基于SRAP分子标记的冰草遗传连锁图谱构建

构建高密度遗传连锁图谱是冰草抗性、品质、产量等重要性状QTL精细定位及标记辅助育种研究的基础。该试验以四倍体杂交冰草F2群体的202个分离单株及其亲本为材料,利用SRAP分子标记技术和Join Map 4.0作图软件对冰草的遗传连锁图谱进行了构建。结果表明:(1)共筛选出22对多态性好、标记位点清晰稳定的SRAP适宜引物,对冰草杂种F2分离单株的基因组DNA进行PCR扩增,共获得510个SRAP多态性标记位点,其比率占88.2%。(2)偏分离分析表明,偏分离标记比率仅为14.12%,符合遗传作图的要求。(3)成功构建了冰草的SRAP分子标记遗传连锁图谱,该图谱有14个连锁群、510个标记,连锁群间长度范围86.4～179.0cM,覆盖基因组总长度1 912.9cM,标记间平均间距3.75cM,为高密度遗传图谱。     

基于SRAP和SSR标记构建的杏扁遗传连锁图谱

以杏扁品种‘龙王帽’授粉‘优一’获得98个F1代单株为作图群体,采用SRAP和SSR标记进行连锁图谱的构建。采用Join Map 4.0软件进行连锁分析,分别构建了‘龙王帽’和‘优一’的分子连锁框架图,共获得132个SRAP标记和17个SSR标记。其中父本遗传图谱涉及8个连锁群,包含53个SRAP标记和9个SSR标记,图谱总长为694.8 cM,标记间平均图距为11.21 cM,平均每个连锁群上有7.75个标记位点,连锁群平均长度为86.85 cM;母本遗传图谱涉及8个连锁群,包含79个SRAP标记和8个SSR标记,图谱总长为924.8 cM,标记间平均图距为10.63 cM,平均每个连锁群上有10.87个标记位点,连锁群平均长度为115.6 cM。     

甘蓝型油菜花瓣缺失基因的图谱定位

在无花瓣品系APT02和正常有花瓣品种中双4号构建的的F2分离群体中,运用AFLP和SRAP两种标记技术对甘蓝型油菜花瓣缺失基因进行分子标记和图谱定位。在两亲本间筛选20对AFLP引物和170对SRAP 引物,进一步通过BSA法筛选,获得了与甘蓝型油菜花瓣缺失基因WHB连锁的1个SRAP标记e8m3_4（600bp）和1个AFLP标记E3247_15（150bp）,标记与基因WHB之间的遗传距离分别为5 cM和13.5cM;构建了一个甘蓝型油菜(Brassica napus.L )的分子标记遗传连锁图谱,该图谱共包含213个AFLP标记、56个SRAP标记和1个形态标记,分布于17个主要连锁群、两个三联体和4个连锁对中,遗传图距总长2487.1cM,标记间平均距离为10.09 cM。通过图谱定位,控制花瓣缺失性状的基因WHB被定位到第4连锁群(LG4)上。     

不结球白菜分子遗传图谱的构建及分析

以不结球白菜'常州乌塌菜'和'二青'杂交产生的181株F2代分离材料为作图群体,利用ISSR、RAPD、SSR、SRAP等分子标记来构建不结球白菜分子遗传连锁图谱.结果表明,构建的连锁图谱包含11个连锁群,由139个标记组成,其中包括4个ISSR标记、19个RAPD标记、22个SSR标记和94个SRAP标记.其中偏分离标记37个,占26.6%;每条连锁群上的标记数在4～33个之间,连锁群长度在61～164 cM的范围,覆盖基因组950 cM,总平均图距6.8 cM.     

