the genic male sterility (GMS) materials in chili pepper ( Capsicum annuum L.), GMS1 has been used for commercial F₁ hybrid seed production. The male sterility of GMS1 is controlled by a recessive nuclear gene, named ms ₁ . In this study, we developed DNA markers linked to the ms ₁ locus using a combination of bulked segregant analysis and amplified fragment length polymorphism (AFLP) in a segregating sibling population. From the screening of 1024 AFLP primer combinations, the AFLP marker E-AGC/M-GTG (514 bp) was identified as being linked to the ms ₁ locus at a distance of about 3 cM. Based on internal sequencing analysis of the E-AGC/M-GTG marker between male fertile and sterile plants, we identified three small deletions with a size of altogether 42 bp in the male-fertile plant and developed a codominant sequence characterized amplified region (SCAR) marker. This SCAR marker may be valuable for marker-assisted breeding in the hybrid seed production system of chili pepper using the GMS1 line.     

小麦抗条锈病基因YrZH84的RGAP标记及其应用

利用RGAP标记及基于标记基因型和表型选择的分离群体分组分析法,对小麦抗条锈病骨干亲本周8425B和感病品种中国春及其F₂分离群体进行分析,获得了与抗条锈基因YrZH84紧密连锁的RGAP标记Xrga-1,连锁距离为0.8 cM。其RGA片段长度为343 bp,BLAST分析结果表明,该RGA序列与已克隆的大麦抗秆锈病基因Rpg1核苷酸序列同源性为93%,与大麦抗白粉病基因Mla家族的核苷酸序列同源性为92%。用标记Xrga-1对黄淮麦区58个小麦品种(系)进行了检测,结合系谱分析和抗病性鉴定结果,发现周8425B的衍生品种周麦11、周麦17、周麦20、周麦22、矮抗58、04中36、源育3号、05中37、宛抗18、豫展10号携带抗条锈病基因YrZH84。这些研究结果对小麦抗条锈病分子育种和YrZH84基因克隆有重要作用。     

Mapping of male sterility gene ms10 in chilli pepper (Capsicum annuum L.)

Most of the hybrid seed in chilli are produced manually, but the use of male sterility (MS) can reduce the cost of hybrid seed production. MS‐12, a nuclear male‐sterile (NMS) line developed at Punjab Agricultural University, Ludhiana (India), has been utilized to develop commercial F₁ hybrids. A recessive gene, designated as ms10, governs MS in MS‐12. Due to recessive gene control, development of new NMS lines incorporating ms10 gene is tedious and time‐consuming. We identified SSR markers AVRDC‐PP12 and AVRDC_MD997* linked to the ms10 gene. A total of 558 primer pairs were screened following bulked segregant analysis (BSA). Linkage analysis in 210 F₂ plants indicated that the two SSR markers were linked to the ms10 gene and the marker AVRDC‐PP12 was closest to the gene at 7.2 cM distance. The marker was mapped to chromosome 1 at genome position 175 694 513 to 175 694 644. Until more closely linked markers are developed, the marker AVRDC‐PP12 would facilitate transfer of ms10 gene through marker‐assisted selection (MAS). Fine mapping would lead to cloning of the ms10 gene.     

SCAR and RAPD markers associated with 18-carbon fatty acids in rapeseed, Brassica napus

Breeding rapeseed for enhanced oil quality includes the development of varieties with low linolenic acid content. The breeder also aims to develop varieties with a high linoleic acid content because of its nutritional value. Restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers have been developed for linolenic acid content, but they are not best suited for a direct application in marker-assisted selection. The RFLP technique is too complex and time-consuming and RAPD markers lack codominance, precluding the distinction of homozygous from heterozygous individuals. In this report the conversion of a RAPD marker to a codominant sequence characterized amplified region (SCAR) marker named L1L9 is described. One of the alleles consisting of an 899 bp fragment (allele A), is associated with low linolenic acid content. The other allele consists of an 641 bp fragment (allele B) and is associated with high linolenic acid content. This marker explains approximately 25% of the genetic variation for this trait. Linkage analysis in the mapping population indicates that the SCAR marker probably tags an ω-3 desaturase gene in B. napus. Two RAPD markers were found to be associated with oleic/linoleic acid content. Markers M14-350 and I06-650 explained approximately 10% and 7% of the genetic variation for linoleic acid content, respectively. These two markers were found linked at 12.3cM in the segregating B. napus F₂ progeny used for mapping. All the markers reported in this paper should be useful in breeding programmes for developing high linoleic and low linolenic acid rapeseed varieties.     

