Possibilities of direct introgression from Brassica napus to B. juncea and indirect introgression from B. napus to related Brassicaceae through B. juncea

The impact of genetically modified canola (Brassica napus) on biodiversity has been examined since its initial stage of commercialization. Various research groups have extensively investigated crossability and introgression among species of Brassicaceae. B. rapa and B. juncea are ranked first and second as the recipients of cross-pollination and introgression from B. napus, respectively. Crossability between B. napus and B. rapa has been examined, specifically in terms of introgression from B. napus to B. rapa, which is mainly considered a weed in America and European countries. On the other hand, knowledge on introgression from B. napus to B. juncea is insufficient, although B. juncea is recognized as the main Brassicaceae weed species in Asia. It is therefore essential to gather information regarding the direct introgression of B. napus into B. juncea and indirect introgression of B. napus into other species of Brassicaceae through B. juncea to evaluate the influence of genetically modified canola on biodiversity. We review information on crossability and introgression between B. juncea and other related Brassicaseae in this report.     

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。     

Development and validation of DNA markers linked to Sdvy-1, a common bean gene conferring resistance to the yellowing strain of Soybean dwarf virus

The yellowing strain of Soybean dwarf virus (SbDV-YS) causes yellowing and yield loss in common bean (Phaseolus vulgaris). The most effective control is achieved through breeding for resistance. An indeterminate climbing cultivar with a white seed coat, ‘Oofuku’, is resistant to SbDV-YS in inoculation tests. We crossed ‘Oofuku’ with an elite cultivar, ‘Taisho-Kintoki’, which is SbDV-YS-susceptible, determinate dwarf with a red-purple seed coat, and performed amplified-fragment-length polymorphism analysis of F₃ lines. From nucleotide sequences of the resistant-specific fragments and their flanking regions, we developed five DNA markers, of which DV86, DV386, and DV398 were closely linked to Sdvy-1, a resistance gene. Using the markers, we developed ‘Toiku-B79’ and ‘Toiku-B80’, the near-isogenic lines (NILs) incorporating Sdvy-1 in the background of ‘Taisho-Kintoki’. The NILs had similar growth habit, maturity date and seed shape to those of ‘Taisho-Kintoki’. The quality of boiled beans was also similar, except that the NILs had more seed coat cracking than ‘Taisho-Kintoki’. The NILs showed no SbDV-YS infection in inoculation tests. We suggest that Sdvy-1 is a useful source of SbDV-YS resistance in common bean.     

Identification of AFLP fragments linked to one recessive genic male sterility (RGMS) in rapeseed (Brassica napus L.) and conversion to SCAR markers for marker-aided selection

117AB is a recessive genic male sterility (RGMS) line in which the sterility is controlled by a duplicate recessive gene named ms, located at two separate loci. In the RGMS line, the genotype of the sterile plant (117A) is msmsmsms, and that of the fertile plant (117B) is Msmsmsms. The present study was aimed to identify DNA markers linked to the ms locus by amplified fragment length polymorphism (AFLP). From the survey of 512 AFLP primer combinations, 6 AFLP fragments (y1, k1, k2, k3, k4, k5) were identified as being tightly linked to the Ms locus. The genetic distances between the markers and the Ms locus were all less than 8 cM, among which two fragments, designated as k2 and k3, co-segregated with the target gene in the tested population. Fragment k2 was successfully converted into a sequence characterized amplified region (SCAR) marker. The markers detected could be valuable in marker-assisted breeding of RGMS in Brassica napus.     

Persistent C genome chromosome regions identified by SSR analysis in backcross progenies between Brassica juncea and B. napus

Given that feral transgenic canola (Brassica napus) from spilled seeds has been found outside of farmer’s fields and that B. juncea is distributed worldwide, it is possible that introgression to B. juncea from B. napus has occurred. To investigate such introgression, we characterized the persistence of B. napus C genome chromosome (C-chromosome) regions in backcross progenies by B. napus C-chromosome specific simple sequence repeat (SSR) markers. We produced backcross progenies from B. juncea and F₁ hybrid of B. juncea × B. napus to evaluate persistence of C-chromosome region, and screened 83 markers from a set of reported C-chromosome specific SSR markers. Eighty-five percent of the SSR markers were deleted in the BC₁ obtained from B. juncea × F₁ hybrid, and this BC₁ exhibited a plant type like that of B. juncea. Most markers were deleted in BC₂ and BC₃ plants, with only two markers persisting in the BC₃. These results indicate a small possibility of persistence of C-chromosome regions in our backcross progenies. Knowledge about the persistence of B. napus C-chromosome regions in backcross progenies may contribute to shed light on gene introgression.     

