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.     

Molecular mapping of a new red mutant gene (Rs) in upland cotton

In recent years, there has been slow progress in improving cotton yield. It is known that the F₁ generation from the cross of the new red mutant and the normal green leaf plant has high photosynthetic efficiency. Therefore, cloning the new red mutant gene and further introducing it into other crops through transgenic techniques is a promising approach for achieving high photosynthetic efficiency through breeding. To map this new mutant gene, tentatively named R _s , the authors constructed an F₂ generation containing 1214 individual plants from mutant EH083 ( Gossypium hirsutum ) and Hai 7124 ( Gossypium barbadense ). Fifty-five pairs of simple sequence repeats and sequence-related amplified polymorphism (SRAP) primers on chromosome 7 were selected to screen the two parents. Finally, the R _s gene was mapped at the 0.3 cM interval flanked by markers NAU3735 and NAU1048.     

Molecular mapping of a fertility restorer gene for cytoplasmic male sterility in soybean

In this study, we report the mapping of the Rf locus in soybean by microsatellite simple sequence repeat (SSR) genetic markers. A cross was made between cytoplasmic male sterility (CMS) line JLCMS82A and restorer line JIHUI 1 based on the DNA polymorphisms revealed by 109 SSR markers. A F₂ population derived from a single F₁ plant containing 103 individuals was used for mapping the Rf locus. The Rf gene of JIHUI 1 gametophytically restores male fertility to JLCMS82A. Fertile and semi-fertile DNA bulks and parental DNAs were screened with 219 SSR markers, and Satt215 which was previously mapped to soybean LG J, was found linked to the Rf gene. Five additional polymorphic SSR markers from LG J were used for analysis and a regional linkage map around the Rf locus was established. SSR markers, Sctt011 and Satt547, flanked the Rf locus at 3.6 cM and 5.4 cM, respectively. The availability of these SSR markers will facilitate the selection of restorer lines in hybrid soybean breeding.     

Cytogenetic analysis of F1, F2 and BC1 plants from intergeneric sexual hybridization between Sinapis alba and Brassica oleracea by genomic in situ hybridization

By intergeneric sexual hybridization between Sinapis alba and Brassica oleracea , F₁, F₂ and BC₁ progeny plants were produced. S. alba plants (genome SS, 2n = 24) were pollinated with B. oleracea (genome CC, 2n = 18), and the fertile F₁ plants were pollinated with B. oleracea to obtain BC₁ plants. GISH analysis showed that 10 out of 12 F₁ plants had 12 S. alba chromosomes (one full S chromosome set) and nine B. oleracea chromosomes (one C chromosome sets), representing the expected hybrids. However, two F₁ plants had 12 S chromosomes and 18 C chromosomes (two C chromosome sets), indicating unexpected hybrids. A maximum of three trivalents between C and S chromosomes were identified at metaphase I of semi-fertile F₁ pollen mother cells (PMCs), which indicates homology and chromosome pairing between these two genomes. The C genome had obviously been doubled in two F₂ plants from selfed semi-fertile F₁ plants. BC₁ plants consisted of 18 C chromosomes and different numbers of one, five and six additional S chromosomes, respectively. Monosomic alien addition lines developed in the present study can be used for B. oleracea breeding and Sinapis alba gene mapping.     

Identification and molecular tagging of a stripe rust resistance gene in wheat line P81

Stripe rust, caused by Puccinia striiformis f. sp. tritici (PST), is one of the most devastating diseases in common wheat ( Triticum aestivum L.). With the objective of identifying and tagging a new gene for resistance to stripe rust in wheat line P81, F₁, F₂ and F_2:3 populations from the cross 'Chuanmai 28'/P81 were inoculated with Chinese PST race CYR32 in greenhouse and field trials. P81 carried a single dominant gene for resistance (designated YrP81 ) to CYR32. Tests of allelism showed that YrP81 was different from Yr5 , Yr10 , Yr15 and Yr26 . Simple sequence repeat (SSR) and resistance gene-analogue polymorphism (RGAP) between the parents were used for genotyping the F₂ populations. YrP81 was closely linked to four SSR loci on chromosome 2BS with genetic distances of 18.3 cM ( Xwmc25 ), 1.8 cM ( Xgwm429 ), 4.1 cM ( Xwmc770 ) and 5.3 cM ( Xgwm148 ). Two RGAP markers RGA1 (NLRR/XLRR) and RGA2 (Pto kin4/NLRR-INV2) were also closely linked to YrP81 with genetic distances of 4.7 and 6.3 cM, respectively. The linkage map of YrP81 and molecular markers was established in the order Xwmc25 - RGA2 - RGA1 - Xgwm429 - YrP81 - Xwmc770 - Xgwm148 . Pedigree analysis, response patterns with Chinese PST races and associations with markers suggested that YrP81 is a novel stripe rust resistance gene. The PCR-based microsatellite and RGAP markers identified here could be applied in selection of YrP81 in wheat breeding.     

