The length of the intact donor chromosome segment around a target gene in marker-assisted backcrossing

Recurrent backcrossing is an established procedure to transfer target genes from a donor into the genetic background of a recipient genotype. By assessing the parental origin of alleles at markers flanking the target locus one can select individuals with a short intact donor chromosome segment around the target gene and thus reduce the linkage drag. We investigated the probability distribution of the length of the intact donor chromosome segment around the target gene in recurrent backcrossing with selection for heterozygosity at the target locus and homozygosity for the recurrent parent allele at flanking markers for a diploid species. Assuming no interference in crossover formation, we derived the cumulative density function, probability density function, expected value, and variance of the length of the intact chromosome segment for the following cases: (1) backcross generations prior to detection of a recombinant individual between the target gene and the flanking marker; (2) the backcross generation in which for the first time a recombinant individual is detected, which is selected for further backcrossing; and (3) subsequent backcross generations after selection of a recombinant. Examples are given of how these results can be applied to investigate the efficiency of marker-assisted backcrossing for reducing the length of the intact donor chromosome segment around the target gene under various situations relevant in breeding and genetic research.     

Stability over genetic backgrounds, generations and years of quantitative trait locus (QTLs) for organoleptic quality in tomato

The efficiency of marker-assisted backcross for the introgression of a quantitative trait locus (QTL) from a donor line into a recipient line depends on the stability of QTL expression. QTLs for six quality traits in tomato (fruit weight, firmness, locule number, soluble solid content, sugar content and titratable acidity) were studied in order to investigate their individual effect and their stability over years, generations and genetic backgrounds. Five chromosome regions carrying fruit quality QTLs were transferred following a marker-assisted backcross scheme from a cherry tomato line into three modern lines with larger fruits. Three sets of genotypes corresponding to three generations were compared: (1) an RIL population, which contained 50% of each parental genome, (2) three BC3S1 populations which segregated simultaneously for the five regions of interest but were almost fully homozygous for the recipient genome on the eight chromosomes carrying no QTL and (3) three sets of QTL-NILs (BC3S3 lines) which differed from the recipient line only in one of the five regions. QTL detection was performed in each generation, in each genetic background and during 2 successive years for QTL-NILs. About half of the QTLs detected in QTL-NILs were detected in both years. Eight of the ten QTLs detected in RILs were recovered in the QTL-NILs with the genetic background used for the initial QTL mapping experiment, with the exception of two QTLs for fruit firmness. Several new QTLs were detected. In the two other genetic backgrounds, the number of QTLs in common with the RILs was lower, but several new QTLs were also detected in advanced generations.     

Resistance QTL confirmed through development of QTL-NILs for barley leaf rust resistance

Near-isogenic lines (NILs) differing with regard to disease QTLs provide valuable material for a more detailed study into the genetic basis of quantitative resistance. Previously obtained information on QTLs that show an effect on leaf rust (Puccinia hordei) in barley was used in a marker-assisted backcross programme. The genome origin in backcross plants was controlled through AFLP marker analysis and graphical genotyping. Plants obtained after the third generation of backcrossing sufficiently resembled the recurrent parent. For one QTL, BC₃S₁ plants were evaluated in a disease test and genotyped. NILs containing the desired QTL in homozygous condition in a recipient background were finally obtained. A disease test and verification of the marker genotype confirmed the identity of the NILs. Simultaneous with the backcross programme a simulation study on efficiency of marker-assisted backcrossing was performed.     

花椰菜与黑芥种间体细胞杂种的获得和鉴定

利用非对称体细胞杂交技术, 获得芸薹属花椰菜(Brassica oleracea var. botrytis)与黑芥(B. nigra)的种间杂种, 实现了野生种质抗病基因向甘蓝类蔬菜作物的渗透。以具有良好再生能力的花椰菜下胚轴原生质体作为融合受体, 具有抗黑腐、黑胫和根肿病优良性状的黑芥叶肉原生质体作为融合供体, 用不同强度的UV射线处理后, 利用PEG方法诱导供、受体原生质体融合。培养后获得170棵再生植株, 选取来自40个不同愈伤组织的40棵单株进行形态学观察及RAPD和SRAP分子标记检测, 结果表明其中30棵为体细胞杂种。染色体计数显示, 约23%杂种植株的染色体数目小于供、受体染色体数之和。用流式细胞仪测定DNA含量显示, 杂种植株DNA含量是受体的2-4倍, 20%杂种植株DNA含量小于供、受体之和。     