Development of SCAR markers linked to the Ns locus in potato

A random amplified polymorphic DNA marker OPG17₄₅₀ linked to the Ns gene that confers resistance of potato to potato virus S (PVS), was used to develop sequence‐characterized amplified region (SCAR) markers. After cloning and sequencing of OPG17₄₅₀ new polymerase chain reaction (PCR) primers were designed to generate dominant (SCG17₃₂₁) and codominant (SCG17₄₄₈) markers. For SCG17₄₄₈, polymorphism between susceptible and resistant genotypes was recovered after digestion of the marker with the restriction enzyme Muni. In addition to the band corresponding to ‘susceptible’ allele that does not contain the Muni cleavage site, two bands of approximately 251 bp and 197 bp were observed in the resistant genotypes. The usefulness of these SCAR markers was verified in diploid potatoes possessing the Ns locus from clone G‐LKS 678147/60, and in tetraploid potatoes derived from G‐LKS 678147/60 and from clone MPI 65118/3.     

杂交油菜"蜀杂6号”特异序列扩增标记(SCAR)建立及其在杂种鉴定中的应用

用100个随机引物对"蜀杂6号”父、母本进行RAPD扩增,选出5个能在父、母本中产生多态性扩增的引物.在杂种F1代中验证它们的特异性和稳定性,从中筛选出能从杂种F1代中稳定扩增"蜀杂6号”父、母本特异标记的随机引物GE204和GE222,并对它们扩增的父、母本特异标记片段进行克隆和测序.根据测序结果设计的特异序列扩增引物,将"蜀杂6     

Development of SCAR markers linked to a scald resistance gene derived from wild barley

The F₂ progeny of a third backcross(BC₃) line, BC line 240, derived from a Turkish accession of wild barley (Hordeum vulgare ssp. spontaneum),segregated for resistance to scald (Rhynchosporium secalis) in a manner indicating the presence of a single dominant resistance gene. Two SCAR marker slinked to this resistance were developed from AFLP markers. Screens of disomic and ditelosomic wheat-barley addition lines with the SCAR markers demonstrated that the scald resistance gene is located in the centromeric region of barley chromosome 3H,a region previously reported to contain a major scald resistance locus, Rrs1. Markers that flank the Rrs1 locus were used to screen the wild barley-derivedBC₃F₂ population. These markers also flank the wild barley-derived scald resistance, indicating that it maps to the same locus as Rrs1; it may beallelic, or a separate gene within a complex locus. However, BC line 240 does not respond to treatment with the Rhynchosporium secalis avirulence factorNIP1 in the same way as the Rrs1-carrying cultivar Atlas46. This suggests that the scald resistance gene derived from wild barley confers a different specificity of response to theRrs1 allele in Atlas46.In order to increase the durability of scald resistance in the field, we suggest that at least two scald resistances should be combined into barley cultivars before release. The scald resistance gene described here will be of value in the Australian environment, and the several markers linked to it will facilitate pyramiding. This revised version was published online in July 2006 with corrections to the Cover Date.     

A pubescence color gene enhances tolerance to cold-induced seed cracking in yellow soybean

In yellow soybean, severe cold weather causes seed cracking on the dorsal side. Yellow soybeans carry the I or iⁱ allele of the I locus and have a yellow (I) or pigmented (iⁱ) hilum. We previously isolated an additional allele, designated as Ic, of the I locus, and reported that yellow soybeans with the IcIc genotype may be tolerant to cold-induced seed cracking. The Ic allele by itself, however, does not confer high tolerance. The association of a pubescence color gene (T) with suppression of low-temperature-induced seed coat deterioration has been previously reported. In the present study, we tested whether T is effective for the suppression of cold-induced seed cracking using two pairs of near-isogenic lines for the T locus in the iⁱiⁱ or IcIc background. In both backgrounds, the cracked seed rate of the near-isogenic line with the TT genotype was significantly lower than that with the tt genotype, which indicates that T has an inhibitory effect on cold-induced seed cracking. Furthermore, we also showed that gene pyramiding of Ic and T can improve tolerance to cold-induced seed cracking. Our findings should aid the development of highly SC-tolerant cultivars in soybean breeding programs.     