利用染色体片段代换系定位棉花抗黄萎病QTL

通过陆地棉与海岛棉杂交并与陆地棉回交,获得1个染色体片段代换系苏VR043,抗江苏非落叶型黄萎病菌系BP2。分子检测表明,苏VR043兼有陆地棉苏棉8号的遗传背景和海7124的D4染色体片段。以苏VR043和苏棉8号为亲本构建包含176个单株的F2群体,通过标记分析和F2:3家系抗病鉴定,发现抗病性状与标记NAU3392连锁,p值为2.3×10-12。根据棉花D组染色体测序结果,参照置换区段的基因组序列,合成92对SSR引物;PCR扩增分析发现,有5对引物在苏棉8号和苏VR043之间表现多态性,并将抗病主效QTL定位在标记ZHX32和NAU3392之间,标记间遗传距离为3.2 c M,QTL解释表型变异64.8%。同时参照棉花D组测序结果,提取对应的置换区段序列,预测包含63个基因,基因聚类分析表明7个基因参与抗逆反应,其中1个基因与抗病相关。     

Molecular mapping of two recessive genes controlling resistance to bacterial leaf pustule disease in soybean (Glycine max)

Bacterial leaf pustule (BLP) caused by Xanthomonas axonopodis pv. glycines (Xag) is a serious soybean disease. A BLP resistant genotype ‘TS-3’ was crossed with a BLP susceptible genotype ‘PK472’, and a segregating F₂ mapping population was developed for genetic analysis and mapping. The F₂ population segregation pattern in 15:1 susceptible/resistance ratio against Xag inoculum indicated that the resistance to BLP in ‘TS-3’ was governed by two recessive genes. A total of 12 SSR markers, five SSR markers located on chromosome 2 and seven SSR markers located on chromosome 6 were identified as linked to BLP resistance. One of the resistance loci (r1) was mapped with flanking SSR markers Sat_183 and BARCSOYSSR_02_1613 at a distance of 0.9 and 2.1 cM, respectively. Similarly, SSR markers BARCSOYSSR_06_0024 and BARCSOYSSR_06_0013 flanked the second locus (r2) at distances of 1.5 and 2.1 cM, respectively. The identified two recessive genes imparting resistance to BLP disease and the SSR markers tightly linked to these loci would serve as important genetic and molecular resources to develop BLP resistant genotypes in soybean.     

Genetic linkage map of cassava (Manihot esculenta Crantz) based on AFLP and SSR markers

To generate a genetic linkage map of cassava ( Manihot esculenta Crantz), 58 F₁ progenies from a cross between Rayong 90 (female) and Rayong 5 (male) were examined in amplification fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses. A total of 469 polymorphic markers consisting of 378 AFLPs generated from 76 primer combinations and 91 SSRs were identified. These markers were analyzed using the joinmap ^® 3.0 package program to construct a genetic linkage map. A total of 33 linkage groups of a common map were constructed from 119 AFLPs and 18 SSRs, spanning 1095 cM with an average of 7.99 cM between markers. The genetic linkage map generated in this study will be useful for genetic studies in cassava particularly for the identification of genetic markers linked to traits of interest, although the complex cassava genome suggests that maybe a long term objective.     

A CAPS marker TAO1902 diagnostic for the I-2 gene conferring resistance to Fusarium oxysporum f. sp. lycopersici race 2 in tomato

Fusarium oxysporum f. sp. lycopersici inhabits most tomato-growing regions worldwide, causing tomato production yield losses. A molecular marker linked to resistance would be useful for tomato improvement programmes. Thus, a cleaved amplified polymorphic sequence (CAPS) marker TAO1₉₀₂ was developed to identify tomato genotypes possessing the I-2 gene, which confers resistance to F. o. lycopersici race 2. The Rsa I or Fok I restriction fragments corresponded to the presence or absence of the I-2 allele in a segregating 100 F₂ progeny, tomato cultivars, 16 resistant and 20 susceptible to Fusarium wilt, respectively, lines and F₁ hybrids, representing various tomato gene pools. TAO1₉₀₂ may be helpful for selection of F. o. lycopersici -resistant tomato germplasm.     

