三色堇与角堇种间遗传连锁图谱构建

该研究以二倍体三色堇和角堇为亲本杂交产生的66株F2代分离群体为作图群体,采用SRAP标记技术进行基因分型,利用JoinMap4.0软件构建了首张三色堇与角堇的种间遗传连锁图谱。结果表明:(1)从256对SRAP引物组合中筛选获得50对多态性好、标记位点清晰且稳定的引物组合。(2)通过对三色堇F2代群体的PCR扩增,共获得118个SRAP多态性标记位点,其中偏分离标记率为24.6%,符合遗传作图需要。(3)成功构建了三色堇和角堇的种间分子遗传连锁图谱,该图谱有15个连锁群,67个SRAP标记,连锁群长度范围1.6~52.2 cM,覆盖基因组总长度327.9 cM,标记间平均图距为4.9 cM。研究结果为三色堇和角堇高密度遗传图谱构建和重要性状的基因定位及分子标记辅助选择育种奠定了基础。     

大粒裸燕麦(Avena nuda L.)遗传连锁图谱的构建

以“元莜麦”和“555”杂交得到的281个F2单株为作图群体,利用20对AFLP引物、3对SSR引物和1个穗型性状构建了一张大粒裸燕麦遗传连锁图。该图谱全长1544.8cM,包含19个连锁群,其上分布有92个AFLP标记、3个SSR标记和1个穗型形态标记,不同连锁群标记数为2-14个,长度在23.7-276.3cM之间,平均长度为81.3cM,标记间平均距离为20.1cM。穗型标记分离比符合3：1,11个AFLP标记表现为偏分离,偏分离比为11.5%。该图谱符合遗传连锁框架图的要求,为今后大粒裸燕麦的QTL定位、分子标记辅助育种和比较基因组学等研究奠定基础。     

甘蓝型油菜A基因组候选BAC克隆的筛选及其插入片段大小分析

选择甘蓝型油菜A基因组10个连锁群上特有的85个SSR分子标记,合成其引物序列,采用四维PCR法筛选甘蓝型油菜-新疆野生油菜二体异附加系BAC文库,成功筛选到甘蓝型油菜A基因组39个BAC克隆,其插入片段介于50～300 kb之间,平均为120 kb。甘蓝型油菜A基因组10个连锁群BAC克隆的获得,对后续开展甘蓝型油菜A基因组染色体识别、基因染色体定位、遗传距离与物理距离间关系分析等均具有重要价值。     

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。     

甘蓝型油菜分子标记连锁图谱的构建及显性细胞核雄性不育基因的图谱定位

在显性细胞核雄性不育系Rs1046A和双低油菜品种Samourai构建的回交分离群体中,运用AFLP和SSR两种标记技术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138个AFL．P标记、83个SSR标记和1个形态标记,分布于18个主要连锁群、2个三联体和1个连锁对上,图谱总长度为2646cM,偏分离标记的比例为11．7％。显性细胞核雄性不育基因Ms被定位到第10连锁群(LG10)上。同时,偏分离标记聚集于第8连锁群(LG8)和第16连锁群(LGl6)的末端,形成了十分明显的偏分离标记密集区域。研究结果对于油菜核不育两型系的分子标记辅助选择育种具有重要意义,同时也为克隆和分离核不育基因以及研究核不育的分子机理打下了良好的基础。     

甘蓝型油菜千粒重性状的QTL初步定位研究

千粒重是油菜重要的产量相关性状之一,构建油菜遗传连锁图谱是研究其产量性状基因的前提。本研究利用小孢子培养技术,选育出了甘蓝型油菜大粒品系(G-42)和小粒品系(7-9)的纯合DH系DH-G-42和DH-7-9,其千粒重分别为6.24 g和2.42 g,二者比值达2.58。以DH-G-42为母本、DH-7-9为父本,构建了含190个单株的F2遗传作图群体,利用SSR和SRAP标记技术绘制遗传连锁图谱,该图谱共包含20个连锁群,涉及128个SSR标记和100个SRAP标记,图谱总长1546.6cM,标记间平均图距为6.78cM。本研究共检测到3个与千粒重性状相关的QTL,分别位于A9和C1连锁群,其中qSW-A9-1和qSW-A9-2贡献率分别达到10.98%和27.45%,均可视为控制粒重的主效QTL。本研究为后续进行油菜千粒重性状QTL的精细定位分析、分子标记辅助选择育种及新基因的克隆等奠定了基础。     

