以分子标记辅助连续回交快速提高花生品种油酸含量及对其后代农艺性状的评价

高油酸是花生重要的品质性状,高油酸花生及其制品具有较好的品质稳定性和较高的营养和保健价值。我国高油酸花生的育成品种类型较少,遗传背景不够丰富,育种手段比较单一。针对上述问题,本研究开发了AS-PCR-MP高油酸分子标记检测方法,优化了KASP分子标记检测体系,利用分子标记辅助连续回交,结合近红外品质快速检测技术及南繁加代技术,以河南省大面积推广的豫花15、远杂9102、豫花9327、豫花9326四个不同类型品种为轮回亲本, 5年内连续回交4代、自交4代,定向获得了4个轮回亲本遗传背景的BC4F4和BC4F5稳定高油酸改良材料24个。调查分析了BC4F4和BC4F5单株的13个农艺性状与轮回亲本的相似度,并利用轮回亲本与非轮回亲本之间的差异SNP的KASP分子标记进行了BC4F4和BC4F5株系的轮回亲本遗传背景检测。结果表明,轮回亲本的遗传背景在BC4F5的比例为79.49%～92.31%。本研究为快速高效改良花生油酸含量探索了新的方法,获得的新品系拓展了高油酸花生的遗传背景,获得的一系列近等基因系可作为遗传研究材料进一步加以利用。     

番茄高色素基因hp1和hp2分子标记的筛选

高色素基因hp1和hp2能显著提高番茄果实的类胡萝卜素总量,对于以提高番茄红素含量为目标的品质育种具有重要应用价值。以这2个基因的突变位点为目标,成功地筛选出了对hp1和hp2分别具有高度特异性的2对引物。由于这样的分子标记实际上就是功能基因本身局部片断的扩增结果,所以能非常可靠地直接鉴别该功能基因,F2群体中分子标记的分离比例与2对基因的独立分配比例完全一致。以这两对引物所产生的分子标记为依据,已经合成了含有hp1和hp2的双突变体。     

小麦品系天95HF2抗叶锈基因定位

苗期基因推导表明,小麦品系天95HF2高抗我国目前多数叶锈菌生理小种。为了确定这一品系所携带的抗病基因,以天95HF2和感病小麦品种郑州5389杂交,获得F1和F2代群体,用叶锈菌小种FHTT和PHTS分别对双亲及其杂交后代进行叶锈抗性鉴定并进行分子标记分析。结果表明,用叶锈菌小种FHTT接种F2代群体时呈现1对显性基因的抗感分离比例,经过亲本和抗感池间标记筛选以及F2代群体的标记检测,Lr1的STS标记WR003和位于5DL的SSR标记wmc443与该抗病基因连锁,遗传距离分别为2.9cM和3.1cM,根据抗性特点和染色体位置推断该基因可能为Lr1。用叶锈菌小种PHTS接种F2代群体时呈现2对基因的抗感分离,分子标记分析结果表明,其中一个基因为Lr1,另一个基因可能为LrZH84。     

水稻温敏雄性核不育基因tms5功能分子标记的开发与应用

在水稻的杂种优势利用中,两系法因为不需要保持系、不育系,育性由单一核基因控制、配组不受恢保关系制约等优点而更受青睐。tms5类型的两系不育系在目前的两系杂交稻中占有绝对主导的地位,针对该基因的功能分子标记辅助育种将为两系法的应用提供一个更加准确和高效的方法。本研究将tms5基因的功能变异开发成基于高分辨率熔解曲线分析(high resolution melting analysis,HRM)检测的特异性分子标记,并对各种类型亲本进行了HRM分型检测及测序验证。结果表明,该标记能够准确区分tms5基因的各种功能变异,且与测序结果完全吻合。将各种变异亲本与突变体‘广占63S’杂交,并用所开发的功能分子标记对其相应的后代植株进行检测,发现该标记能够很好地区分各种类型亲本与突变体的F1代个体及后续的BC1F1和BC3F2代植株。因此,所开发的功能性分子标记可以用于tms5基因的资源鉴定和分子标记辅助改良,从而为两系不育系的准确高效分子育种提供技术支持。     