5 个籼稻背景的高代回交置换系的置换片段分析

以轮回亲本籼稻品种9311(Oryz a sativa ssp. indica ‘Yangdao 6’)为对照, 选用132个亲本间有多态性的SSR标记, 对以粳稻品种日本晴(Oryz a sativa ssp. japonica 'Nipponbare’)为供体的5个高代回交置换系的农艺性状及置换片段进行分析。5个置换系在粒长、粒宽、千粒重、剑叶长、株高及落粒性等方面与籼稻品种9311之间有极显著差异, 其余性状与籼稻品种9311间的差异不显著; 从置换系中检测出8个置换片段, 总长度为236.0 cM, 平均长度为29.5 cM; 从置换片段上检出包括2个千粒重、1个粒长、1个粒宽、1个剑叶长、1个株高和1个落粒性共7个QTLs , 分别分布在水稻第1、3、5、6和第10染色体上。其中, 第1染色体上控制剑叶长的QTL和第6染色体上控制株高的QTL可能是新发现的QTLs。实验结果进一步丰富了置换系群体的数量和质量, 也为QTLs 的精细定位及分子设计育种奠定了基础。     

Assigning Brassica microsatellite markers to the nine C-genome chromosomes using Brassica rapa var. trilocularis–B. oleracea var. alboglabra monosomic alien addition lines

Brassica rapa var. trilocularis-B. oleracea var. alboglabra monosomic alien addition lines (MAALs) were used to assign simple sequence repeat (SSR) markers to the nine C-genome chromosomes. A total of 64 SSR markers specific to single C-chromosomes were identified. The number of specific markers for each chromosome varied from two (C3) to ten (C4, C7 and C9), where the designation of the chromosomes was according to Cheng et al. (Genome 38:313-319, 1995). Seventeen additional SSRs, which were duplicated on 2-5 C-chromosomes, were also identified. Using the SSR markers assigned to the previously developed eight MAALs and recently obtained aneuploid plants, a new Brassica rapa-B. oleracea var. alboglabra MAAL carrying the alien chromosome C7 was identified and developed. The application of reported genetically mapped SSR markers on the nine MAALs contributed to the determination of the correspondence between numerical C-genome cytological (Cheng et al. in Genome 38:313-319, 1995) and linkage group designations. This correspondence facilitates the integration of C-genome genetic information that has been generated based on the two designation systems and accordingly increases our knowledge about each chromosome. The present study is a significant contribution to genetic linkage analysis of SSR markers and important agronomic traits in B. oleracea and to the potential use of the MAALs in plant breeding.     

5个籼稻背景的高代回交置换系的置换片段分析

以轮回亲本籼稻品种9311（Oryza sativassp.indica‘Yangdao6’）为对照,选用132个亲本间有多态性的SSR标记,对以粳稻品种日本晴（Oryzasativassp.japonica‘Nipponbare’）为供体的5个高代回交置换系的农艺性状及置换片段进行分析。5个置换系在粒长、粒宽、千粒重、剑叶长、株高及落粒性等方面与籼稻品种9311之间有极显著差异,其余性状与籼稻品种9311间的差异不显著;从置换系中检测出8个置换片段,总长度为236.0cM,平均长度为29.5cM;从置换片段上检出包括2个千粒重、1个粒长、1个粒宽、1个剑叶长、1个株高和1个落粒性共7个QTLs,分别分布在水稻第1、3、5、6和第10染色体上。其中,第1染色体上控制剑叶长的QTL和第6染色体上控制株高的QTL可能是新发现的QTLs。实验结果进一步丰富了置换系群体的数量和质量,也为QTLs的精细定位及分子设计育种奠定了基础。     

Generation and characterization of Brassica rapa ssp. pekinensis – B. oleracea var. capitata monosomic and disomic alien addition lines

Journal of Genetics - Five monosomic alien addition lines (MAALs) of Brassica rapa ssp. pekinensis – B. oleracea var. capitata were obtained by hybridization and backcrossing between B. rapa...     

The association of flowering time quantitative trait loci with duplicated regions and candidate loci in Brassica oleracea.

A population of 150 doubled haploid lines of rapid cycling Brassica oleracea, derived from an F1 from a var. alboglabra x var. italica cross, was scored for flowering time in two trials. Using information on 82 mapped molecular markers, spread evenly across the nine linkage groups, QTL were identified at six locations; one each on linkage groups O2 and O3 and two each on linkage groups O5 and O9. In total, these QTL explained 58 and 93% of the genetical variation in the two trials. Three of these QTL, on linkage groups O2, O3, and O9, were situated in regions showing considerable homology both with each other and with chromosome regions of B. nigra that have been shown to affect flowering time. These same regions are all homologous to a single tract of Arabidopsis chromosome 5, which contains a number of the flowering-related genes, one or more of which may be candidates for the QTL found in Brassica.     