甘蓝型黄籽油菜种皮色泽QTL作图

甘蓝型黄籽油菜具有低纤维、高蛋白及高含油量的优点,因而己成为广大油菜育种工作者研究的重点之一。利用甘蓝型黑籽品系油研2号作父本,计蓝型黄籽品系GH06为母本,获得132个单株的F2群体;以AFLP和SSR为主要分析方法,构建了包括164个标记的甘蓝型油菜遗传连锁图谱,其中包括125个AFLP标记、37个SSR标记及一个RAPD和一个SCAR标记,分布在19个连锁群上,覆盖油菜基因组2549．8cM,标记间平均距离15．55cM。利用多区间作图法,对种皮色泽QTL进行分析,在第5及第19连锁群上各检测到一个QTL位点,分别解释表型变异46％及30．9％。     

大豆遗传图谱的构建和分析

利用大豆栽培品种科丰1号和南农1138－2杂交得到的重组近交系NJRIKY,通过RFLP,SSR,RAPD和AFLP4种分子标记的遗传连锁分析,构建了包含24个连锁群,由792个遗传标记组成的大豆较高密度连锁图谱,该图谱覆盖2320．7cM,平均图距2．9cM,SSR标记的多态性较高,在基因组中的位置相对稳定,可以作为锚定标记,有利于连锁群的归并和不同图谱的比较整合;而AFLP标记对于增加图谱密度效率较高,但其容易出现聚集现象,从而造成连锁群上有很大的空隙（gap),另外,在连锁群中有21．7％的分子标记出现偏分离,该图谱为基因定位,比较基因组学和重要农艺性状的QTL定位等研究打下了基础。     

''百农64''×''京双16''小麦遗传连锁图谱构建

通过对小麦品种‘百农64’ב京双16’F3家系群体的SSR和AFLP分析,构建了含100个SSR标记(91个引物)和58个AFLP标记(12个引物)的小麦遗传连锁图,158个标记组成20个连锁群,覆盖小麦基因组3 114cM,标记间平均间距为19.7 cM.将前人未定位的12个SSR标记定位到了小麦遗传连锁图谱上.为小麦慢白粉病性等农艺性状的QTL分析打下了良好基础.     

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。     

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。     

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。     

Fine mapping of the yellow seed locus in Brassica juncea L

The yellow mustard plant in Northern Shaanxi is a precious germplasm, and the yellow seed trait is controlled by a single recessive gene. In this report, amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) techniques were used to identify markers linked to the brown seed locus in an F(2) population consisting of 1258 plants. After screening 256 AFLP primer combinations and 456 pairs of SSR primers, we found 14 AFLP and 2 SSR markers that were closely linked to the brown seed locus. Among these markers, the SSR marker CB1022 showed codominant inheritance. By integrating markers previously found to be linked to the brown seed locus into the genetic map of the F(2) population, 23 markers were linked to the brown seed locus. The two closest markers, EA02MC08 and P03MC08, were located on either side of the brown seed locus at a distance of 0.3 and 0.5 cM, respectively. To use the markers for the breeding of yellow-seeded mustard plants, two AFLP markers (EA06MC11 and EA08MC13) were converted into sequence-characterized amplified region (SCAR) markers, SC1 and SC2, with the latter as the codominant marker. The two SSR markers were subsequently mapped to the A9/N9 linkage group of Brassica napus L. by comparing common SSR markers with the published genetic map of B. napus. A BLAST analysis indicated that the sequences of seven markers showed good colinearity with those of Arabidopsis chromosome 3 and that the homolog of the brown seed locus might exist between At3g14120 and At3g29615 on this same chromosome. To develop closer markers, we could make use of the sequence information of this region to design primers for future studies. Regardless, the close markers obtained in the present study will lay a solid foundation for cloning the yellow seed gene using a map-based cloning strategy.     

Generation and mapping of SCAR and CAPS markers linked to the seed coat color gene in Brassica napus using a genome-walking technique.

The yellow seed coat trait in No. 2127-17, a resynthesized purely yellow Brassica napus line, is controlled by a single partially dominant gene, Y. A double-haploid population derived from the F1 of No. 2127-17 x 'ZY821' was used to map the seed coat color phenotype. A combination of AFLP analysis and bulked segregant analysis identified 18 AFLP markers linked to the seed coat color trait. The 18 AFLP markers were mapped to a chromosomal region of 37.0 cM with an average of 2.0 cM between adjacent markers. Two markers, AFLP-K and AFLP-H, bracketed the Y locus in an interval of 1.0 cM, such that each was 0.5 cM away from the Y locus. Two other markers, AFLP-A and AFLP-B, co-segregated with the seed color gene. For ease of use in breeding programs, these 4 most tightly linked AFLP markers were converted into reliable PCR-based markers. SCAR-K, which was derived from AFLP-K, was assigned to linkage group 9 (N9) of a B. napus reference map consisting of 150 commonly used SSR (simple sequence repeat) markers. Furthermore, 2 SSR markers (Na14-E08 and Na10-B07) linked to SCAR-K on the reference map were reversely mapped to the linkage map constructed in this study, and also showed linkage to the Y locus. These linked markers would be useful for the transfer of the dominant allele Y from No. 2127-17 to elite cultivars using a marker-assisted selection strategy and would accelerate the cloning of the seed coat color gene.     

