Identification of AFLP and SCAR markers linked to the male fertility restorer gene of <Emphasis Type="Italic">pol</Emphasis> CMS (<Emphasis Type="Italic">Brassica napus</Emphasis> L.)

The pol cytoplasmic male-sterility system has been widely used as a component for utilization of heterosis in Brassica napus and offers an attractive system for study on nuclear–mitochondrial interactions in plants. Genetic analyses have indicated that one dominant gene, Rfp, was required to achieve complete fertility restoration. As a first step toward cloning of this restorer gene, we attempted molecular mapping of the Rfp locus using the amplified fragment length polymorphism (AFLP) technique combined with bulked segregant analysis (BSA) method. A BC₁ population segregating for Rfp gene was used for tagging. From the survey of 1,024 AFLP primer combinations, 13 linked AFLP markers were obtained and five of them were successfully converted into sequence characterized amplified region (SCAR) markers. A population of 193 plants was screened using these markers and the closest AFLP markers flanking Rfp were at the distances of 2.0 and 5.3 cM away, respectively. Further the AFLP or SCAR markers linked to the Rfp gene were integrated to one doubled-haploid (DH) population derived from the cross Quantum × No.2127-17 available in our laboratory, and Rfp gene was mapped on N18, which was the same as the previous report. These molecular markers will facilitate the marker-assisted selection (MAS) of pol CMS restorer lines.     

甘蓝型油菜遗传图谱的构建及芥酸含量的QTL分析

一个由甘蓝型油菜品种Quantum (黄花、低芥酸)和人工合成的甘蓝型油菜品系No.2127-17（白花、高芥酸）为亲本材料建立的DH群体中芥酸呈现单基因的遗传模式。为了发展与芥酸紧密连锁的分子标记对其实行有效的控制,随机选择121个结实正常的DH系为作图群体,利用SSR和RAPD标记构建了一张甘蓝型油菜的遗传连锁图谱。在亲本间检测     

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。     

Identification of RAPD and SCAR markers linked to northern leaf blight resistance in waxy corn (Zea mays var. ceratina)

Exserohilum turcicum causes northern corn leaf blight (NCLB), an important disease occurring in maize producing areas throughout the world. Currently, the development of cultivars resistant to E. turcicum seems to be the most efficient method to control NCLB damage. Marker-assisted selection (MAS) enables breeders to improve selection efficiency. The objective of this work was to identify random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) markers associated with NCLB resistance. Bulked segregant analysis (BSA) was used to search for RAPD markers linked to NCLB resistance genes, using F₂ segregating population obtained by crossing a susceptible inbred ‘209W’ line with a resistant inbred ‘241W’ line. Two hundred and twenty-two decamer primers were screened to identify four RAPD markers: OPA07₅₂₁, OPA16₄₅₇, OPB09₅₂₀, and OPE20₅₃₆ linked to NCLB resistance phenotype. These markers were converted into dominant SCAR markers: SCA07₄₉₆, SCA16₄₂₀, SCB09₄₆₄, and SCE20₄₂₉, respectively. The RAPD and SCAR markers were developed successfully to identify NCLB resistant genotypes in segregating progenies carrying NCLB resistant traits. Thus, the markers identified in this study should be applicable for MAS for the NCLB resistance in waxy corn breeding programs.     

水稻显性半矮秆基因的SCAR标记及初步定位

粳稻品系Y98149是从离子束诱变的后代中获得的显性半矮秆突变体，与野生型Y98148是一对株高近等基因系。将已经获得的3个与水稻显性半矮秆基因紧密连锁的RAPD标记分别克隆、测序，根据测序结果设计了3对特异性PCR引物，成功地将RAPD标记S1041₅₂₅、S1076₅₄₉和S1272₄₀₃转化成更稳定的SCAR标记SCS1041₄₉₈、SCS1076₅₁₀和SCS1272₃₈₈。通过Y98148×Y98149的F₂代分离群体的分析，这3个SCAR标记与显性半矮秆基因的遗传距离分别为12.6 cM、7.5 cM和16.3 cM, 且位于基因的同一侧。序列同源性比较表明，标记S1272₄₀₃为单拷贝，其核苷酸序列与水稻第7染色体上两个BAC克隆B1249D05(AP006451)和OJ1212-C12(AP005604)同源性为99%，B1249D05与OJ1212-C12有23 kb的重叠区域，标记S1272₄₀₃位于这个重叠区域，据此初步将显性半矮秆基因定位，为进一步精确定位和图位克隆奠定了基础。     

Identification and inheritance of a partially dominant gene for yellow seed colour in Brassica napus

