四倍体马铃薯熟性连锁SCAR标记的开发与验证

熟性是马铃薯的重要数量性状之一。本研究以马铃薯早熟品种中薯3号和晚熟品种中薯19及其熟性分离群体(221份)为材料,高通量简化基因组测序和集群分离分析(BSA)相结合,开发获得一个SCAR标记,命名为SCAR5-8。进一步利用该标记对分离群体的53份早熟和63份晚熟子代及70份四倍体马铃薯品种进行验证,结果表明,该标记在分离群体和四倍体品种中的检测结果与表型鉴定结果总体吻合度分别达87.1%与81.4%,Pearson’s双侧相关分析显示,其相关性均达极显著水平。该SCAR标记可被用于标记辅助选择,对加速四倍体马铃薯育种进程具有重要意义。     

马铃薯块茎形状CAPS标记的开发与验证

马铃薯块茎形状(薯形)是选育马铃薯品种尤其是加工品种最重要的性状之一。前人研究表明薯形是由位于第10染色体的单基因Ro控制的,并且圆形对长形为显性。本研究以二倍体马铃薯长薯形基因型10618-01和圆薯形基因型320-02及其213个F₁代薯形分离群体为材料,基于马铃薯基因组相关序列信息结合分离群体分组分析法开发标记,获得了一个CAPS标记1137-CAPSVI。利用该标记对53份不同薯形的四倍体马铃薯高代品系进行验证,结果表明该标记的选择准确率达83.02%,进一步的Pearson双侧相关分析显示其相关性达极显著水平,且该标记对圆薯形材料的选择准确率更高,达91.42%。该CAPS标记的获得对于四倍体马铃薯分子标记辅助育种,加速马铃薯品种尤其是加工专用型品种的选育具有重要意义。     

马铃薯块茎形状基因CAPS标记的开发与验证

马铃薯块茎形状(薯形)是选育马铃薯品种尤其是加工品种最重要的性状之一。前人研究表明薯形是由位于第10染色体的单基因Ro控制的,并且圆形对长形为显性。本研究以二倍体马铃薯长薯形基因型10618-01和圆薯形基因型320-02及其213个F1代薯形分离群体为材料,基于马铃薯基因组相关序列信息结合分离群体分组分析法开发标记,获得了一个CAPS标记1137-CAPSVI。利用该标记对53份不同薯形的四倍体马铃薯高代品系进行验证,结果表明该标记的选择准确率达83.02%,进一步的Pearson双侧相关分析显示其相关性达极显著水平,且该标记对圆薯形材料的选择准确率更高,达91.42%。该CAPS标记的获得对于四倍体马铃薯分子标记辅助育种,加速马铃薯品种尤其是加工专用型品种的选育具有重要意义。     

影响四倍体马铃薯块茎中糖生物碱含量的上位性互作的鉴定

糖生物碱是一种次生代谢产物，其特点是味道不好，大量食用有毒。应该对马铃薯块茎中的糖生物碱含量进行选择，且DNA标记可以显著促进这一过程。本试验旨在研究马铃薯块茎中糖生物碱总含量（TGA）的遗传控制以及DNA标记。从TGA含量趋异的四倍体马铃薯无性系构建分离群体，该群体有一个高TGA含量的亲本LT7和一个低TGA含量的亲本NT8。我们应用342个ISSR单引物或者是这种引物组合以及36个CAPS引物对，用不同限制性酶进行降解，     

基于2b-RAD测序的四倍体马铃薯熟性相关的分子标记开发

马铃薯熟性是由多基因控制的数量性状,是我国不同栽培区划选择适宜品种的重要指标之一。本研究以晚熟品种中薯18号和早熟品种中薯5号及其F₁分离群体为材料, 2018—2019年连续2年对“中薯18号(母本)×中薯5号(父本)”杂交分离群体进行熟性评价,从中筛选出极端晚熟和极端早熟的基因型各30个,并分别构建极端晚熟和极端早熟基因组DNA混池。利用简化基因组2b-RAD (2b-restriction site-associated DNA)技术测序,寻找差异标签开发出3个与熟性连锁的分子标记SCARA2-2、SCARA4-21和SCARA5-16,3个分子标记联合使用对熟性分离群体子代进行验证,晚、早熟表型符合率分别到达了87.5%和93.0%,这些分子标记的开发和联合使用对辅助马铃薯熟性选择具有重要的参考价值。     