Construction of a genetic linkage map in <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> (Osbeck) using RAPD and SSR markers

The primary genetic linkage maps of Fenneropenaeus chinensis (Osbeck) were constructed by using the “two-way pseudo-testcross” strategy with RAPD and SSR markers. Parents and F1 progeny were used as segregating populations. Sixty-one RAPD primers and 20 pairs of SSR primers were screened from 460 RAPD primers and 44 pairs of SSR primers. These primers were used to analyze the parents and 82 progeny of the mapping family. About 146 primers (128 RAPDs, 18 microsatellites) in the female and 127 primers (109 RAPDs, 18 microsatellites) in the male were segregating markers. The female linkage map included eight linkage groups, nine triplets and 14 doublets, spanning 1,173 cM with the average marker density of 11.28 cM, and the observed coverage was 59.36%. The male linkage map included 10 linkage groups, 12 triplets and seven doublets, spanning 1,144.6 cM with the average marker density of 12.05 cM, and the observed coverage was 62.01%. The construction of the F. chinensis genetic linkage maps here opened a new prospect for marker-assisted selection program, comparative genomics and quantitative trait loci (QTL) gene location and cloning.     

The genetic basis of flowering time and photoperiod sensitivity in rapeseed (Brassica napus L.)

The objective of this study was to dissect the genetic control of days to flowering (DTF) and photoperiod sensitivity (PS) into the various components including the main-effect quantitative trait loci (QTLs), epistatic QTLs and QTL-by-environment interactions (QEs). Doubled haploid (DH) fines were produced from an F1 between two spring Brassica napus cultivars Hyola 401 and Q2. DTF of the DH lines and parents were investigated in two locations, one location with a short and the other with a long photoperiod regime over two years. PS was calculated by the delay in DTF under long day as compared to that under short day. A genetic linkage map was constructed that comprised 248 marker loci including SSR, SRAP and AFLP markers. Further QTL analysis resolved the genetic components of flowering time and PS into the main-effect QTLs, epistatic QTLs and QEs. A total of 7 main-effect QTLs and 11 digenic interactions involving 21 loci located on 13 out of the 19 linkage groups were detected for the two traits. 3 main-effect QTLs and 4 pairs of epistatic QTLs were involved in QEs conferring DTF. One QTL on linkage group (LG) 18 was revealed to simultaneously affect DTF and PS and explain for the highest percentage of the phenotypic variation. The implications of the results for B. napus breeding have been 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.     

Mapping and validation of chromosome regions conferring a new boron-efficient locus in <Emphasis Type="Italic">Brassica napus</Emphasis>

Considerable genotypic variation exists in the response of different cultivars of rapeseed (Brassica napus) to B deficiency. This raises the possibility of genetic improvement of a B nutrition trait that will make the plant more tolerant to low B stress. The results of our study showed that B-efficient backcross plants had lower B concentration and more dry matter when grown at low levels of B when compared with the recurrent parent. Accordingly, we proposed that the improved B efficiency was attributed to either a high B utilization efficiency or less demand for B. The results of the genetic analysis showed that B efficiency is a dominant trait that is controlled by a single locus, namely BnBE2. By using bulked segregant analysis (BSA) in combination with amplified fragment length polymorphism (AFLP) and sequence related amplified polymorphism (SRAP) techniques, five SRAP markers and one converted single strand conformation polymorphism (SSCP) marker were identified to be linked to BnBE2 after screening 1,800 primer combinations. The six markers together with BnBE2 were mapped in a region that covered a genetic distance of 6.9 cM on a linkage group using a BC₆ population. This region was located on linkage group N14 after mapping these markers in two doubled haploid (DH) populations (TNDH and BQDH). The SRAP and AFLP markers were sequenced and found to be homologous to a BAC sequence from Brassica oleracea (CC). This finding suggested that the segment containing BnBE2 locus originated from the C genome of Brassica oleracea. Three SSR markers were identified to be linked to BnBE2 through comparative mapping. All these markers might have potential value for facilitating the pyramiding of the BnBE2 gene with other B efficient genes in order to improve the B efficiency trait and for further fine mapping of the BnBE2 gene in Brassica napus.