洋葱CMS-T型育性分子标记的筛选与鉴定

为筛选便于鉴定洋葱细胞质雄性不育类型及育性相关基因型的分子标记,采用cob(s)、cob(n)、orfA501、Cpp1和orf725共5个与CMS相关分子标记,jnurf13、AcSKP1、DNF-566、RNS-357、OPT和PsaO共6个与育性Ms位点相关分子标记分别对洋葱CMS类型和育性基因型筛选与鉴定。在洋葱不育类型分子标记鉴定中,Cpp1标记不适合本材料CMS分类,cob(s)、cob(n)和orfA501 3个标记联合鉴定洋葱CMS-T类型,筛选出orf725标记适合区分不育株及其不育类型;在34份洋葱育性相关基因型鉴定中,DNF-566、RNS-357、OPT和PsaO4个标记鉴定结果与表型不一致,筛选出jnurf13和AcSKP1 2个分子标记能够鉴定洋葱保持系基因型msms、不育系基因型msms、育性恢复基因型MsMs及杂合子(Msms)。结果表明洋葱CMS-T材料由单核基因Ms位点控制,并且首次报道AcSKP1标记用于CMS-T材料验证,但AcSKP1标记采用混合PCR,鉴定成本相对较高。所以,orf725和jnurf13标记适合田间筛选洋葱不育株类型和育性相关基因型。     

基于ovine SNP 50K芯片对策勒黑羊繁殖和抗逆性状的遗传性分析

为筛选出策勒黑羊种质资源繁殖和抗逆性状分子标记和相关基因，本研究以100只无亲缘关系的策勒黑羊为研究对象，基于Illumina ovine SNP 50 K bead chip进行基因分型；用Plink1.90对基因组数据进行质量控制；对有效SNPs计算哈代-温伯格值，选择从小到大排列后前1%的分子标记，参考绵羊基因组(Oar＿v4.0)进行注释，对注释到的基因进行GO和KEGG分析。结果表明：1)共获得480个SNPs, 417个候选基因，其中，繁殖性状基因13个、抗逆性状相关基因30个；2)繁殖性状相关的基因有SOX9、GTF2A1、GNAQ等，抗逆性相关的基因有TMEM154、ZNF70、CREB3L2等。综上，本研究解析了策勒黑羊繁殖和抗逆性状基因的遗传规律，特别是对抗逆性相关候选基因研究，发现策勒黑羊群体携带抗肺炎相关的分子标记，为策勒黑羊保种和改良提供了分子水平的参考。     

Identification and molecular tagging of two Arabidopsis resistance genes to Botrytis cinerea

To map Arabidopsis resistance genes to Botrytis cinerea, Arabidopsis Col-0 ecotype resistant to B. cinerea BC18 isolate and Arabidopsis Ler ecotype susceptible to B. cinerea BC18 isolate were crossed. According to the resistant responses of the F₁, BC₁ and F₂ populations to B. cinerea, we identified two genes, named BC1 and BC2, responsible for the resistance of Arabidopsis Ler ecotype to B. cinerea. Through the method of map-based cloning, BC1 was linked to DNA markers CCR1 and DHS1 on the fourth chromosome of Arabidopsis with genetic distances of 1.2 cM and 1.6 cM for CCR1 and DHS1, respectively, and BC2 was linked to DNA markers CA72/NGA151 and NGA106 on the fifth chromosome with genetic distances of 1.4 cM and 2.4 cM for CA72/NGA151 and NGA106, respectively. Our results are beneficial for chromosome walking so that we can obtain the whole gene sequences, which will facilitate the understanding of their roles and manners of resistance to B. cinerea.     