白桦RAPD遗传连锁图谱的构建

以80个来自欧洲白桦(Betula pendula Roth)×中国白桦(Betula platyphylla Suk)的F1个体为作图群体。利用2个亲本和10个F1个体对1,200个10 bp的随机寡核苷酸引物进行筛选, 确定了208个多态性引物。利用RAPD标记, 按照拟测交的作图策略, 分别构建了欧洲白桦和中国白桦的分子标记连锁图谱。对2个亲本和80个F1代作图群体进行随机扩增, 共获得了364个多态性位点。χ2检验结果表明有307个位点符合1∶1分离的拟测交分离, 26个位点符合3∶1分离, 31个位点属偏分离位点。拟测交位点中有145个位点来自欧洲白桦, 有162个位点来自中国白桦。利用2点连锁分析, 欧洲白桦中的145个连锁标记构成了14个不同的连锁群(4个以上标记), 6个三连体和6个连锁对, 37个为非连锁位点, 连锁标记覆盖的总图距为955.6 cM (centimorgan), 平均图距14.9 cM。而来自中国白桦的162个标记构成了15个连锁群(4个以上标记), 4个三连体和6个连锁对, 21个为非连锁位点, 连锁标记覆盖的总图距为1,545.8 cM (centimorgan), 平均图距15.2 cM。该图谱的建立为进一步将两个图谱整合为一个高密度图谱及重要基因的定位奠定了基础。     

中国明对虾AFLP分子标记遗传连锁图谱的构建

以中国明对虾抗WSSV(白斑综合症病毒,WhiteSpotSyndromeVirus)选育群体第四代为母本,野生中国明对虾为父本,采用单对杂交方式产生F1代,F1代个体姊妹交产生F2代共42个个体为做图群体。62对AFLP选择性引物组合共产生529个分离位点,符合1∶1孟德尔分离类型位点共253个,3∶1孟德尔分离类型位点共276个。利用拟测交理论分别构建中国明对虾雌虾、雄虾的遗传连锁图谱,利用F2自交模型构建共同的AFLP分子标记连锁图谱。三张连锁图上分别有31、25和44个连锁群,图谱分辨率为分别为2.4cM、2.4cM和2.1cM。标记间隔距离分别为12.20cM、11.45cM和11.12cM图谱覆盖率分别达到50.21%、51.93%和48.08%。能够基本满足进行QTL(数量性状位点,QuantitativeTraitLocus)定位的需要。将该图谱和其他对虾类遗传连锁图谱进行了比较分析,探讨了利用相关分子标记将已有图谱进行整合的可能。     

白桦AFLP遗传连锁图谱的构建

以80个中国白桦(Betula platyphylla Suk)×欧洲白桦(Betula pendula Roth)的F1个体为作图群体, 利用扩增片段长度多态性(Amplified fragment length polymorphism, AFLP)标记, 按照拟测交作图策略, 分别构建了中国白桦和欧洲白桦的分子标记遗传连锁图谱。从64对AFLP引物组合中筛选出34对多态性丰富的引物组合, 这些入选的引物组合在分离群体中共检测到451个多态性位点。χ2检验结果表明, 有362个位点符合1∶1分离(拟测交分离位点), 41个位点符合3∶1分离, 20个位点符合1∶3分离, 28个位点属偏分离位点。在符合拟测交分离的位点中, 201个位点来自中国白桦, 161个位点来自欧洲白桦。利用2点连锁分析, 来自中国白桦的201个标记构成了14个连锁群(4个以上标记), 10个三连体和14个连锁对, 45个为非连锁位点, 连锁标记覆盖的总图距为1 296.1 cM, 平均图距15.5 cM。而来自欧洲白桦的161个标记构成了17个不同的连锁群(4个以上标记), 8个三连体和4个连锁对, 15个为非连锁位点, 连锁标记覆盖的总图距为1 035.8 cM, 平均图距12 cM。     

粳稻SRAP分子标记遗传群的构建与分析

用超级稻品种‘沈农606’和普通粳稻‘丽江新团黑谷’为亲本杂交获得的102份F_2代单株,通过SRAP分子标记遗传分析,构建了包含14个连锁群,由129个多态性位点组成的水稻连锁图谱,此图谱覆盖基因组长度1671.5 cM,平均图距13.0 cM。连锁群上有17.2%的多态性位点表现偏分离,偏分离标记在连锁群上存在热点区域。     