A yellow‐seeded doubled haploid (DH) line no. 2127‐17, derived from a resynthesized Brassica napus L., was crossed with two black‐seeded Brassica cultivars ‘Quantum’ and ‘Sprint’ of spring type. The inheritance of seed colour was investigated in the F₂, and BC1 populations of the two crosses and also in the DH population derived from the F1 of the cross ‘Quantum’× no. 2127‐17. Seed colour analysis was performed with the colorimeter CR‐300 (Minolta, Japan) together with a visual classification system. The immediate F1 seeds of the reciprocals in the two crosses had the same colour as the self‐pollinated seeds of the respective black‐ and yellow‐seeded female parents, indicating the maternal control of seed colour. The F1 plants produced yellow‐brown seeds that were darker in colour than the seeds of no. 2127‐17, indicating the partial dominance of yellow seed over black. In the segregating BC1 progenies of the two crosses, the frequencies of the black‐ and yellow‐seeded plants fit well with a 1 : 1 ratio. In the cross with ‘Quantum’, the frequencies of yellow‐seeded and black‐seeded plants fit with a 13 : 3 ratio in the F₂ progeny, and with a 3 : 1 ratio in the DH progeny. However, a 49 : 15 segregation ratio was observed for the yellow‐seeded and black‐seeded plants in the F₂ progeny of the cross with ‘Sprint’. It was postulated from these results that seed colour was controlled by three pairs of genes. A dominant yellow‐seeded gene (Y) was identified in no. 2127‐17 that had epistatic effects on the two independent dominant black‐seeded genes (B and C), thereby inhibiting the biosynthesis of seed coat pigments.     

青海大黄油菜粒色性状分子标记的开发和图谱整合

利用青海大黄油菜和褐籽白菜型油菜09A-126构建BC₄和F₂分离群体, 结合AFLP与群体分离分析法(bulked segregant analysis, BSA)筛选引物, 获得5个与黄籽基因Brsc1紧密连锁的分子标记Y11~Y15。5个AFLP特异片段的序列, 均与白菜型油菜的A9染色体部分序列表现同源。将5个AFLP标记成功转化为5个SCAR标记(SC11~SC15)。利用目标基因所在染色体区段序列筛选到7个与目标基因紧密连锁的SSR标记(BrID10607、KS10760、B089L03-3和A1~A4)。利用SCAR和SSR标记扫描F₂群体中部分单株, 发现SC14和A1为共显性标记。用BC₄群体将Brsc1定位在标记Y06和A4之间1.7 Mb的区间内, 遗传距离分别为0.115 cM和0.98 cM。标记Y05和Y12与Brsc1共分离。本研究为黄籽油菜分子标记辅助选择育种体系的建立及目标基因的进一步精细定位和图位克隆奠定了基础。     

Identification of SCAR markers linked to <Emphasis Type="Italic">or</Emphasis>, a gene inducing beta-carotene accumulation in Chinese cabbage

The or mutation in Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a recessive, single-locus mutation that causes the head leaves of the plant to accumulate carotenoids and turn orange. In China, considerable attention has been focused in recent years on breeding the variety with orange head leaves. In this study, sequence-characterized amplified region (SCAR) markers linked to the or gene were identified based on random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) by performing a bulked segregant analysis (BSA) using a doubled haploid (DH) population derived from the F₁ cross between 91-112 (white head leaves) and T12-19 (orange head leaves) via microspore culture. Two RAPD markers—OPB01-845 and OPAX18-656—and 1 AFLP marker, namely, P67M54-172, were identified to be linked to the or gene, and they were successfully converted into the SCAR markers SCR-845, SCOR204, and SCOR127, respectively. In a linkage analysis, these 3 SCAR markers and 2 previously published simple sequence repeat markers, namely, BRMS-51 and Ni4D09 (located on R9 linkage group), were mapped to the same linkage group with the or gene at a LOD score of 6.0, indicating that the or gene should be located on the linkage group R9 of the A genome. In addition, accuracies of 92%, 90%, and 89.1% were obtained when 110 different inbred breeding lines of Chinese cabbage were used for investigation with these 3 SCAR markers, indicating that these makers could be used in marker-assisted selection in orange head leaf breeding programs for Chinese cabbage.     