Improving Fiber Quality Traits of Xinluzao 45 Using Gossypium barbadense Chromosome Segment Substitution Lines

Gossypium barbadense chromosome segment substitution lines could be used as an important germplasm resource for improving fiber quality traits of upland cotton. In this study, we used TM-1 background substitution lines CSSL-122, the Xinjiang upland cotton Xinluzao 45 and hybrid, backcross progeny BC₂F₁ populations comprising 120 individual plants as test materials. Nineteen SSR markers located in Gossypium barbadense chromosomes land inked with fiber length and strength were used to screen out obvious polymorphic primers between the parents. We then traced and detected chromosome segments of Gossypium barbadense in the BC₂F₁ populations. Simultaneously, we compared fiber quality traits of positive plants that contained Gossypium barbadense chromosome segments with non-positive plants in the BC₂F₁ populations. The results showed that two markers, NAU2987 and BNL3145, which were linked with fiber length, could accurately identify the positive plants. In addition, compared with non-positive plants, the increased fiber length and strength of the positive plants were very significant (P < 0.01). Our research suggested that alien Gossypium barbadense chromosome segments significantly improved the fiber quality traits of Xinluzao 45. Thus, the Gossypium barbadense chromosome segment substitution lines will provide a vital theoretical basis and practical reference for improving Upland Cotton fiber quality traits.     

A high-resolution map of the rice blast resistance gene Pi15 constructed by sequence-ready markers

The gene Pi15 for resistance of rice to Magnaporthe grisea was previously mapped to a ≈0.7-cM region on chromosome 9. To further define the chromosomal region of the Pi15 locus, a contig spanning the locus was constructed, in silico , through bioinformatics analysis using a reference sequence of the cultivar 'Nipponbare'. One simple sequence repeat marker adopted from the International Rice Microsatellite Initiative and six candidate resistance gene (CRG) markers, developed from gene annotation of the reference sequence of the contig, were used for linkage analysis in a mapping population consisting of 504 extremely susceptible F₂ plants. The Pi15 locus was delimited to a ≈0.5-cM region flanked by the markers CRG5 and CRG2 and co-segregated with the markers BAPi15₇₈₂, CRG3 and CRG4, which was physically converted to a 44-kb interval.     

Inheritance and gene tagging of male fertility restoration of cytoplasmic-nuclear male-sterile line NJCMS1A in soybean

The F₁, F₂ and F_2:3 of the NJCMS1A × 'Zhongdou 5' cross were used to analyse the inheritance of the male fertility restoration of the cytoplasmic-nuclear male-sterile line NJCMS1A in soybean. The results of genetic analysis showed two pairs of dominant genes conferring the male fertility restoration of NJCMS1A, which further confirmed previous results. The F₂ population from the NJCMS1A × 'Zhongdou 5' cross was used for tagging the restorer genes for NJCMS1A with 664 pairs of simple sequence repeat primers selected randomly from the genetic linkage map of soybean published by Cregan et al. (1999) . Satt626 on linkage group M and Satt300 on linkage group A1 of the integrated linkage map by Song et al. (2004) were found to link to the two restorer genes of NJCMS1A. The maximum-likelihood estimates of the genetic distance between the two markers, Satt626 and Satt300, and the two restorer genes of 'Zhongdou 5' were 9.75 and 11.18 cM, respectively.     

QTL mapping of maize (Zea mays) stay-green traits and their relationship to yield

A maize genetic linkage map derived from 115 simple sequence repeat (SSR) markers was constructed from an F₂ population. The F₂ was generated from a cross between a stay-green inbred line (Q319) and a normal inbred line (Mo17). The map resolved 10 linkage groups and spanned 1431.0 cM in length with an average genetic distance of 12.44 cM between two neighbouring loci. A total of 14 quantitative trait loci (QTL) were detected for stay-green traits at different postflowering time intervals and identified by composite interval mapping. The respective QTL contribution to phenotypic variance ranged from 5.40% to 11.49%, with trait synergistic action from Q319. Moreover, maize stay-green traits were closely correlated to grain yield. Additional QTL analyses indicated that multiple intervals of stay-green QTL overlapped with yield QTL.     