Marker-assisted introgression of five QTLs controlling fruit quality traits into three tomato lines revealed interactions between QTLs and genetic backgrounds

The evaluation of organoleptic quality of tomato fruit requires physical, chemical and sensory analyses, which are expensive and difficult to assess. Therefore, their practical use in phenotypic selection is difficult. In a previous study, the genetic control of several traits related to organoleptic quality of fresh-market tomato fruit was investigated. Five chromosome regions strongly involved in organoleptic quality attributes were then chosen to be introgressed into three different recipient lines through marker-assisted selection. A marker-assisted backcross (MABC) strategy was performed, as all the favorable alleles for quality traits were provided by the same parental tomato line, whose fruit weight (FW) and firmness were much lower than those of the lines commonly used to develop fresh market varieties. Three improved lines were obtained after three backcrossing and two selfing generations. The implementation of the MABC scheme is described. The three improved lines were crossed together and with the recipient lines in a half-diallel mating scheme, and the simultaneous effect of the five quantitative trait locus (QTL) regions was compared in different genetic backgrounds. Significant effects of the introgressed regions and of the genetic backgrounds were shown. Additive effects were detected for soluble solid and reducing sugar content in two genetic backgrounds. A partially dominant effect on titratable acidity was detected in only one genetic background. In contrast, additive to dominant unfavorable effects of the donor alleles were detected for FW and locule number in the three genetic backgrounds. Recessive QTL effects on firmness were only detected in the two firmest genetic backgrounds. Comparison of the hybrids in the half-diallel gave complementary information on the effects of: (1) the alleles at the selected regions, (2) the genetic backgrounds and (3) their interaction. Breeding efficiency strongly varied according to the recipient parent, and significant interactions between QTLs and genetic backgrounds were shown for all of the traits studied.     

Marker-Assisted Introgression of Quantitative Trait Loci

The use of molecular markers for the introgression of one or several superior QTL alleles into a recipient line is investigated using analytic and simulation results. The positions of the markers devoted to the control of the genotype at the QTLs in a ``foreground selection' step are optimized given the confidence interval of the QTL position. Results demonstrate that using at least three markers per QTL allows a good control over several generations. Population sizes that should be recommended for various numbers of QTLs are calculated and are used to determine the limit in the number of QTLs that can be monitored simultaneously. If ``background selection' devoted to accelerate the return to the recipient parent genotype outside the QTL regions is applied, the positions of the markers devoted to the control of the QTLs have to be reconsidered. When several QTLs are monitored simultaneously, background selection among the limited number of individuals resulting from the foreground selection step accelerates the increase in genomic similarity with the recipient parent, with only limited costs. Background selection is even more efficient in a pyramidal backcross program where QTLs are first monitored one by one.     

Inheritance of oilseed rape (Brassica napus) RAPD markers in a backcross progeny with Brassica campestris

Different cultivars/transgenic lines of oilseed rape (Brassica napus) were crossed (as females) with different cultivars/populations of Brassica campestris. All cross combinations produced seed, with an average seed set per pollination of 9.8. Backcrossing of selected interspecific hybrids (as females) to B. campestris resulted in a much lower seed set, average 0.7 seed per pollination. In the single backcross progeny where a large enough population (92 plants) was obtained for analysis, 33 B. napus specific RAPD markers were investigated to determine the extent of transfer of oilseed rape genetic material into this population. Markers were transferred to the backcross generation with frequencies ranging from 26% to 91%. Almost all of the markers (30/33) were transferred in a frequency not significantly different from 50%. Analysis of the pairwise segregation of markers revealed that 23 markers could be assigned to six linkage groups, most probably reflecting six B. napus C-chromosomes. The presence of backcross plants with recombinant genotypes suggests that complex genetic processes can take place during interspecific hybridisation and backcrossing in these Brassica species. The implications of our results for the possible choice of integration sites of transgenes in oilseed rape are discussed.     