芥菜型油菜黄籽性状的遗传、基因定位和起源探讨

油菜种皮颜色既是一个形态指示性状, 又与种子休眠和品质有关。以芥菜型油菜种皮颜色分离的2个BC₆F₂群体为作图群体,用微卫星(SSR)等标记进行连锁定位, 并用定位标记对22份材料进行关联分析, 通过反转录-聚合酶链反应(RT-PCR)分析12份材料种皮中4-二氢黄酮醇还原酶(DFR)、花色素合酶(ANS)和花色素还原酶(ANR)基因的表达, 对6份黄籽材料的种皮颜色基因等位性进行测定, 结果将芥菜型油菜控制种皮颜色的2个基因位点分别定位到A9和B3连锁群, 并找到其两侧紧密连锁标记, 发现黄籽材料种皮颜色基因位点附近0.9 cM和1.5 cM区域高度保守, 所有黑色种皮中DFR、ANS和ANR基因均表达, 所有黄色种皮中DFR和ANS均不表达,但ANR基因表达或不表达,黄籽材料的种皮颜色基因等位。根据这些结果结合前人研究, 认为芥菜型油菜种皮颜色基因是调控基因,黄籽为单一起源。     

AFLP and SCAR markers linked to the suppressor gene (Rf) of a dominant genetic male sterility in rapeseed (Brassica napus L.)

Rs1046AB is a line which is true breeding for a dominant genetic male sterility gene (Ms) but which is a mixture of male fertile and sterile individuals (a two-type line) because it is segregating for a dominant suppressor gene (Rf). This system provides a promising alternative to the CMS system for hybrid breeding in Brassica napus. In order to identify molecular markers linked to the rf gene, a near-isogenic line (NIL) population from the cross between a sterile individual (MsMsrfrf) and a fertile individual (MsMsRfrf) in Rs1046AB was subjected to amplified fragment length polymorphism (AFLP) analysis, with a combination of comparing near isogenic lines (NILs) and bulked segregant analysis (BSA). From 2,816 pairs of AFLP primers, six fragments showing polymorphism between the fertile and sterile bulks as well as the individuals of the bulks were identified. Linkage analysis indicated that the six AFLP markers are tightly linked to the Rf gene and all are distributed on the same side. The minimum genetic distance between the Rf gene and a marker was 0.7 cM. Since the AFLP markers are not suitable for large-scale application in MAS (marker-assisted selection), our objective was to develop a fast, cheap and reliable PCR-based assay. Consequently, three of the four closest AFLP markers were converted directly to sequence characterized amplified region (SCAR) markers. For the other marker a corresponding SCAR marker was successfully obtained after isolating the adjacent sequences by PCR Walking. The available SCAR markers of the Rf gene will greatly facilitate future breeding programs using dominant GMS to produce hybrid varieties.     

芥菜型油菜多室基因Bjmc2的精细定位

芥菜型多室油菜的产量比普通两室油菜更高,定位乃至克隆多室基因可为油菜遗传改良及解释多室角果形成机制创造条件。本研究通过验证JD11-2家系衍生群体仅在BjMc2位点上存在差异,可用于BjMc2的定位。采用AFLP结合BSA法分析BC₅和BC₆群体,筛选到1个与BjMc2连锁的AFLP标记并转化为SCAR标记SC1。基于该AFLP标记序列信息,利用白菜同源序列设计SSR引物和SCAR引物,获得11对SSR标记和1对SCAR标记。通过在芥菜型油菜BAC文库中的挑选,获得2个覆盖目标区域的单克隆,由此开发1个SSR标记。将获得的SCAR和SSR标记扫描BC₇群体,构建了两室性状基因BjMc2的遗传连锁图,两侧最近标记ZX17和BACsr96与目标基因之间的遗传距离分别为0.048 cM和0.340 cM,并定位到白菜A7 scaffold000019的946~1014 kb之间,约68 kb物理距离。     

Construction of an integrated genetic map based on maternal and paternal lineages of tea (Camellia sinensis)

Genetic study on important traits of tea is difficult because of its self-incompatibility in nature. Moreover, development of a new variety usually needs more than 20 years, since it takes many years from seedling to matured plants for trait investigation. Genetic map is an essential tool for genetic study and breeding. In this study, we have developed an integrated genetic map of tea (Camellia sinensis) using a segregating F1 population derived from a cross between two commercial cultivars (‘TTES 19’ and ‘TTES 8’). A total of 574 polymorphic markers (including SSR, CAPS, STS, AFLP, ISSR and RAPD), 69 markers with highly significant levels of segregation distortion (P < 0.001) (12.0 %) were excluded from further analyses. Of the 505 mapped markers, there were 265 paternal markers (52.5 %), 163 maternal markers (32.3 %), 65 doubly heterozygous dominant markers (12.9 %), and 12 co-dominant markers (2.4 %). The co-dominant markers and doubly heterozygous dominant markers were used as bridge loci for the integration of the paternal and maternal maps. The integrated map comprised 367 linked markers, including 36 SSR, 3 CAPS, 1 STS, 250 AFLP, 13 ISSR and 64 RAPD that were assigned to 18 linkage groups. The linkage groups represented a total map length of 4482.9 cM with a map density of 12.2 cM. This genetic map has the highest genetic coverage so far, which could be applied to comparative mapping, QTL mapping and marker assisted selection in the future.     