2个抗虫棉的外源Bt基因分子鉴定及其染色体定位

以中美2个抗虫棉品种GK19与33B为试验材料,利用检测中美Bt基因的特异性引物,分别对抗虫棉亲本GK19和33B进行PCR扩增,并通过SSR分子标记技术对其Bt基因进行分子鉴定与染色体定位,旨在从外源基因转化事件的视角探究中美转基因抗虫棉差异的分子基础。结果表明, GK19为中国转Bt基因抗虫棉, 33B为美国转Bt基因抗虫棉; GK19的Bt基因被定位在棉花Chr.20上,共16对SSR多态性标记与其Bt基因连锁,两侧的分子标记为NAU3907和NAU2579,其遗传距离分别为2.4 cM和1.5 cM; 33B的Bt基因被定位在棉花Chr.26上,共20对SSR多态性标记与Bt基因连锁,目标Bt基因位于标记NAU460和dc40260之间,其遗传距离分别为3.6 cM和2.0 cM。以上结果表明GK19和33B属于不同的遗传转化事件。     

Development of STS markers and QTL validation for common bacterial blight resistance in common bean

Common bacterial blight (CBB) of common bean ( Phaseolus vulgaris L.), is one of the major diseases that decrease yield and quality. A major quantitative trait locus (QTL) for CBB resistance from line XAN 159 was transferred into two bean lines, HR45 and HR67. Previous studies identified that two markers are linked to this QTL but the chromosome location was not consistent. To identify more tightly linked markers and to verify the chromosome location, 65 additional markers were mapped using 81 recombinant inbred lines (RILs) derived from a cross HR67 × OAC95-4. The QTL was mapped to a 13 cM region on chromosome 1 and defined by eight molecular markers that explained 25–52% of the phenotypic variation. Six tightly linked amplified fragment length polymorphism markers (0.6–9.7 cM from the QTL peak) were converted into seven sequence tagged site markers, three of which were mapped to this QTL. Five tightly linked markers were used to screen 907 F₂ plants derived from a cross HR45 × 'OAC Rex' and four of them were linked to each other within 4.2 cM. These markers may be useful in marker-assisted selection and map-based cloning of this major QTL.     

Fine‐mapping a fibre strength QTL QFS‐D11‐1 on cotton chromosome 21 using introgressed lines

An initial F₂ mapping population of 223 plants of the cross between TM‐1 (Gossypium hirsutum L.) × H102 (Gossypium barbadense L.) was used to map QTLs controlling fibre strength in cotton. A genetic linkage map with 408 SSR markers was constructed with a total length of 3872.6 cM. Multiple‐QTL model of the software MapQTL version 5.0 was used to map QTLs related to fibre strength of the F_2 : 3 population. QTL QFS‐D11‐1 conferring fibre strength was mapped between NAU2950 and NAU4855 on chromosome 21 (Chr. 21) which explained 23.4% of phenotypic variation. Introgressed lines (ILs), that is, IL‐D11‐1, IL‐D11‐2 and IL‐D11‐3 were obtained through marker‐assisted backcrossing in TM‐1 background. An F₂ population of 758 plants derived from cross IL‐D11‐2 × TM‐1 was used for fine‐mapping QTL QFS‐D11‐1. QFS‐D11‐1 was mapped between markers NAU2110 and NAU2950, adjacent to its initial interval NAU2950–NAU4855 with phenotypic variation explaining 35.8%. QFS‐D11‐1 was further mapped to 0.6 cM from the flanking marker NAU2950. The results will give a basis for marker‐assisted selection of QFS‐D11‐1 in cotton breeding and to lay the foundation for cloning QFS‐D11‐1.     