番茄茄红素β-环化酶基因高效沉默载体构建

番茄茄红素是番茄果实中含量最多的一种类胡萝卜素,也是植物类胡萝卜素代谢过程中的一个重要中间产物.它的环化是在茄红素β-环化酶(Lyc-b)和茄红素ε-环化酶(Lyc-e)催化下完成的,其中茄红素β-环化酶在番茄茄红素转变为β-胡萝卜素的过程中起主要作用.我们利用RT-PCR方法从番茄叶片中克隆出Lyc-b基因,将PCR产物与pGEM-T载体连接后进行测序,扩增的DNA序列与发表的茄红素β-环化酶序列一致.以此为模板,克隆了400 bp左右的目的片段,利用Gateway系统构建Lyc-b基因目的片段的pJawoh18-2b ihpRNA表达载体.经PCR与酶切鉴定的阳性克隆转化农杆菌LBA1100,利用农杆菌叶盘法转化番茄获得了抗性愈伤及再生苗,以检测ihpRNA沉默载体能否降低茄红素β-环化酶基因表达,并为进一步了解该基因在番茄类胡萝卜素代谢过程中的作用奠定基础.这些数据未在本文列出.     

番茄果实中番茄红素含量的遗传分析

以5个番茄自交系为材料,完全双列杂交设计,对番茄果实中番茄红素配合力及其遗传参数进行了分析和估算.试验表明:番茄红素的一般配合力为-0.608～0.734,特殊配合力为-0.302～1.208;番茄红素的广义遗传力为57.35%,狭义遗传力为46.36%,其遗传符合加性-显性模型.所以在育种中采用系谱选择以提高和固定高番茄红素性状是有效的,有性杂交育种可在早期世代进行选择.     

蔬菜分子育种获重要进展——自主开发实用分子标记技术并逐步应用

我国蔬菜作物分子标记辅助育种研究取得重要进展。自主开发了一些实用性较强的分子标记技术。先后建立了ERPAR、多重PCR和共显性SCAR等简单PCR标记技术。利用这些技术,成功地对甘蓝显性雄性不育基因,番茄线虫、ToMV等多种抗病性基因,番茄红素相关基因等进行了标记辅助选择。     

由番茄果实表面色差估计的番茄红素含量遗传分析

本试验选择两个果实表面色差a/b显著不同的番茄品系,通过P1、P2、F1, F2、B1和B2六世代的分析方法,研究了由番茄果实表面色差a/b估计番茄红素含量的遗传规律。结果表明：由番茄果实表面色差估计番茄红素含量的遗传符合两对加性-显性-上位主基因遗传模型(B_1_1),主基因效应在B1、B2和F2 三个世代的遗传率分别为50.31％、65.15％和32.77％。     

鲜食番茄果肉中番茄红素含量性状的遗传分析

为更快更好地进行番茄果肉颜色的评定,定位相关QTLs以改良果肉的颜色品质,本研究选用果肉番茄红素含量显著不同的2个番茄品系,利用分光光度计法测定番茄红素含量,并通过P1、P2、F1、F2、B1和B2六世代联合分析法,研究分析了果肉番茄红素含量的遗传规律。结果表明：F2、B1、B2分离世代群体呈双峰或单峰偏态分布,主基因的加性效应明显,多基因对番茄红素含量分布有较大影响,存在明显的主基因多基因特征,显示番茄果肉番茄红素含量的遗传为多基因控制的数量性状;果肉番茄红素含量的遗传符合一个加性主基因和加性-显性多基因模型(D-2模型)。主基因遗传率较高,为27.39%~66.93%;多基因遗传率较低,为12.09%~48.44%。所以,利用有性杂交育种提高和固定高番茄红素性状,在早期世代进行选择有利于发现新QTLs位点。     