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 Aegilops speltoides derived leaf rust resistance gene Lr28 in wheat

In a segregating homozygous F₂ population of bread wheat involving a leaf rust resistance gene Lr28 derived from Aegilops speltoides, six randomly amplified polymorphic DNA (RAPD) markers, three each in coupling and repulsion phase were identified as linked to Lr28, mapped to a region spanning 32 cM including the locus. The F₂ and F₃ populations were studied in the phytotron challenged with the most virulent pathotype 77-5 of leaf rust. A coupling phase linked RAPD marker S464₇₂₁ and a repulsion phase linked RAPD marker S326₅₅₀ flanked the gene Lr28 by a distance of 2.4± 0.016 cM on either side. The flanking markers genetically worked as co-dominant markers when analyzed together after separate amplification in the F₂ population by distinguishing the homozygotes from the heterozygotes and increased the efficiency of marker assisted selection by reducing the false positives and negatives. One of the three RAPD markers, S421₆₄₀ was converted to locus specific SCAR marker SCS421₆₄₀ which was further truncated by designing primers internal from both ends of the original RAPD amplicon to eliminate a non-specific amplification of nearly same size. The truncated polymorphic sequence characterized amplified region marker (TPSCAR) SCS421₅₇₀ was 70 bp smaller, but resulted in a single band polymorphism specific to Lr28 resistance. The TPSCAR marker was validated for its specificity to the gene Lr28 in nine different genetic backgrounds and on 43 of the 50 Lr genes of both native and alien origin, suggesting the utility of the SCAR markers in pyramiding leaf rust resistance genes in wheat.     

水稻抗白叶枯病基因Xa23的PCR分子标记定位及辅助选择

用Xa23的近等基因系CBB23及其感病轮回亲本JG30构建了包含2562个单株的F2作图群体.通过分析571个感病单株,找到2个新的与Xa23基因连锁的SSR标记RM187和RM206,它们与Xa23之间的遗传图距分别为7.1 cM和1.9 cM.通过筛选1200个RAPD引物,获得2个与Xa23基因连锁的RAPD标记RpdH5和RpdS1184,与Xa23之间的遗传图距分别为7.0 cM和7.6 cM     

Development of SCAR and CAPS markers linked to a recessive male sterility gene in lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L.)

Genetic male sterility (GMS) has been a useful system for the production of hybrid varieties in self-pollinated plants. We obtained a GMS line developed from a spontaneous mutation in lettuce (Lactuca sativa L.). Genetic analysis in our previous study revealed that the sterility was controlled by a recessive gene which was named ms-S. For simple and quick screening of individuals showing male sterility, we attempted molecular mapping of the ms-S locus using an amplified fragment length polymorphism (AFLP) technique. From the examination of 4,096 AFLP primer combinations, 63 AFLP markers were found to be linked to the gene and nine of them were successfully converted into sequence characterized amplified region (SCAR) markers and cleaved amplified polymorphic sequence (CAPS) markers. Linkage analysis indicated that these nine markers were closely linked to the ms-S gene and all were located on the same side of the gene. The minimum genetic distance between the ms-S gene and a marker was 3.1 cM. These results provide additional information for map-based cloning of the ms-S gene and will be of great help for lettuce breeding using GMS to produce F₁ hybrids.     

High-resolution mapping of the nud locus controlling the naked caryopsis in barley

The hulled or naked caryopsis character of barley is an important agronomic trait because of the direct link to its use. A single recessive gene, nud, located on the long arm of chromosome 7H, controls the naked caryopsis character. Previously, linked amplified fragment length polymorphism (AFLP) bands from bulked segregant analysis were screened, and the nud gene was mapped in a population of 151 F₂ plants. In the present study, the aim was to construct a high‐resolution map of the nud gene towards its positional cloning. Two AFLP bands were converted into sequence‐characterized amplified region (SCAR) markers (sKT5 and sKT9), and a previously reported SCAR marker sKT3 was improved for more reliable detection of polymorphism. A total of 2380 segregants derived from five cross‐combinations were analysed, and the nud gene was flanked by sKT3 and sKT9, at the 0.6‐cM proximal and the 0.06‐cM distal side, respectively. The SCAR markers developed in this study should be useful for marker‐assisted selection in naked barley breeding employing crosses between naked and hulled accessions.     