黄淮南片麦区区试小麦品种(系)的遗传多样性及Pm21和1BL/1RS分子检测

为了解当前黄淮麦区区试品种(系)的遗传特征,利用分布于小麦基因组的96对SSR标记分析2016年参加黄淮南片麦区品种试验的78个小麦品种(系)的遗传多样性,同时,对Pm21基因和1BL/1RS易位系进行分子检测。结果表明,96对SSR引物中有80对(83.3%)在所有材料表现多态,共检测到307个等位变异,变幅为1~8,平均每个引物扩增3.84个等位变异;位点多态性信息含量(PIC)变幅为0.10~0.83,平均0.62;供试材料的遗传距离变幅为0.14~0.16,平均0.58。78个材料均不含有Pm21基因,69个(88.5%)材料属于1BL/1RS易位系。     

中梁12小麦抗条锈病基因遗传分析与SSR分子定位

中梁12具有抗逆性强、适应性广、抗条锈性强等许多优良的生物学特性。为明确其抗条锈性及遗传规律,利用当前流行的中国条锈菌小种CYR30对抗病品种中梁12与感病品种铭贤169及其杂交后代代F1、F2、F3和BC1代进行苗期抗条锈性遗传分析,并对其抗条锈基因进行SSR分子标记。结果表明,中梁12对CYR30小种具有良好的抗性,由1对显性基因控制,暂命名为YrZh12。该基因与位于小麦7AL染色体上的4个SSR位点Xwmc695、Xcfd20、Xbarc121和Xbarc49连锁,其中最近的侧翼位点为Xcfd20和Xbarc121,其遗传距离分别是3.1cM和4.9cM。系谱分析YrZh12基因可能来自抗引655,由于7AL染色体上没有其他抗条锈病基因,YrZh12可能是一个抗条锈病的新基因。     

应用单核苷酸多态性(SNP)标记鉴定短颌鲚、湖鲚和刀鲚

为了快速区分长江流域各水体中的鲚属鱼类,利用单核苷酸多态性(Single nucleotide polymorphism,SNP)标记对短颌鲚(Coilia brachygnathus)、湖鲚(C. nasus taihuensis)和刀鲚(C. nasus)进行物种鉴定。从120个在短颌鲚和刀鲚之间分化指数(Fst)为1的SNP位点中随机挑选出20个用于引物设计,从21个在湖鲚和刀鲚之间分化指数较高的SNP位点中随机挑选出10个设计引物。随机挑20尾短颌鲚、20尾湖鲚和12尾刀鲚用设计的引物扩增、测序。结果表明:洞庭湖和鄱阳湖的短颌鲚与刀鲚可以使用以上19个SNP分子标记中的任何一个完全分开,剩下1个SNP标记在所扩增的样本中没有发现多样性变异而被弃用。用于区分湖鲚与刀鲚的10个SNP分子标记中有8个在所测样本中可以扩增并用于物种鉴定。其中单独使用Ct-Cn_wtap位点时,湖鲚和刀鲚的基因频率相差最大,当使用Ct-Cn_wtap和Ct-Cn_eif2b4位点时,对湖鲚和刀鲚的鉴别准确率可以达到100%。本研究提供了稳定、高效和低成本识别短颌鲚和刀鲚、湖鲚和刀鲚的方法,为今后鲚属鱼类的物种鉴定和资源保护提供了有效的工具。     

交链格孢菌EST-SSR信息分析与分子标记通用性评价

为了弄清交链格孢菌(Alternaria alternata) EST数据库中的SSR信息资源,并开发可用于研究近缘种细极链格孢(Alternaria tenuissima)遗传多样性的EST-SSR分子标记。从NCBI数据库下载A.alternata表达序列标签(Expressed sequence tag,EST)共727条,经过研究分析发现,A.alternata EST中SSR含量非常丰富,每5.343 kb含有1个SSR位点。根据SSR两端保守序列,设计合成13对EST-SSR引物,对来自新疆不同地区不同寄主的38株A.tenuissima进行通用性检测。检测结果显示共有5对引物能扩增出多态性条带,扩增多态率为55.56%。因此,基于A.alternata EST数据库开发的SSR分子标记,在开展近缘种A.tenuissima遗传多样性研究时具有较好的通用性。     