Genetic mapping for the Rf1 (fertility restoration) gene in sunflower (Helianthus annuus L.) by SSR and TRAP markers

The Rf1 gene in sunflower can effectively restore the pollen fertility of PET1 cytoplasm in male-sterile lines and has been widely used in commercial hybrid production. Identifying molecular markers tightly linked to this gene will be useful in marker-assisted selection to develop maintainer and restorer lines. Rf1 has been mapped to Linkage Group (LG) 13 of the public sunflower simple sequence repeat (SSR) map by aligning maps constructed from different populations and only one SSR marker was reported to be loosely linked to Rf1 . This paper reports the result of applying target region amplification polymorphism (TRAP) and SSR markers to map and develop a sequence-tagged site (STS) marker tightly linked to Rf1 using two populations derived from a cross between two U.S. public sunflower lines, RHA439 and cmsHA441. An SSR marker, ORS511, was 3.7 cM from the Rf1 gene and a TRAP marker, K11F05Sa12-160, was linked to Rf1 at a distance of 0.4 cM. This TRAP marker was converted to an STS marker for using in sunflower breeding.     

A genetic linkage map of rhodesgrass based on an F1 pseudo-testcross population

The linkage relationships between 164 polymorphic amplified fragment length polymorphism (AFLP) and 25 restriction fragment length polymorphism (RFLP) fragments assayed in a pseudo‐testcross population generated from the mating of single genotypes from two divergent cultivars were used to construct female, ‘Katambora’ (‘Kat’) and male, ‘Tochirakukei’ (‘Toch’) parental genetic maps for rhodesgrass. The ‘Kat’ genetic map consists of 84 marker loci (72 AFLP and 12 RFLP markers) distributed on 14 linkage groups and spans a total length of 488.3 cM, with an average distance of 7.8 cM between adjacent markers. The ‘Toch’ genetic map consists of 61 marker loci (52 AFLP and nine RFLP) mapped on 12 linkage groups spanning a total length of 443.3 cM, with an average spacing of 9.0 cM between adjacent markers. About 23% of the markers remained unassigned. The level of segregation distortion observed in this cross was 11.1%. In both maps, linked duplicated RFLP loci were found. These linkage maps will serve as a starting point for linkage studies in rhodesgrass with potential application for marker‐assisted selection in breeding programmes.     

Molecular markers linked to genes for specific rust resistance and indeterminate growth habit in common bean

Bulked segregant analysis was utilized to identify random amplified polymorphic DNA (RAPD) markers linked to genes for specific resistance to a rust pathotype and indeterminate growth habit in an F2 population from the common bean cross PC-50 (resistant to rust and determinate growth habit) × Chichara 83-109 (susceptible to rust and indeterminate growth habit). Six RAPD markers were mapped in a coupling phase linkage with the gene ( Ur-9) for specific rust resistance. The linkage group spanned a distance of 41 cM. A RAPD marker OA4.1050 was the most closely linked to the Ur-9 gene at a distance of 8.6 cM. Twenty-eight RAPD markers were mapped in a coupling phase linkage with the gene ( Fin) for indeterminate growth habit. The linkage group spanned a distance of 77 cM. RAPD markers OQ3.450 and OA17.600 were linked to the Fin allele as flanking markers at a distance of 1.2 cM and 3.8 cM, respectively. The RAPD markers linked to the gene for specific rust resistance of Andean origin detected here, along with other independent rust resistance genes from other germplasm, could be utilized to pyramid the different genes into a bean cultivar for durable rust resistance.