基于侯选基因标记的四倍体马铃薯休眠QTL关联分析

休眠期是马铃薯(SolanumtuberosumL.)重要的块茎性状之一,寻找调控马铃薯块茎休眠的关键基因,揭示其分子机制以选育具有适宜休眠期长度的马铃薯品种,对于解决当前马铃薯产业中过长或过短休眠期带来的经济损失和食品安全隐患等问题十分关键。前期研究在二倍体马铃薯连锁群体中定位了6个加性休眠QTL,本研究拟在四倍体马铃薯育种材料中验证这些休眠QTL。基于休眠QTL连锁的候选基因标记,采用混合线性模型(MLM),模型中考虑群体结构和亲缘关系(Q+K),在四倍体马铃薯自然群体St-hzau中对马铃薯块茎休眠期进行了关联分析。5号染色体上休眠QTL DorB5.3连锁的候选基因标记S199_300和GWD (根据葡聚糖水双激酶α-glucan water dikinase基因设计)与马铃薯块茎休眠期具有显著的关联(P0.05),分别解释了休眠期表型变异的7.8%和3.2%,分别能增加休眠期7.1 d和4.5 d,即在二倍体马铃薯连锁群体中定位的稳定主效休眠QTL DorB5.3在四倍体马铃薯关联群体St-hzau中也表现显著, DorB5.3的稳定性在关联分析结果中得到了验证,表明候选基因标记策略在马铃薯块茎休眠QTL关联分析中是一种有效的策略。本研究所验证的主效休眠QTL DorB5.3及相应连锁标记可以直接用于马铃薯休眠育种。据此可以推测GWD可能在控制还原糖含量和块茎休眠2个方面均发挥作用,马铃薯块茎休眠机制与还原糖含量变化机制可能存在着部分交叉。     

普通菜豆抗炭疽病基因SCAR标记鉴定

利用12个菜豆品种（鉴别寄主）评价了7个抗炭疽病基因SCAR标记的可靠性和实用性,其中SBB14_1150/1050标记引物扩增没有特异性,SAS13₉₅₀没有扩增带。用5个可靠的菜豆抗炭疽病基因SCAR标记（SCAreoli₁₀₀₀、SH18₁₁₀₀、SAB3₄₀₀、SB12₃₅₀ 和SCF10₁₀₇₂）,对127份普通菜豆抗炭疽病品种进行抗炭疽病基因分子标记鉴定,82份未检测到SCAR标记,45份分别含有1~3个SCAR标记;检测到SCAR标记的资源中,13份含有SCAreoli₁₀₀₀标记,13份含有SH18₁₁₀₀标记,5份含有SAB3₄₀₀标记,9份含有SB12₃₅₀标记,11份含有SCF10₁₀₇₂标记。分析表明抗病品种含有的抗病基因标记与品种来源存在相关性。     

二倍体马铃薯富利亚和窄刀薯杂种块茎中铁和锌的含量及广义遗传力

为了解二倍体马铃薯块茎中铁和锌含量变化范围,并估算铁和锌含量的广义遗传力,为今后马铃薯铁和锌的生物强化提供基础材料和数据。32个适应长日照的二倍体马铃薯富利亚(Solanumphureja)和窄刀薯(S.stenotomum)杂种无性系(S.phureja-S.stenotomum)以及四倍体品种克新13号(对照)采用随机区组设计,3次重复,在黑龙江省钠河市种植2年,分析块茎铁和锌的含量。二倍体马铃薯铁含量的变化范围是36.9~54.1μg/gDW,其中28个二倍体无性系铁含量显著高于四倍体品种;锌含量的变化范围是17.7~34.2μg/gDW,亦有28个二倍体无性系锌含量显著高于四倍体品种。二倍体马铃薯铁的广义遗传力估值为0.8068,95%的置信区间为0.6043~0.9057;锌的广义遗传力估值为0.6236,95%的置信区间为0.2290~0.8163。二倍体块茎铁和锌含量不存在相关性。这些结果表明,S.phureja-S.stenotomum杂种是改善马铃薯品种铁和锌含量宝贵的资源材料。     