利用2个F_2群体整合中国豌豆高密度SSR遗传连锁图谱

豌豆(Pisum sativum L.)是一种重要的食用豆类作物,在全世界范围内广泛种植,既可作为人类食物,也可作为牲畜饲料。用SSR标记构建的遗传连锁图谱在豌豆和其他作物的标记辅助育种中发挥着重要的作用。尽管对豌豆遗传连锁作图的研究已有悠久历史,但公众可获得且可转移的SSR标记以及基于遗传独特的中国豌豆种质的高密度遗传连锁图谱仍然有限。为了获得更多可转移的SSR标记和中国豌豆的高密度遗传连锁图谱,本研究首先从自主开发和文献获取的12,491个全基因组SSR标记中筛选了617个多态性SSR标记,并用于G0003973×G0005527 F_2群体遗传连锁图谱的加密。加密后的图谱全长扩展到5330.6 cM,包含603个SSR标记,标记平均间距离8.8 cM,相比之前的图谱有明显改善。基于上述结果,我们又筛选了119个具有多态性的SSR标记,用于构建大样本W6-22600×W6-15174 F_2群体的遗传连锁图谱,新图谱累积长度为1127.1 cM,包含118个SSR标记,装配在7条连锁群上。最后,将来自以上2个遗传图谱的数据进行整合,得到了一张覆盖范围6592.6 cM的整合图谱,包含668个SSR标记,由509个基因组SSR、134个EST-SSR和25个锚定标记组成,分布在7条连锁群上。这些SSR标记和遗传连锁图谱将为豌豆的遗传研究和标记辅助育种提供有力工具。     

与亚洲棉光籽突变体DPL972中光籽基因紧密连锁的SSR分子标记的开发与应用

对棉纤维相关性状基因的克隆及遗传分析可为阐明纤维形成机制奠定良好基础.以野生型材料DPL971及其光籽突变体DPL972为亲本构建F2群体,其中光籽与毛籽性状分离比符合3∶1,表明突变体DPL972中的光籽基因为显性单基因(将其命名为GaFzl).结合棉花基因组数据,合成1567对SSR(Simple sequence repeat)引物,覆盖了A组13条染色体,标记多态性分析和群体连锁分析发现在染色体A08上存在15对与光籽基因GaFzl连锁的标记,其中最近的标记为SSR82,遗传距离为6.6 cM.本实验完成了GaFzl基因的染色体初步定位,开发了15个与其连锁的SSR标记,为下一步图位克隆奠定了基础.     

Identification of AFLP and RAPD markers linked to anthracnose resistance in grapes and their conversion to SCAR markers

Resistance to anthracnose or black spot ( Elsinoe ampelina ), a serious fungal pathogen in viticulture and table grape production, was investigated on 25 grape cultivars. Bioassays performed with culture filtrates produced by the pathogen revealed 14 resistant genotypes. In most plants resistance originated from Vitis labrucsa but also genotypes with V. rupestris and V. riparia  ×  V. rupestris background showed resistance. Genetic analysis was conducted in F₁, S₁ and BC₁ plants developed from various cultivars. In total, 326 F₁ plants were evaluated, 172 genotypes proofed to be resistant, whereas 154 were susceptible to anthracnose. A Mendelian segregation ratio of 1 : 1 (χ² = 0.30–0.65) indicating that anthracnose resistance is controlled by a single dominant gene. To facilitate the use of marker-assisted selection in grape-breeding PCR-based markers were developed by random amplified polymorphic DNA and amplified fragment length polymorphism in bulk segregant analysis. Finally, OPB 15₁₂₄₇ was developed as a sequence characterized amplified region marker being diagnostic for the locus of resistance to anthracnose in all resistant genotypes tested. Within the 25 grape cultivars OPB 15₁₂₄₇ is diagnostic in the genetic background of both V. labrucsa and V. rupestris and V. riparia  ×  V. rupestris .     

大豆品种郑97196抗疫霉病基因RpsZheng精细定位

大豆疫霉病是由大豆疫霉引起的一种重要大豆病害,可造成严重的经济损失。种植含有抗疫霉病基因的大豆品种是控制该病害最有效的途径。前人在大豆品种郑97196的3号染色体上鉴定了一个抗疫霉病基因RpsZheng。本研究的目的是验证幵精细定位抗疫霉病基因RpsZheng。以Williams和郑97196杂交衍生的188个F_(2:3)家系为作图群体,用大豆3号染色体上的SSR标记构建RpsZheng遗传连锁图,获得与RpsZheng紧密连锁的侧翼SSR标记SattWM82_39 (2.5 cM)和BARCSOYSSR_03_0269 (1.0 cM)。基于亲本间全基因组重测序数据鉴定和开发多态性InDel标记,进一步将RpsZheng候选区域缩小至105.2 kb,通过检测RpsZheng候选区域内的共分离标记特异性,获得了能够有效检测RpsZheng的分子标记WZInDel11。本研究明确了RpsZheng的候选基因组区间,鉴定出了能够有效用于基因功能研究和辅助选择育种的共分离分子标记。