Development of molecular markers linked to the Leptosphaeria maculans resistance gene Rlm6 and inheritance of SCAR and CAPS markers in Brassica napus × Brassica juncea interspecific hybrids

Leptosphaeria maculans causes blackleg disease on Brassica napus, an economically important oilseed crop. Brassica juncea has high resistance to blackleg and is a source for the development of resistant B. napus varieties. To transfer the Rlm6 resistance gene from B. juncea into B. napus, an interspecific cross between B. napus “Topas DH16516” and B. juncea “Forge” was produced, followed by the development of F₂ and F₃ generations. Sequence characterized amplified region (SCAR) and cleaved amplified polymorphic sequence (CAPS) markers linked to the L. maculans resistance gene Rlm6 were developed. Segregation of SCAR and CAPS markers linked to Rlm6 were confirmed by genotyping of F₂ and F₃ progeny. Segregation of CAPS markers and phenotypes for blackleg disease severity in F₂ plants had a Mendelian ratio of 3:1 in resistant vs. susceptible plants, respectively, supporting the assumption that genetic control of resistance was by a single dominant gene. The molecular markers developed in this study, which show linkage with the L. maculans resistance gene Rlm6, would facilitate marker‐assisted backcross breeding in a variety development programme.     

RAPD and SCAR markers linked to sex determination in Eucommia ulmoides Oliv.

Eucommia ulmoides Oliv. is strictly a dioecious perennial tree native to China. The pistillate plants are economically more useful than the staminate plants. The random amplified polymorphic DNA (RAPD) technique was used to screen markers of sex determination in this species. A 569 bp RAPD marker, marker linked to sex determination in E. ulmoides (MSDE), was found in all the pistillate but not in the staminate plants; its exclusiveness to pistillate plants was confirmed by Southern blotting. MSDE was sequenced and specific primers were synthesized to generate a 569bp pistillate-specific SCAR marker, SCARmr. SCARmr could be useful for screening E. ulmoides plants for gender even before they reach reproductive maturity, resulting in considerable saving of time and economic resources.     

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。     

Molecular markers for yield components in Brassica juncea – do these assist in breeding for high seed yield?

A population of 112 F₁-derived doubled haploid lines was produced from a reciprocal cross of Brassica juncea. The parents differed for seed quality, seed color and many agronomic traits. A detailed RFLP linkage map of this population, comprising 316 loci, had been constructed, and was used to map quantitative trait loci (QTL) for seed yield and yield components, viz. siliqua length, number of seeds per siliqua, number of siliques per main raceme and 1000-seed weight. Stable and significant QTLs were identified for all these yield components except seed yield. For yield components, a selection index based on combined phenotypic and molecular data (QTL effects) could double up the efficiency of selection compared to the expected genetic advance by phenotypic selection. Selection indices for high seed yield, based on the phenotypic data of yield and yield components, could only improve the efficiency of selection by 4% of the genetic advance that can be expected from direct phenotypic selection for yield alone. Inclusion of molecular data together with the phenotypic data of yield components in the selection indices did not improve the efficiency of selection for higher seed yield. This is probably due to often negative relationships among the yield components. Most of the QTLs for yield components were compensating each other, probably due to linkage, pleiotropy or developmentally induced relationships among them. The breeding strategy for B. juncea and challenges to marker assisted selection are discussed.     

Salt-tolerance in Brassica juncea L. II. Salt-stress induced changes in polypeptide pattern of in vitro selected NaCl-tolerant plants

Summary The effect of salt-stress was studied on SDS-PAGE pattern of polypeptides in seedling, leaf and siliqua tissues of four genetically stable lines (SR2P1-2, SR3P2-1, SR3P6-1 and SR3P6-2) of in vitro selected NaCl-tolerant plants, a non-selected somaclone (CP5-2) and parent variety Prakash of Brassica juncea L. Seedlings were raised in 0, 50, 100 and 150 meq/l NaCl solutions and plants were irrigated with nutrient solution with 0, 30, 60 & 90 meq/l NaCl. Salinity induced distinct genotype specific changes in polypeptide pattern of leaf and siliqua tissues, while it had no effect on the polypeptide pattern of seedlings, radicle or hypocotyl tissues in any of the lines. In leaves, at vegetative stage, a high molecular weight protein of 53.2 kD while disappered at 60 mM and higher NaCl level in cv. Prakash and SR3P2-1, it appeared in SR2P1-2 and CP5-2 only at these higher salt levels and in SR3P6 lines it was present irrespective of stress conditions. Differences were also observed for a 93.8 kD protein which appeared anew under stress in cv. Prakash, CP5-2 and SR2P1-2, while it was absent in SR-3 lines. Intensity of the 57.3 kD protein decreased in cv. Prakash, increased in SR-2 and CP-5 lines whereas remained unchanged in SR-3 lines under salt-stress. In siliquae, salt stress induced the expression of four new polypeptides (56.1–70.8 kD) at 60 mM NaCl in cv. Prakash, and at 30 mM in SR2P1-2, SR3P2-1 and SR3P6-1 lines, while these were present in CP5-2 and SR3P6-2 even in the absence of NaCl stress.     