结球甘蓝抗霜霉病基因连锁的SCAR标记开发

结球甘蓝霜霉病是由十字花科寄生霜霉菌引发的严重病害。本研究利用高代自交系‘R103’抗病亲本和‘S101’感病亲本及其F2分离群体,于霜霉病病发高峰期田间自然诱发抗性鉴定,由单基因显性控制。运用AFLP分子标记技术,结合BSA法,对双亲和2对抗、感基因池的筛选和验证,获得2个与结球甘蓝抗霜霉病基因相关的AFLP标记。204 bp BoRAAC/CAC标记转化为SCAR标记,发现在抗病亲本和抗病池中与感病亲本和感病池中条带只存在量上的差异,在抗性选择起中只能起辅助作用;191 bp BoRAAG/CTC标记(EU-635443.1),转化为SCAR标记,仅在抗病亲本和2个抗病池中获得目的条带,经F2群体单株验证后,与抗性位点的遗传距离为5.7cM。利用BoRAAG/CTC113标记对F1和BC1群体与多年苗期霜霉病抗性鉴定的20份结球甘蓝种质资源进行验证和筛选,该标记与品种(系)的霜霉病抗性吻合率达到95%,初步表明该标记可应用于早期结球甘蓝抗霜霉病抗性辅助选择。     

8个水稻抗条纹叶枯病分子标记有效性的验证及评价

利用已报道的8个与水稻条纹叶枯病抗性基因Stv-bi连锁的标记,即SCAR标记ST-10;STS标记H11-8、H11-12;InDel标记M68.4和M79.1以及SSR标记RM21-8、RM21-15、H21,结合田间抗性鉴定,对24份水稻品种(系)(包括2份抗、感对照)以及2个抗感组合的F2分离世代进行抗病性分析、筛选和评价,旨在有效利用抗病资源选育抗病新品种,并对这8个分子标记的有效性和选择效率进行验证比较,筛选出高效的辅助选择分子标记。结果表明,仅有SCAR标记ST-10和STS标记H11-8的分子检测与田间表型鉴定的吻合率超过或接近90%,其余分子标记均低于80%,其中,3对SSR标记在抗感材料中几乎没有多态性。比较而言,SCAR标记ST-10更能有效地用于条纹叶枯病抗性材料的筛选,结合使用STS标记H11-8更能准确有效地检测Stv-bi基因的存在,进一步提高选择效率。     

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.     

鲜食番茄茄红素基因的AFLP-SCAR分子标记

番茄红素(Lycopene)是功能性天然色素,因其高抗氧化功能而备受关注.为筛选高番茄红素品种,加速鲜食番茄分子标记辅助育种进程,本试验以高茄红素番茄骨干系F-516F8和低茄红素骨干系Nor作为试验材料,研究了鲜食番茄高茄红素基因的AFLP-SCAR分子标记.利用杂交F1代、自交F2代遗传群体,通过64对E/M引物组...     

Identification of a SCAR marker linked to a recessive male sterile gene (Tems) and its application in breeding of marigold (Tagetes erecta)

In marigold, an F₂ segregation population of 167 plants was constructed from a cross of a line (M525A) carrying the male sterility trait × an inbred line (f53f). In line M525A, the male sterility trait was controlled by the recessive gene, Tems . The intersimple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) techniques combined with bulked segregant analysis were used to develop markers linked to the trait. From a survey of the 38 ISSR primers and 170 SRAP primer combinations, only one SRAP marker that was closely linked to the target trait was identified and successfully converted into sequence characterized amplified region (SCAR) marker that was located within 2.4 cM from Tems locus. The marker was validated with five other two-type lines and in each case the male fertile plants were reliably identified. This SCAR marker therefore permits the efficient marker-assisted selection of male sterile individuals in breeding programmes of marigold and will greatly facilitate the breeding of F₁ cultivars.     