EST-SSR标记应用于麦冬类植物遗传多样性研究

为研究麦冬类植物材料的遗传多样性,利用非变性聚丙烯酰胺凝胶电泳技术,用8对EST-SSR引物在32份麦冬类植物材料进行扩增检测,并对样本进行聚类分析。结果表明:8对引物的PIC(Polymorphic information content)位于0~0.869,平均值0.567,引物1扩增出最多的等位基因数,占总值的20.41%;遗传距离矩阵研究发现,个体1和个体23间的遗传距离最大为2.48,说明二者遗传分化程度最大,个体18和个体23地理来源虽然不同,但二者间遗传距离最小,表明二者间有一定程度的基因交流现象;同时,居群c(野生杭麦冬)内的平均遗传距离最小为0.03,居群d(阔叶山麦冬)内的平均遗传距离最大为0.93。在聚类分析中,32份材料被分为2大类,在主坐标分析中,32份材料被分为3大类,2种分析结果具有相似性。研究表明EST-SSR标记可以很好地对麦冬类植物材料进行划分。     

Construction of a genetic linkage map and QTL analysis for some leaf traits in pear (Pyrus L.)

The major incompatibility barriers to specific inbred lines and the long generation duration in Pyrus L. may hinder the Pyrus breeding process. A genetic linkage map provides the foundation for quantitative trait loci (QTL) mapping and molecular marker-assisted breeding. In this study, we constructed a genetic map with 145 F₁ populations from a cross of two cultivars, Yali and Jingbaili, using AFLP and SSR markers. The map consisted of 18 linkage groups which included 402 genetic markers and covered 1395.9 cM, with an average genetic distance of 3.8 cM. The interval mapping was used to identify quantitative trait loci associated with four leaf agronomic traits in the F₁ population. The results indicated that four QTLs were associated with leaf length, two QTLs with leaf width, two with leaf length/leaf width, and three with petiole length. The eleven QTLs were associated with 9.9%–48.5% of the phenotypic variation in different traits. It is considered that the map covers almost the whole genome, and molecular markers will be greatly helpful to the related breeding.     

Construction of a molecular map for melon (Cucumis melo L.) based on SRAP

A molecular map of melon (Cucumis melo L.) was constructed with SRAP (Sequence-Related Amplified Polymorphism) markers using a population consisting of 114 F₂ individuals derived from the cross of 4G21 (C. melo var. chinensis) and 3A832 (C. melo var. saccherinus). Twenty-nine primer pairs were used and 187 polymorphic loci were produced. The map consists of 12 linkage groups that include 152 genetic markers and cover 2077.1 cM with an average genetic distance of 13.67 cM. Every linkage group has 6–32 genetic markers with average genetic distance of 9.72–19.19 cM. The length of linkage group is 85.3–496.1 cM. __________ Translated from Acta Horticulturae Sinica, 2007, 34(1): 135–140 [译自 : 园艺学报]     

小麦品种辽春10抗叶锈病基因LrLC10的比较基因组学定位

为明确小麦品种辽春10对叶锈菌小种PHT抗病的遗传基础,利用感病小麦材料87-1与抗病小麦材料辽春10构建的F_(2:3)群体,对其进行成株期抗病性鉴定和遗传分析。结果表明,辽春10中含有1个显性抗叶锈病基因,暂命名为LrLC10。利用BSA法和比较基因组学策略对该抗病基因进行分子标记分析,将LrLC10定位于小麦2BS染色体上。共构建了含有8个分子标记的LrLC10基因的连锁图,其中:CAUT253位于LrLC10的远着丝粒侧,距离为0.1cM;CAUT163和CAUT131与LrLC10共分离;CAUT239位于LrLC10的近着丝粒侧,遗传距离为0.5cM。     