利用MAS手段改良恢复系R838稻瘟病抗性研究

为了提高杂交水稻恢复系R838稻瘟病的抗性水平,利用MAS手段将抗稻瘟病基因pigm导入目标受体,通过连续多代回交并对其回交后代进行分子标记检测或稻瘟病抗性表型鉴定。筛选回交高世代群体自交,并用三系不育系进行测配。结果表明：F1目标基因占有率为52.4%,随着回交世代的增加,其所含目标基因的占有率稳定在50%左右,通过回交高世代群体苗瘟表型鉴定,苗叶瘟抗性3~9级,株系间差异明显;用三系不育系配组,F1苗叶瘟抗性随入选父本抗性提高而提高,且农艺性状与R838基本保持一致。因此,通过分子标记辅助育种技术,用pigm稻瘟病抗性基因改良目标受体的稻瘟病抗性是一个有效途径。     

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.     

EST辅助的甘蓝型油菜显性核不育AFLP标记转化

甘蓝型油菜显性核不育广泛应用于轮回选择和杂种优势利用,不育基因标记的开发与应用对于基因克隆和育种实践具有重要意义。基于AFLP标记SA12MG14的序列信息,从拟南芥整合数据库中,检索与标记序列同源的甘蓝型油菜EST,结合标记和EST序列设计特异引物,转化成新的SCAR标记。获得的SCAR标记S6B3,具有很高的检测稳定性,在回交群体Popu2上分析验证,结果与AFLP标记完全一致。该标记与不育基因相距0.3 cM,将其用于临保系同源的纯合型不育系选育,可有效提高育种工作效率。     

黄瓜黄色子叶突变体遗传及相关AFLP标记

以黄色子叶突变体B180H为母本,颜色正常子叶黄瓜Q12为父本,配制F1、F2、BC1,并通过对6世代子叶叶色特征的观察和统计分析,研究该黄色子叶性状的遗传规律,并利用BSA法、AFLP技术筛选与黄色子叶性状基因紧密连锁的分子标记。结果表明,黄瓜黄色子叶性状是由1对核基因控制的稳定遗传的隐性性状,绿色子叶相对于黄色为完全显性。通过BSA法和AFLP分子标记,应用1 024对引物组合EcoRⅠ-NN/MseⅠ-NNN对黄色、绿色子叶亲本进行筛选,筛选到了1个与黄瓜黄色子叶性状相关的共显性标记E-AG/M-AAG。测序结果表明,片段长度分别为258 bp和257 bp,为发生插入/缺失或突变的同源序列;在黄色子叶个体中只扩增出了258 bp的特异片段,绿色子叶个体中扩增出了257 bp的特异片段或同时扩增出258 bp和257 bp两个特异片段。经组外其他F2单株验证发现,鉴定结果符合率高达96.74%,以Mapmaker 3.0软件分析,该标记与子叶黄色突变位点的连锁距离为3.2 cM。该性状可作为苗期遗传标记性状,在杂交育种和品种纯度鉴定上有极大的利用价值。     

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.     

Identification of two SCAR markers co-segregated with the dominant Ms and recessive ms alleles in onion (Allium cepa L.)

Cytoplasmic genetic male-sterility is used to produce hybrid onion (Allium cepa L.) seeds worldwide. In this paper, we present the results of research aimed toward identifying PCR-based markers linked to the Ms locus through amplified fragment length polymorphism (AFLP). After screening 512 AFLP primer combinations, only one AFLP fragment was identified as being flanking linked to the dominant Ms allele. Subsequently, the AFLP marker was converted into a sequence-characterized amplified region (SCAR) marker, designated as DNF-566, co-segregated with the dominant Ms allele in first backcross (BC₁) segregated populations. Furthermore, we designed another molecular marker (RNS-357) co-segregated with the ms allele to identify different genotypes (i.e., MsMs, Msms, or msms). Both markers could be used for evaluating onion lines with different genetic backgrounds (including male-sterile lines, maintainer lines, male-fertile lines, and commercial based F₁ hybrid cultivars). The results of this study indicate that maintainer plants could be directly selected by using these 2 SCAR markers in the onion breeding process, and this may contribute significantly toward breeding onion F₁ hybrid cultivars.     