利用向日葵重组自交系构建遗传图谱

以向日葵自选系K55为母本、K58为父本杂交组合,通过单粒传得到的187个F_5：6代重组自交系群体为作图材料,联合应用SSR和AFLP标记构建遗传连锁图谱。经过78对SSR引物和48对AFLP引物组合选择性扩增,分别得到341和1119条带,共1460条,分别获得多态性条带184条和393条,共577条多态性条带,占所有条带的39.52%。SSR和AFLP标记各有84个和108个多态性标记偏离孟德尔分离比例(P=0.05),共192个偏分离标记。采用JoinMap4.0软件进行连锁分析,构建了1张总长度为2759.4 cM、包含17个连锁群、连锁495个多态性标记的遗传图谱,其中偏分离标记170个,标记间的平均图距为5.57 cM。每个连锁群上分布有5~72个标记,长68.88~250.17 cM。本图谱为向日葵永久性图谱,为向日葵重要性状QTL定位和基因克隆奠定基础。     

RAPD和SSR两种标记构建的中国对虾遗传连锁图谱

利用RAPD和SSR分子标记结合拟测交策略,对中国对虾(Fenneropenaeuschinensis)“黄海1号”雌虾与野生雄虾作为亲本进行单对杂交产生的F1代,采用RAPD和SSR两种分子标记技术初步构建了中国对虾雌、雄遗传连锁图谱。对460个RAPD引物和44对SSR引物进行筛选,共选出61个RAPD引物和20对SSR引物,用于对父母本和82个F1个体进行遗传分析。共得到母本分离标记146个(RAPD标记128个,微卫星标记18个)和父本分离标记127个(RAPD标记109个,微卫星标记18个)。雌性图谱包括8个连锁群、9个三联体和14个连锁对,标记间平均间隔为11·28cM,图谱共覆盖1173cM,覆盖率为59·36%;雄性图谱包括10个连锁群、12个三联体和7个连锁对,标记间平均间隔为12·05cM,图谱共覆盖1144·6cM,覆盖率为62·01%。中国对虾遗传图谱的构建为其分子标记辅助育种、比较基因组作图及数量性状位点的定位与克隆奠定了基础。     

Constructing genetic linkage maps for Pinus elliottii var. elliottii and Pinus caribaea var. hondurensis using SRAP, SSR, EST and ISSR markers

A total of 122 F₁ individuals from a single full-sib interspecific hybrid family crossed between Pinus elliottii var. elliottii (PEE) and P. caribaea var. hondurensis (PCH) were used to construct a detailed genetic linkage maps using four types of molecular markers: sequence-related amplified polymorphism (SRAP), microsatellite (SSR), expressed sequence tag polymorphism (ESTP) and inter-simple sequence repeat (ISSR). There were 381 SRAP, 108 SSR, 25 ESTP and 32 ISSR loci, segregating in the interspecific F₁ hybrid individuals. Framework maps were constructed at a LOD score threshold of 4.0 using the JoinMap^® 3.0. The map for the male parent (PCH) had 108 markers in 16 linkage groups (LGs), with a total length of 1,065.9 cM (Kosambi) and an average marker interval of 9.87 cM. The map for the female parent (PEE) contained 93 markers in 19 LGs, with a total length of 1,006.7 cM (Kosambi) and an average marker interval of 10.82 cM. The maps for PCH and PEE covered 56.5 and 70.3 % of their respective genomes. Based on the position of 36 loci segregating in both parents, 8 homologous LGs between PEE and PCH were identified.     

Genetic linkage maps of barfin flounder (<Emphasis Type="Italic">Verasper moseri</Emphasis>) and spotted halibut (<Emphasis Type="Italic">Verasper variegatus</Emphasis>) based on AFLP and microsatellite markers

Barfin flounder (Verasper moseri) and spotted halibut (Verasper variegatus) are two economically important marine fish species for aquaculture in China, Korea and Japan. Construction of genetic linkage maps is an interesting issue for molecular marker-assisted selection (MAS) and for better understanding the genome structure. In the present study, we constructed genetic linkage maps for both fish species using AFLP and microsatellite markers based on an interspecific F₁ hybrid family (female V. moseri and male V. variegatus). The female genetic map comprised 98 markers (58 AFLP markers and 40 microsatellite markers), distributing in 27 linkage groups, and spanning 637 cM with an average resolution of 8.9 cM. Whereas the male genetic map consisted of 86 markers (48 AFLP and 38 microsatellite markers) in 24 linkage groups, covering a length of 625 cM with an average marker spacing of 10 cM. The expected genome length was 1,128 cM in female and 1,115 cM in male, and the estimated coverage of genome was 56% for both genetic maps. Moreover, five microsatellite markers were observed to be common to both genetic maps. This is the first time to report the genetic linkage maps of V. moseri and V. variegatus that could serve as the basis for genetic improvement and selective breeding, candidate genes cloning, and genome structure research.     