小麦品种渝麦13号细胞质类型的分子标记鉴定

为从分子水平对小麦渝麦13号进行细胞质类型鉴定,本研究以野生燕麦、普通小麦(中国春和薛早)及30份具有小麦属和山羊草属不同细胞质类型的核质代换系为参照,利用24个小麦叶绿体SSR分子标记对小麦品种渝13号及其衍生后代进行了分析。结果发现,5个标记的扩增带型在渝麦13号及其衍生后代和普通小麦材料之间表现出明显的多态性,说明小麦品种渝麦13号及其衍生后代的细胞质类型与普通小麦材料不同。通过比对野生燕麦细胞质和T型细胞质的带型发现,在24个标记中,有15个分子标记在野生燕麦细胞质和T型细胞质间表现出明显的多态性。在这些多态性分子标记中,渝麦13号及其衍生材料细胞质的扩增带型与T型细胞质的带型完全一致,而与野生燕麦的带型不一致。通过比对渝麦13号和其他29份小麦核质代换系的细胞质扩增带型后发现,渝麦13号与这些代换系均不同。因此,小麦品种渝麦13号及其衍生系的细胞质类型很可能为T型。     

芸薹属植物细胞质特异分子标记的开发

开发芸薹属植物细胞质特异分子标记,为芸薹属植物种质资源鉴定、保护及育种利用奠定基础。33份芸薹属植物中,20份甘蓝型油菜,2份芥菜型油菜,2份埃塞俄比亚芥,3份甘蓝,2份白菜,2份黑芥,以及芸芥和板蓝根各1份作为对照,利用细胞质特异分子标记进行PCR扩增分析。笔者前期研发的一步多重PCR技术不仅能将pol、ogu、IP-ogu、nap、cam等5种细胞质类型鉴别开来,而且在埃塞俄比亚芥、黑芥、甘蓝、芸芥和板蓝根中扩增出特异的条带。引物Indel-F/R在具有nap细胞质的甘蓝型油菜材料中特异扩增出1个长度为344 bp的条带,该扩增产物比pol和cam细胞质材料中扩增产物多1个长度为93 bp的InDel,可作为区分nap细胞质的特异分子标记。引物mtSSR2-F/R和rsp3F/R-1在埃塞俄比亚芥和黑芥中扩增产物与其他参试芸薹属植物不同,可用作区分埃塞俄比亚芥和黑芥细胞质类型的分子标记。     

甘蓝种和芥菜型油菜细胞质的遗传多样性

对36份供试材料（15份甘蓝种材料、12份芥菜型油菜、4份不同细胞质类型的甘蓝型油菜、2份白菜型油菜及黑芥、埃塞俄比亚芥、芸芥各1份）进行叶绿体和线粒体SSR分析。7对多态性叶绿体特异的SSR引物在参试材料中共检测到31条多态性条带,每个位点等位基因为3~8个,平均4.43个,PIC值为0.234~0.711,平均为0.550。 2对线粒体特异的SSR引物检测到多态性条带数分别为3和5,PIC值分别为0.409和0.558。将线粒体与叶绿体的SSR标记合并,36份材料的遗传相似系数为0.538~1.000,在遗传相似系数 0.700 处,36份材料可明显分为5类,分别为芥菜型油菜、甘蓝型油菜和白菜型油菜、埃塞俄比亚芥和黑芥、甘蓝、芸芥,结果与传统的分类一致。芥菜型油菜内存在4种单倍型,而甘蓝仅有一种单倍型,芥菜型油菜材料间的细胞质遗传多样性高于甘蓝种材料间的细胞质多样性。     

Molecular Mapping of Two Novel Stripe Rust Resistant Genes YrTp1 and YrTp2 in A-3 Derived from Triticum aestivum × Thinopyrum ponticum