家蚕抗核型多角体病毒病分子标记辅助育种

利用我国家蚕种质资源中发现的高抗NPV的材料NB和敏感材料306,组配近等基因系。采用RAPD技术获得分子标记,将标记转换成SCAR(sequence characterized amplified region)标记,利用SCAR标记开展了家蚕抗NPV新品种辅助育种选择,获得了家蚕抗NPV新品种。     

洋葱雄性不育恢复位点Ms座位的SSR标记开发及其应用

本研究旨在开发稳定可靠的洋葱育性位点分子标记,并将其应用于育种实践,筛选育种系,从而缩短育种周期,节省育种成本,加速洋葱杂交种的培育进程。以洋葱育性恢复Ms座位的AFLP序列为基础,采用序列比对分析、PCR检测和表型分析等方法,开发、鉴定、应用了WH-SSR-1分子标记。结果表明:WH-SSR-1标记与Ms位点紧密连锁,Ms位点基因型为纯合显性MsMs时,有1条102 bp的扩增带;纯合隐性msms时,有1条99 bp的扩增带;杂合Msms时,有102 bp和99 bp两条扩增带。32份洋葱材料的验证结果证实了Ms座位的标记类型与基因型完全相符。随后从4个OP群体中直接获得了2个配套的不育系与保持系,‘吊玉’和‘天正红玉’因未检测到保持株或不育株,无法简单实现配套。WH-SSR-1标记与Ms位点紧密连锁,能够将其用于育种系筛选的育种实践,从OP群体中直接选择保持株和不育株,同时实现两系配套。     

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.     

甘蓝品种的AFLP指纹鉴别图谱分析

本试验采用AFLP技术分析了来自全国9个栽培地区的44个甘蓝主栽品种,共筛选了40对E 3/M 3引物组合,多态性条带的数量从0条到15条不等。其中引物组合E-AAC/M-CTA是甘蓝品种中多态性最高的引物,有15条多态性条带,多态性条带的百分率为30%,但该引物不足以区分44个供试的甘蓝品种。同时筛选了11组E 2/M 3对引物组合,其中引物组合E-AG/M-CTC产生了13条清晰的多态性条带。以E-AAC/M-CTA和E-AG/M-CTC这两对引物组合的多态性条带构建了44份甘蓝品种材料的指纹图谱,此指纹图谱可以将供试的44份材料一一区分。     

Development of SCAR and CAPS Markers for a Partially Dominant Yellow Seed Coat Gene in Brassica Napus L

Summary We have previously identified 2 RAPD and 8 AFLP markers that were associated with yellow seed coat gene in a stable and pure yellow-seeded DH line No. 2127-17, however, they were not suitable in large-scale MAS. In this paper, it reported our efforts in developing rapid and reliable SCAR and CAPS markers from the RAPD and AFLP markers. Based on the sequence information in the regions of the most closely linked 4 AFLP and 2 RAPD markers, one SCAR (SCS1130) and one CAPS (SCA1) maker were successfully developed. Linkage analysis on a 127 DH lines population derived from the cross Hui5148-2 × No. 2127-17 revealed that they were tightly flanked (3.2 cM, 3.8 cM respectively). The resulting SCS1130 and SCA1 were further evaluated in backcross breeding families and demonstrated more accurate and valuable in MAS breeding. The development of these new markers would facilitate and accelerate the yellow seed coat breeding in Brassica napus.