An interspecific (Capsicum annuum ×C. chinese) F2 linkage map in pepper using RFLP and AFLP markers

We have constructed a molecular linkage map of pepper (Capsicum spp.) in an interspecific F₂ population of 107 plants with 150 RFLP and 430 AFLP markers. The resulting linkage map consists of 11 large (206–60.3 cM) and 5 small (32.6–10.3 cM) linkage groups covering 1,320 cM with an average map distance between framework markers of 7.5 cM. Most (80%) of the RFLP markers were pepper-derived clones, and these markers were evenly distributed across the genome. By using 30 primer combinations, we were able to generate 444 AFLP markers in the F₂ population. The majority of the AFLP markers clustered in each linkage group, although PstI/MseI markers were more evenly distributed than EcoRI/MseI markers within the linkage groups. Genes for the biosynthesis of carotenoids and capsaicinoids were mapped on our linkage map. This map will provide the basis of studying secondary metabolites in pepper. Received: 20 October 1999 / Accepted: 3 July 2000     

GENETIC MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES,PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS1

To establish a molecular‐marker‐assisted system of breeding and genetic study for Laminaria japonica Aresch., amplified fragment length polymorphism (AFLP) was used to construct a genetic linkage map of L. japonica featuring 230 progeny of F₂ cross population. Eighteen primer combinations produced 370 polymorphic loci and 215 polymorphic loci segregated in a 3:1 Mendelian segregation ratio (P ≤ 0.05). Of the 215 segregated loci, 142 were ordered into 27 linkage groups. The length of the linkage groups ranged from 6.7 to 90.3 centimorgans (cM) with an average length of 49.6 cM, and the total length was 1,085.8 cM, which covered 68.4% of the estimated 1,586.9 cM genome. The number of mapped markers on each linkage group ranged from 2 to 12, averaging 5.3 markers per group. The average density of the markers was 1 per 9.4 cM. Based on the marker density and the resolution of the map, the constructed linkage map can satisfy the need for quantitative trait locus (QTL) location and molecular‐marker‐assisted breeding for Laminaria.     

RAPD-based genetic linkage map of blueberry derived from a cross between diploid species (Vaccinium darrowi and V. elliottii)

An initial genetic linkage map for blueberry has been constructed from over 70 random amplified polymorphic DNA (RAPD) markers that segregated 11 in a testcross population of 38 plants. The mapping population was derived from a cross between two diploid blueberry plants: the F₁ interspecific hybrid (Vaccinium darrowi Camp x V. elliottii Chapm.) and another V. darrowi plant. The map currently comprises 12 linkage groups (in agreement with the basic blueberry chromosome number) and covers a total genetic distance of over 950cM, with a range of 3–30cM between adjacent markers. The use of such a map for identifying molecular markers linked to genes controlling chilling requirement and cold hardiness is discussed.     

CONSTRUCTION OF A GENETIC LINKAGE MAP FOR PORPHYRA HAITANENSIS (BANGIALES,RHODOPHYTA) BASED ON SEQUENCE‐RELATED AMPLIFIED POLYMORPHISM AND SIMPLE SEQUENCE REPEAT MARKERS1