Loss of variety resistance to stripe rust (Puccinia striiformis Westend f. sp.tritici) is an important factor causing massive periodical epidemic of rust in wheat production. Creation and development of new races of rust pathogen have led to serious crisis of resistance loss in widely planted varieties. This has quickened the search for new resistance resources. Molecular marker could facilitate the identification of the location of novel genes. A line A-3 with high resistance (immune) to currently epidemic yellow rust races (CY29, 31, 32) was screened out in offspring of Triticum aestivum × Thinopyrum ponticum. Segregation in F₂ and BC₁ populations indicated that the resistance was controlled by two independent genes: one dominant and one recessive. SSR markers were employed to map the two resistant genes in the F₂ and BC₁ populations. A marker WMC477-_167bp located on 2BS was linked to the dominant gene with genetic distance of 0.4 cM. Another marker WMC364-_{208 bp} located on 7BS was linked to the recessive-resistant gene with genetic distance of 5.8 cM. The two genes identified in this paper might be two novel stripe rust resistant genes, which were temporarily designated as YrTp1 and YrTp2, respectively. The tightly linking markers facilitate transfer of the two resistant genes into the new varieties to control epidemic of yellow rust.     

Identification of disease resistances in wheat-<Emphasis Type="Italic">Leymus multicaulis</Emphasis> derivatives and characterization of <Emphasis Type="Italic">L. multicaulis</Emphasis> chromatin using microsatellite DNA markers

To identify resistance to Fusarium head blight (FHB), cereal yellow dwarf virus (CYDV), stem rust (Sr), and powdery mildew (Pm) in 24 common wheat (Triticum aestivum)-Leymus multicaulis addition/translocation lines that were developed cytogenetically and to verify the authenticity of these lines using microsatellite (SSR) DNA markers. Resistance to FHB was identified in the wheat-L. multicaulis addition lines, Line 9 and Line 26, which both contained L. multicaulis-specific fragments as shown by SSR markers. The translocation line, Trans 1, and the addition lines, Line 5 and Line 29, have resistance to stem rust (IT 0). Resistance to CYDV was evaluated based on virus titers measured by enzyme linked immunosorbent assay (ELISA). The addition line, Line 23, showed low virus titer (0.15), indicating resistance to CYDV. The segregation distribution of CYDV resistance in 98 F₂ plants of Line 23/CS showed a significant deviation from 3:1. Inoculation with a set of 14 differential Blumeria graminis f. sp. tritici (Bgt) isolates did not detect powdery mildew resistance in translocation line Trans 1, addition line Line 9 and the amphiploid of wheat-L. multicaulis. However, Line 26 exhibited the resistance response pattern of Kavkaz, which carries Pm8, indicating that Line 26 most likely has the powdery mildew resistance gene Pm8 inherited from its parent lines Feng Kang 7 or Feng Kang 10. Twelve SSR markers, distributed on different homeologous chromosome groups of wheat, which distinguished L. multicaulis addition/translocation chromosomes, were used to verify the presence of L. multicaulis chromatin in the putative wheat-L. multicaulis addition/translocation lines. Of the 24 addition/translocation lines investigated using the 12 polymorphic SSR markers, 18 wheat-L. multicaulis derivatives showed the expected L. multicaulis-specific fragments, indicating that all of these 18 addition/translocation lines would most likely have the introgressed L. multicaulis chromosome(s). Chromosomal rearrangements also were detected in some of the wheat-L. multicaulis introgression lines.     

Identification of QTLs Related to Bolting in Brassica rapa ssp. pekinensis （syn. Brassica campestris ssp. pekinensis）

A genetic linkage map of Brassica rapa ssp. pekinensis was constructed with 186 AFLP （amplified fragment length polymorphism） markers by using a doubled-haploid （DH） population with 183 individuals. The individuals were derived from F1 which was developed by crossing a bolting resistant DH line Y-177-12 and an easy bolting DH line Y195-93a. AFLPs were generated by the use of restriction enzymes EcoR Ⅰ and Mse Ⅰ . The segregation of each marker and linkage was analyzed by using JoinMap version 3.0. Mapped markers were aligned in ten linkage groups which covered 887.8 cM with an average marker interval of 4.47 cM. Markers showing skewed segregation ratio were clustered in six LGs. Quantitative trait loci （QTL） were mapped for bolting resistance by using MAPQTL 4.0 package. Four QTLs explaining from 7.0 to 9.4% of the total variation were detected, all of them increase bolting resistance. These mapped QTLs could be used to develop a marker assisted selection programme for bolting resistance breeding.     