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.     

Inheritance of seed colour in Brassica campestris L. and breeding for yellow-seeded B. napus L.

Summary Seed colour inheritance was studied in five yellow-seeded and one black-seeded B. campestris accessions. Diallel crosses between the yellow-seeded types indicated that the four var. yellow sarson accessions of Indian origin had the same genotype for seed colour but were different from the Swedish yellow-seeded breeding line. Black seed colour was dominant over yellow. The segregation patterns for seed colour in F₂ (Including reciprocals) and BC₁ (backcross of F₁ to the yellow-seeded parent) indicated that the black seed colour was conditioned by a single dominant gene. Seed colour was mainly controlled by the maternal genotype but influenced by the interplay between the maternal and endosperm and/or embryonic genotypes. For developing yellow-seeded B. napus genotypes, resynthesized B. napus lines containing genes for yellow seed (Chen et al., 1988) were crossed with B. napus of yellow/brown seeds, or with yellow-seeded B. carinata. Yellow-seeded F₂ plants were found in the crosses that involved the B. napus breeding line. However, this yellow-seeded character did not breed true up to F₄. Crosses between a yellow-seeded F₃ plant and a monogenomically controlled black-seeded B. napus line of resynthesized origin revealed that the black-seeded trait in the B. alboglabra genome was possibly governed by two independently dominant genes with duplicated effect. Crossability between the resynthesized B. napus lines as female and B. carinata as male was fairly high. The sterility of the F₁ plants prevented further breeding progress for developing yellow-seeded B. napus by this strategy.     

Salt-tolerance in Brassica juncea L. I. In vitro selection,agronomic evaluation and genetic stability

Summary In vitro selection of salt tolerant plants of Brassica juncea L. (Indian mustard) cv. Prakash has been accomplished by screening highly morphogenic cotyledon explant cultures on high NaCl media. Out of a total of 2,620 cotyledons cultured on high salt medium, 3 survived, showed sustained growth and regenerated shoots. They were multiplied by axillary bud culture on NaCl free medium. The salt-selected shoots retained salt tolerance following 3 month of growth and multiplication on control medium. While two of these somaclones flowered and set seeds, third one grew slowly, had abnormal leaf morphology and was sterile. The seed of the two fertile plants were sown in the field to raise R₁ segregating generation. Data were recorded for field, other agronomic components and oil content. The somaclonal lines, both selected salt-tolerant and non-selected, showed tremendous amount of variation for all the characters studied. One of the two tolerant somaclones invariably showed reduced height, longer reproductive phase and higher 1000 seed weight. Based on the agronomic performance of R₁ plants of these somaclones, some plants were selected and their progeny were evaluated for agronomic performance under standard field conditions and salt-tolerance in the greenhouse using sand pot culture method. Most of the lines bred true for their specific characteristics. In the greenhouse, selected salt-tolerant somaclones (SR-2 and SR-3) performed better for plant growth, yield and other agronomic traits at higher salt treatments, indicating thereby that salt-tolerance trait selected in vitro was expressed in the whole plants and is genetically stable and transmitted onto the progeny. The two tolerant lines, however, differed in their salt-tolerance during vegetative and reproductive phases as indicated by their salt-tolerance and stress susceptibility indices. The mechanism of salt-tolerance is not clear and needs to be further investigated.     

A study on disease variation in the populations of an interspecific cross of Brassica juncea L. x B. napus L.

Summary F₁ behaviour and F₂ variation in disease reaction were studied in the interspecific cross Brassica juncea x B. napus. Gene(s) for adult resistance to blackleg (Leptosphaeria maculans) were found to be present in the A genome of B. juncea and could be transferred to B. napus. Gene(s) for complete (seedling plus adult) resistance in B. juncea appeared to be located in the B genome. The chance of their transfer to the oilseed rapes (B. napus or B. campestris) would therefore seem to be remote.     

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.