Molecular markers and molecular genetic maps are prerequisites for molecular breeding in any plant species. A comprehensive genetic linkage map for cultivated Porphyra haitanensis T. J. Chang et B. F. Zheng has not yet been developed. In this study, 157 double haploid (DH) lines [derived from a YSIII (wildtype) × RTPM (red‐type artificial pigmentation mutant) cross] were used as a mapping population in P. haitanensis. A total of 60 pairs of sequence‐related amplified polymorphism (SRAP) primers and 39 pairs of simple sequence repeat (SSR) primers were used to detect polymorphisms between the two parents. Fifteen SRAP and 16 SSR polymorphic primer pairs were selected to analyze the DH population. A linkage genetic map comprising 67 SRAP markers and 20 SSR markers in five linkage groups, with a total length of 830.6 cM and an average of 10.13 cM between markers, was constructed. The markers were distributed evenly in all linkage groups without clustering. The linkage groups comprised 12–23 markers ranging in length from 134.2 to 197.3 cM. The estimated genome length of P. haitanensis was 942.4 cM, with 88.1% coverage. This is the first report of a comprehensive genetic map in P. haitanensis. The map presented here will provide a basis for the development of high‐density genetic linkage maps and lay the foundation for molecular breeding work in P. haitanensis.     

A genetic linkage map of tef [Eragrostis tef (Zucc.) Trotter] based on amplified fragment length polymorphism

A genetic linkage map of tef was constructed with amplified fragment length polymorphism (AFLP) markers using F₅ recombinant inbred lines (RILs) derived by single seed descent from the intraspecific cross of ’Kaye Murri’×’Fesho’. A total of 192 EcoRI/MseI primer combinations were screened for parental polymorphism. Around three polymorphic fragments per primer combination were detected, indicating a low polymorphism level in tef. Fifty primer combinations were selected to assay the mapping population, and 226 loci segregated among 85 F₅ RILs. Most AFLP loci behaved as dominant markers (presence or absence of a band), but about 15% of the loci were codominant. Significant deviations from the expected Mendelian segregation ratio were observed for 26 loci. The genetic linkage map comprised 211 markers assembled into 25 linkage groups and covered 2,149 cM of genome. AFLP is an efficient marker system for mapping plant species with low polymorphism such as tef. This is the first genetic linkage map constructed for tef. It will facilitate the mapping of genes controlling agronomically important traits and cultivar improvement in tef. Received: 27 April 1998 / Accepted: 4 January 1999     

Construction of genetic linkage map of the medicinal and ornamental plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

An integrated genetic linkage map of the medicinal and ornamental plant Catharanthus roseus, based on different types of molecular and morphological markers was constructed, using a F₂ population of 144 plants. The map defines 14 linkage groups (LGs) and consists of 131 marker loci, including 125 molecular DNA markers (76 RAPD, 3 RAPD combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and 37 other PCR based DNA markers), selected from a total of 472 primers or primer pairs, and six morphological markers (stem pigmentation, leaf lamina pigmentation and shape, leaf petiole and pod size, and petal colour). The total map length is 1131.9 cM (centiMorgans), giving an average map length and distance between two markers equal to 80.9 cM and 8.6 cM, respectively. The morphological markers/genes were found linked with nearest molecular or morphological markers at distances varying from 0.7 to 11.4 cM. Linkage was observed between the morphological markers concerned with lamina shape and petiole size of leaf on LG1 and leaf, stem and petiole pigmentation and pod size on LG8. This is the first genetic linkage map of C. roseus.     

Comparative molecular mapping in Ceratotropis species using an interspecific cross between azuki bean (Vigna angularis) and rice bean (V. umbellata)

A genetic linkage map was developed with 86 F₂ plants derived from an interspecific cross between azuki bean (Vigna angularis, 2n=2x=22) and rice bean (V. umbellata, 2n=2x=22). In total, 14 linkage groups, each containing more than 4 markers, were constructed with one phenotypic, 114 RFLP and 74 RAPD markers. The total map size was 1702 cM, and the average distance between markers was 9.7 cM. The loci showing significant deviation from the expected ratio clustered in several linkage groups. Most of the skewed loci were due to the predominance of rice bean alleles. The azuki-rice bean linkage map was compared with other available maps of Vigna species in subgenus Ceratotropis. Based on the lineage of the common mapped markers, 7 and 16 conserved linkage blocks were found in the interspecific map of azuki bean ×V. nakashimae and mungbean map, respectively. Although the present map is not fully saturated, it may facilitate gene tagging, QTL mapping and further useful gene transfer for azuki bean breeding. Received: 20 March 1999 / Accepted: 29 April 1999