应用不同分子标记鉴别入侵广州草地贪夜蛾的寄主型

【目的】利用不同分子标记对入侵广州的草地贪夜蛾Spodoptera frugiperda进行寄主型鉴定,以便监测虫源扩散规律以及预测预警虫情。【方法】对采集自广州花都玉米植株的草地贪夜蛾,利用3个COI基因标记COI-A、COI-B和COI-C,以及Tpi基因标记进行鉴定,并对结果进行比较。【结果】序列比对和寄主型分析显示,COI-A、COI-B和COI-C标记鉴定到入侵广州的草地贪夜蛾为已报道的水稻型,且COI-A标记鉴定到草地贪夜蛾与黏虫Mythimna separate的杂合子。基于Tpi基因的标记鉴定到入侵广州的草地贪夜蛾为玉米型,且在第126和第127位碱基为GA/AT杂合。【结论】COI-A标记具有较强的物种鉴别能力,能够鉴定到草地贪夜蛾与其他近源种的杂交事件;Tpi标记可用于鉴定草地贪夜蛾寄主型。2个标记结合相互验证可以提高准确率。开发微卫星标记或者寄主取食功能性基因标记可以实现寄主型的精准鉴定。     

Correlation between allele sizes of microsatellites and phenotypic variations in rice landraces

Yunnan is one of the largest centers of genetic diversity in the world. Allele size of microsatellites associated with phenotypic traits of rice landraces in Yunnan, Southwest China, was investigated based on 20 SSR markers and 23 phenotypic traits, as well as eight mineral elements in brown rice within the core collection of 629 accessions; and there was a significant correlation for 182 (r = 0.083^* − 0.438^**) of 620 pairs among these markers and traits, as well as elements. Surprisingly, there was a significant correlation for 94 of 180 pairs between the allele size of microsatellites and grain traits, and 48 of 160 pairs between allele size of microsatellites and panicle traits. In these rice landraces, 309 alleles were detected, with an average of 15.5 alleles per marker, ranging from 5 (RM60) to 40 (RM257). There was a significant correlation between the allele size of 20 SSR markers and some phenotypic traits, such as the significant correlation of 17 (r = −0.085^* − 0.438^**) pairs between the allele size of RM224 and 23 phenotypic traits, as well as eight elements. The allele size of microsatellites was more associated with grain or panicle traits than that of plant traits or element contents in brown rice. Grain length/width ratio and 1–2 internode length, as indica-japonica classification traits, in which two traits were closely associated with the allele size of 14 SSR markers ranging from 0.089^* to −0.438^**. Therefore, allele size of SSRs was associated with phenotypic traits (especially in grain traits), as well as elemental contents in brown rice. These authors contributed equally to this work.     

同羊ETAA1基因InDel检测及其与生长性状的关联分析

为寻找同羊中与生长性状相关的分子标记,试验对ETAA1基因InDel的多态性与同羊生长性状进行了关联分析.以166只无亲缘关系的同羊为研究对象,通过Ensembl数据库筛选ETAA1基因潜在的InDel位点,利用PCR和琼脂糖凝胶电泳对筛选到的InDel位点进行检测和分型后,进一步对InDel位点不同基因型的同羊生长性状进行关联分析.结果 显示,位于绵羊3号染色体的ETAA1基因第1内含子存在一个插入12 bp的InDel突变;根据插入的类型,可以分为II,DD和ID 3种基因型;检测出的InDel位点多态性对同羊公羊臀端高有显著影响,该位点Ⅱ基因型个体臀端高显著高于ID型与DD型(P＜0.05).因此,可以把ETAA1基因此位点InDel多态作为同羊臀端高性状选择的候选分子标记.