云南元江普通野生稻株高和抽穗期QTL定位研究

以云南元江普通野生稻为供体亲本,在特青的遗传背景下构建了一套BC3高代回交群体。利用117个SSR标记分析383个BC3F2株系的基因型,采用单标记分析法对控制元江普野株高和抽穗期的QTL进行分析。在北京和合肥两个地点试验结果表明,控制株高的QTL分布在第1染色体上,在RM104附近有一个QTL,与sd-1位置相当,其对表现型变异的贡献率在两个地点分别为27％和28％,其加性效应值分别为26．24cm和26．28cm,来自野生稻的等位基因显著提高回交群体的株高;在第1、3、7、8、11染色体共检测到6个控制抽穗期QTL,其中第8染色体RM25附近控制抽穗期的QTL在两个地点的贡献率分别为13％和15％,加性效应值为4．60d和3．65d,来自野生稻的等位基因使回交群体抽穗期延迟。     

Introgression of quantitative trait loci (QTLs) determining stripe rust resistance in barley: an example of marker-assisted line development

 Genome-analysis tools are useful for dissecting complex phenotypes and manipulating determinants of these phenotypes in breeding programs. Quantitative trait locus (QTL)-analysis tools were used to map QTLs conferring adult plant resistance to stripe rust (caused by Puccinia striiformis f.sp. hordei) in barley. The resistance QTLs were introgressed into a genetic background unrelated to the mapping population with one cycle of marker-assisted backcrossing. Doubled-haploid lines were derived from selected backcross lines, phenotyped for stripe-rust resistance, and genotyped with an array of molecular markers. The resistance QTLs that were introgressed were significant determinants of resistance in the new genetic background. Additional resistance QTLs were also detected. The susceptible parent contributed resistance alleles at two of these new QTLs. We hypothesize that favorable alleles were fixed at these new QTLs in the original mapping population. Genetic background may, therefore, have an important role in QTL-transfer experiments. A breeding system is described that integrates single-copy and multiplex markers with confirmation of the target phenotype in doubled-haploid lines phenotyped in field tests. This approach may be useful for simultaneously producing agronomically useful germplasm and contributing to an understanding of quantitatively inherited traits. Received: 6 May 1997 / Accepted: 1 September 1997     

植物基因组研究与利用的新型工具——异源单体附加系

在高等植物中, 以种间杂交和回交把有益基因从一个物种转移到另一个物种为目的育种项目中, 单个外源染色体常常被附加到含有受体细胞完整一套染色体中, 形成异源单体附加系。这种异源单体附加系是阐明基因组结构和转移基因的有效工具。它可以通过回交形成覆盖整个基因组的渗入系重叠群, 用于建立以受体物种基因组为载体的外源物种基因组文库。另外, 一套完整的异源单体附加系也可看作是一个拥有分散供体基因组成为单个染色体单位的文库, 便于精确高通量地将标记分配到单个供体染色体上, 从而可以比较供体染色体和各自的直向同源受体染色体之间的标记位置和同线性关系。同时, 也便于研究同源染色体的渗入机制和配对状态。文中介绍了异源单体附加系的培育和特性, 并着重阐明了它在遗传育种和基础研究中的应用。     

Physical organization of the major duplication on Brassica oleracea chromosome O6 revealed through fluorescence in situ hybridization with Arabidopsis and Brassica BAC probes.

The close relationship between Brassica oleracea and Arabidopsis thaliana has been used to explore the genetic and physical collinearity of the two species, focusing on an inverted segmental chromosome duplication within linkage group O6 of B. oleracea. Genetic evidence suggests that these segments share a common origin with a region of Arabidopsis chromosome 1. Brassica oleracea and Arabidopsis bacterial artificial chromosome probes have been used for fluorescence in situ hybridization analysis of B. oleracea pachytene chromosomes to further characterize the inverted duplication. This has been highly effective in increasing the local resolution of the cytogenetic map. We have shown that the physical order of corresponding genetic markers is highly conserved between the duplicated regions in B. oleracea and the physical lengths of the regions at pachytene are similar, while the genetic distances are considerably different. The physical marker order is also well conserved between Arabidopsis and B. oleracea, with only one short inversion identified. Furthermore, the relative physical distances between the markers in one segment of B. oleracea and Arabidopsis have stayed approximately the same. The efficacy of using fluorescence in situ hybridization, together with other forms of physical and genetic mapping, for elucidating such issues relating to synteny is discussed.     