Efficiency of PCR-RF-SSCP marker production in Brassica oleracea using Brassica EST sequences

Efficiencies of SCAR, CAPS and PCR-RF-SSCP marker production were investigated using two combinations of breeding lines in Brassica oleracea. Published EST sequences of B. oleracea, Brassica rapa, Brassica napus, and Arabidopsis thaliana and newly determined nucleotide sequences of anther cDNA clones from B. oleracea were used for designing primer pairs to amplify genes. The percentage of primer pairs yielding DNA amplification of a single gene was higher in primer pairs of B. oleracea (91%) than those of B. rapa (56%) and A. thaliana (17%). Single DNA fragments amplified by 9% of the primer pairs showed polymorphism as SCAR markers between a broccoli line and a Chinese kale line by agarose-gel electrophoresis. CAPS analysis showed different band patterns in 32% of the same-sized DNA fragments, and PCR-RF-SSCP analysis revealed DNA polymorphism in 52% of those showing no DNA polymorphism by CAPS. In total, 71% of the single DNA fragments were converted to DNA markers. The frequency of DNA polymorphism between parental lines of a cabbage F₁ hybrid was lower, 5% by SCAR and 12% by CAPS. However PCR-RF-SSCP analysis revealed DNA polymorphism in 21% of the DNA fragments showing no polymorphism by CAPS. These results suggest that PCR-RF-SSCP analysis enables highly efficient DNA marker production for mapping of genes in Brassica using progeny, even progeny of closely related parents. Analysis of selfed seeds of broccoli F₁ cultivars using PCR-RF-SSCP markers indicated that PCR-RF-SSCP analysis is also applicable to seed purity tests.     

RAPD and SCAR markers linked to the sex expression locus M in asparagus

Bulk segregant analysis (BSA), random amplified polymorphic DNA (RAPD) and sequence characterized amplified region (SCAR) methods were used to map molecular markers to the sex locus M of asparagus. Two parents, A19 (male, Mm) and MW25 (female, mm), and 63 progeny were used for the study. Two DNA bulks, one male and one female, were made by pooling equal amounts of DNA from 10 randomly selected progeny of each sex type. A total of 760 arbitrary decamer oligonucleotide primers were used for RAPD analysis. Primer OPC15 produced two RAPD markers, OPC15-98 and OPC15-30, both of which were linked to the M locus at a distance of 1.6 cM. Subsequently, amplified RAPD fragment OPC15-98 was cloned and sequenced. The sequence was then used to design flanking 24-mer oligonucleotide SCAR primers SCC15-1 and SCC15-2. Both of these SCAR primers amplified a single 980 bp fragment; the same size as the cloned RAPD fragment. However, the SCAR marker was dominant as was the original OPC15-98 band from which it was derived. These RAPD and SCAR markers could be used for scoring male and female progeny in the mapping population, but were not found to be applicable to other asparagus germplasm studied. This revised version was published online in July 2006 with corrections to the Cover Date.     

SCAR and CAPS markers flanking the Brassica oleracea L. Pp523 downy mildew resistance locus demarcate a genomic region syntenic to the top arm end of Arabidopsis thaliana L. chromosome 1

We recently mapped the Pp523 locus that includes a single, dominant gene conferring resistance to downy mildew expressed in adult plants to a 75.1 cm long linkage group on a genetic linkage map of Brassica oleracea L. More recently, we identified a new AFLP marker 2.8 cm downstream from the resistance gene. The five DNA markers within an 8.5 cm region encompassing the Pp523 gene were cloned and sequenced. Three of these markers were transformed into SCARs (sequence characterised amplified regions), however, two among them were monomorphic and were analysed as CAPS (cleaved amplified polymorphic sequence) markers among the mapping population. Searched against genomic databases, the five B. oleracea DNA-marker sequences matched Arabidopsis thaliana L. gene sequences that delimit a conserved syntenic region in the top arm end of chromosome 1 of this last species. Considering the close genetic relatedness between both species, the information on this specific genomic region in A. thaliana is particularly useful for the construction of a fine-scale map of the corresponding genomic region in B. oleracea. The identified SCAR and CAPS markers can be used for marker assisted selection (MAS) in breeding programs aimed at the introgression of the Pp523 resistance locus, allowing the reliable indirect identification of plants harbouring the resistance gene with a margin of error of approximately six in ten-thousand selected plants.