Identification and characterization of RAPD and SCAR markers linked to anthracnose resistance gene in sorghum [Sorghum bicolor (L.) Moench]

Anthracnose, one of the destructive foliar diseases of sorghum growing in warm humid regions, is incited by the fungus Colletotrichum graminicola.The inheritance of anthracnose resistance was studied using the parental cultivars of Sorghum bicolor (L.) Moench, HC 136 (susceptible to anthracnose) and G 73 (anthracnose resistant). The F₁ and F₂ plants were inoculated with the local isolates of C. graminicola cultures. The F₂ plants showed a segregation ratio of 3 (susceptible): 1(resistant) indicating that the locus for resistance to anthracnose in sorghum accession G 73 segregates as a recessive trait in a cross to susceptible cultivar HC 136. RAPD (random amplified polymorphic DNA) marker OPJ 01₁₄₃₇ was identified as marker closely linked to anthracnose resistance gene in sorghum by bulked segregant analysis of HC 136 × G73 derived recombinant inbred lines (RILs) of sorghum. A total of 84 random decamer primers were used to screen polymorphism among the parental genotypes. Among these, only 24 primers were polymorphic. On bulked segregant analysis, primer OPJ 01 amplified a 1437 bp fragment only in resistant parent G 73 and resistant bulk. The marker OPJ 01₁₄₃₇ was cloned and sequenced. The sequence of RAPD marker OPJ 01₁₄₃₇ was used to generate specific markers called sequence characterized amplified regions (SCARs). A pair of SCAR markers SCJ 01-1 and SCJ 01-2 was developed using Mac Vector program. SCAR amplification of resistant and susceptible parents along with their respective bulks and RILs confirmed that SCAR marker SCJ 01 is at the same loci as that of RAPD marker OPJ 01₁₄₃₇ and hence, is linked to anthracnose resistance gene. Resistant parent G 73 and resistant bulk amplified single specific band on PCR amplification using SCAR primer pairs. The RAPD marker OPJ 01₁₄₃₇ was mapped at a distance of 3.26 cM apart from the locus governing anthracnose resistance on the sorghum genetic map by the segregation analysis of the RILs. Using BLAST program, it was found that the marker showed 100 per cent alignment with the contig{_}3966 located on the longer arm of chromosome 8 of sorghum genome. Therefore, these identified RAPD and SCAR markers can be used in the resistance-breeding program of sorghum anthracnose by marker-assisted selection.An erratum to this article can be found at     

Marker-assisted selection for oleic acid content in spring turnip rape

The efficiency of marker-assisted selection for oleic acid content was studied in an F₂ population of spring turnip rape, Brassica rapa ssp. oleifera, using a sequence characterized amplified region (SCAR) marker, the distance of which from the oleic acid gene is 11.5 cM, and allele-specific markers which are located at the gene locus. As expected, the allele-specific markers recognized the oleic acid genotypes more precisely than the SCAR marker. A further complication in the use of the SCAR was the existence of null alleles and polymorphism in almost all the lines/cultivars tested. Therefore, parents have to be carefully selected to find informative combinations in different crosses. Allele-specific markers are very efficient in selection since heterozygous plants can be identified. This is not possible when selection is based on the commonly applied gas chromatography analysis of oil composition. The allele-specific detection was further simplified by using direct staining of PCR products instead of electrophoresis.     

Population structure and linkage disequilibrium in diploid and tetraploid potato revealed by genome‐wide high‐density genotyping using the SolCAP SNP array

Genome‐wide association (GWA) mapping in potato requires high‐density genotyping. With the Illumina SolCAP potato single‐nucleotide polymorphism (SNP) array, a first tool for GWA mapping in potato became available. Thirty‐six tetraploid varieties and eight diploid breeding clones were genotyped for 8303 SNPs using this array. The objectives of our study were to examine in this set of germplasm: (i) the degree of polymorphism of the SolCAP SNPs in European germplasm, (ii) the population structure, (iii) temporal trends of genetic diversity and (iv) the genome‐wide extent of linkage disequilibrium (LD). Three‐quarters of the SNPs were polymorphic. In the principal coordinate analysis, a clear separation of tetraploid from diploid genotypes was observed, whereas no distinct subgroups among the tetraploid varieties were detected. The nonlinear trendline of the LD measure vs. the physical map distance decayed within 275 bp to an value of 0.10, indicating that theoretically, about 3 million equally distributed SNPs are required for GWA mapping in this diverse set of germplasm. As the LD decay changes with the population selected for GWA mapping, the number of required markers might be different in other germplasm.     

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.