Generation of B. nigra-B. rapa chromosome addition stocks: cytology and microsatellite markers (SSRs) based characterization

To improve Brassica nigra, the B-genome donor for Brassica juncea through selective introgression of useful variation from A-genome chromosomes, B. nigra-B. rapa chromosome addition stocks were successfully synthesized for the first time. Resynthesized B. juncea was used as B-genome donor species and A-genome addition stocks were developed by hybridizing sesquidiploid plant (ABB) as female and using B. nigra as the male parent. Various cycles of backcrossing and/or selfing were utilized to isolate plants carrying addition of three A-genome chromosomes in the background of B. nigra. These chromosome addition stocks were characterized by chromosome counts, pollen and seed fertility and chromosome specific microsatellite (SSRs) markers. The chromosome number in different backcross/self generations ranged between 2n=26 and 2n=19 with relatively high frequency of univalents (8-10I) at in meiotic configurations observed, suggesting the role of preferential transmission of A-genome chromosomes. SSRs analysis revealed that B. rapa chromosomes 3 and 4 were the first to get eliminated followed by chromosome 10. Remaining chromosomes were maintained till BC(1)F(4). However, second cycle of backcrossing (BC(2)) led to the elimination of chromosome numbers 1 and 2. BC(2)F(2) plants carried the chromosome numbers 6, 7, 8 and 9. Generation BC(3) having plants with 2n=19 carried chromosome numbers 6, 7 and 8. It is possible that chromosomes 6, 7 and 8 had higher transmission frequency and these were better tolerated by the B. nigra genome.     

水稻叶绿素含量动态QTL分析

为剖析水稻不同生育时期叶绿素含量的变化动态及遗传机制,以‘Sasanishiki’（粳稻）、‘Habataki’（籼稻）及其杂交衍生的85个回交重组自交系（BILs）群体为材料,对控制水稻叶片叶绿素含量的数量性状基因位点（QTL）变化动态进行了分析。共检测到39个QTL,包括26个非条件QTL和13个条件QTL,分布在除第7和第11号染色体以外的10条染色体上,平均每个时期检测到3．25个非条件QTL。其中生育前期和生育中后期检测到的QTL位点较少,仅为1—3个;在生育中期（盛期）检测到的QTL位点相对较多,一般为4～5个。在生育期的中期和后期均能在第1和2号染色体上检测到控制叶绿素含量的QTL,并且这些QTL位点在1和2号染色体上呈现聚集现象。本研究同时发现了一些新的QTL位点,这些QTL将有助于我们更全面地了解叶绿素在不同发育时期的遗传基础。     

Marker-assisted introgression of Trypanotolerance QTL in mice

A marker-assisted introgression (MAI) experiment was conducted to use genetic markers to transfer each of the three trypanotolerance QTL from a donor mouse strain, C57BL/6, into a recipient mouse strain, A/J. We used a backcross strategy that consisted of selecting two lines, each carrying two of the donor QTL alleles through the backcross (BC) phase. At the fourth BC generation, single-carrier animals were selected for the production of homozygous animal in the intercross phase. The QTL regions (QTLR) were located on chromosomes MMU1, MMU5, and MMU17. Groups of mice with different genotypes and the parental lines were subjected to a challenge with Trypanosoma congolense. The results show that trypanotolerance QTL was successfully moved into the recipient background genotype, yielding a longer survival time. The mean estimated survival time was 57.9, 49.5, and 46.8 days for groups of mice carrying the donor QTL on MMU1, MMU5, and MMU17 on A/J background. The mean estimated survival time was 29.7 days for the susceptible A/J line and 68.8 days for the resistant C57BL/6 line. The estimated QTLR effects are close to 30% smaller than those in the original mapping population which was likely caused by the difference in the background on which the effects of QTLR are tested. This is the first report of successful marker-assisted introgression of QTL in animals. It is experimental proof of the use of genetic markers for marker-assisted introgression in animal breeding.Institut National des Recherches Agricoles du Bénin, 01 BP 884 COTONOU, République du Bénin     

基于MAS的小麦抗赤霉病育种材料抗性评价

为了提高黄淮海麦区小麦育种材料的赤霉病抗性,采用分子标记辅助选择的方法,将来自望水白的4个抗赤霉病主效QTL 3B-QTL、4B-QTL、5A-QTL和6B-QTL导入不同的感病背景中,在后代BC1F3和BC1F4株系中评价它们的抗病效应和农艺性状回复情况。结果表明:(1)导入4个抗病QTL株系的平均病小穗率和病粒率分别为12.2%和6.3%,而受体亲本则分别达到59.1%和44.2%,抗病性显著提高;(2)病小穗数和病粒率与穗长及株高极显著负相关,但与可育小穗数、百粒重、旗叶长和旗叶宽等农艺性状指标没有显著相关性。因此,通过导入抗病主效QTL可以显著改善感病材料的抗性,为进一步选育高产抗病品种提供基础材料。不良农艺性状的紧密连锁阻碍着抗赤霉病主效QTL的高效利用,需要通过继续回交或与其他品种杂交来打破这种遗传连锁关系。