Efficient Method of Agrobacterium-mediated Transformation for Triticale (x Triticosecale Wittmack)

Transgenic plants of triticale cv. Wanad were obtained after transformation using three combinations of strain/vectors. Two of them were hypervirulent Agrobacterium tumefaciens strains (AGL1 and EHA101) with vectors containing bar under maize ubiquitin 1 promoter (pDM805), and both hpt under p35S and nptII under pnos (pGAH). The third one was a regular LBA4404 strain containing super-binary plasmid pTOK233 with selection genes the same as in pGAH. The efficiency of transformation was from 0 to 16% and it was dependent on the selection factor, auxin pretreatment, and the strain/vector combination. The highest number of transgenic plants was obtained after transformation with LBA4404(pTOK233) and kanamycin selection. Pretreatment of explants with picloram led to the highest number of plants obtained after transformation with both Agrobacterium/vector systems LBA4404(pTOK233) and EHA101(pGAH) and selected with kanamycin. Transgenic character of selected plants was examined by PCR using specific primers for bar, gus, nptII, and hpt and confirmed by Southern blot hybridization analysis. There was no GUS expression in T₀ transgenic plants transformed with gus under p35S. However the GUS expression was detectable in the progeny of some lines. Only 30% of 46 transgenic lines showed Mendelian segregation of GUS expressing to GUS not expressing plants. In the remaining 70% the segregation was non-Mendelian and the rate was much lower than 3:1. Factors that might effect expression of transgenes in allohexaploid monocot species are discussed.     

Protoplast culture and transformation studies of Triticale (x Triticosecale Wittmack)

Plant Cell, Tissue and Organ Culture (PCTOC) -     

Efficient plant regeneration system for immature embryos of triticale (x Triticosecale Wittmack)

A range of tissue culture conditions were tested to improve embryo culture frequency, and to develop an efficient plant regeneration system for triticale. Immature embryos (14–21 days post-anthesis) from two triticale genotypes (Hx87-139 and Tahara) were cultured on a commonly used Murashige and Skoog (MS) and on Lazzeri's (L1) basal medium with varied carbon sources, and two different plant growth regulators; 2,4-Dichlorophenoxyacetic acid (2,4-D) and 3,6-Dichloro-2-methoxybenzoic acid (dicamba). Although embryos could be cultured on both media types, L1 based medium was better than MS basal salts for callus induction and somatic embryogenesis, with plant regeneration frequencies up to 11 fold greater on L1 media types. In the presence of dicamba, callus induction was more rapid, that resulted in subsequent regeneration of up to 2 fold more plantlets than from callus induced on medium containing 2,4-D. Maltose appeared to be a superior carbon source during differentiation of callus. Genotype Tahara showed a better regenerative response than Hx87-138, with up to 23 normal, fertile plants being produced from a single embryo when cultured on L1MDic medium, containing maltose (5%) and dicamba (20 mg l^–1). Applications of this tissue culture procedure in triticale improvement through genetic engineering are also discussed.     

Genetic mapping of a 7R Al tolerance QTL in triticale (x Triticosecale Wittmack)

Triticale (x Triticosecale Wittmack) is a relatively new cereal crop. In Poland, triticale is grown on 12 % of arable land (http://www.stat.gov.pl). There is an increasing interest in its cultivation due to lowered production costs and increased adaptation to adverse environmental conditions. However, it has an insufficient tolerance to the presence of aluminum ions (Al³⁺) in the soil. The number of genes controlling aluminum tolerance in triticale and their chromosomal location is not known. Two F2 mapping biparental populations (MP1 and MP15) segregating for aluminum (Al) tolerance were tested with AFLP, SSR, DArT, and specific PCR markers. Genetic mapping enabled the construction of linkage groups representing chromosomes 7R, 5R and 2B. Obtained linkage groups were common for both mapping populations and mostly included the same markers. Composite interval mapping (CIM) allowed identification of a single QTL that mapped to the 7R chromosome and explained 25 % (MP1) and 36 % (MP15) of phenotypic variation. The B1, B26 and Xscm150 markers were 0.04 cM and 0.02 cM from the maximum of the LOD function in the MP1 and MP15, respectively and were highly associated with aluminum tolerance as indicated by Kruskal–Wallis nonparametric test. Moreover, the molecular markers B1, B26, Xrems1162 and Xscm92, previously associated with the Alt4 locus that encoded an aluminum-activated malate transporter (ScALMT1) that was involved in Al tolerance in rye (Secale cereale) also mapped within QTL. Biochemical analysis of plants represented MP1 and MP15 mapping populations confirmed that the QTL located on 7R chromosome in both mapping populations is responsible for Al tolerance.     

甜瓜遗传图谱的构建及果实与种子QTL分析

以遗传性状差异较大的甜瓜材料日本安农二号与新疆哈密瓜K413杂交产生的143个F2单株为作图群体,以AFLP与SSR分子标记为主构建了包含12个连锁群、142个遗传标记位点的甜瓜遗传图谱,其中包括121个AFLP标记、16个SSR标记、3个STS标记、2个性状标记,连锁群总长度为1 014.2 cM。应用复合区间作图法对甜瓜果实的大小、长宽比、糖度、硬度以及甜瓜种子的长、宽、形状、重量等性状进行遗传定位与分析。基因定位结果显示控制果肉颜色的基因位于C9连锁群AFLP分子标记NDAA与NCFA之间。其他性状表现为数量性状控制,共检测到25个数量性状基因座,不同性状基因座位有重叠分布的特点。其中C5连锁群标记NCA-N73C区间检测到QTLs Sl5.1、Sw5.1和Swt5.1分别控制种子长、宽和千粒重,分别可解释表型变异的17%、19%和23%。该区域包含的来自母本安农二号的基因位点对甜瓜种子的长、宽、千粒重均有明显的抑制作用;位于C8连锁群标记N73A与NFDA间的QTL通过影响种子的宽度从而影响种子的形状与重量;同样位于C8连锁群的果实长宽比QTL Fs8.1在F2和F3中均检测到,分别解释表型变异的25%和19%,表现为部分显性,来自安农二号的等位基因抑制甜瓜果实伸长,生成圆形甜瓜;还发现控制甜瓜果实心糖、边糖、果实硬度的QTL各一个。     

Mapping QTLs for tissue culture response of mature wheat embryos

The mature wheat embryo is arguably one of the best explants for genetic transformation because of its unlimited availability and lack of growth season restriction. However, an efficient regeneration system using mature wheat embryos (Triticum aestivum L.) is still not available. To identify genes related to the tissue culture response (TCR) of wheat, QTLs for callus induction from mature embryos and callus regeneration were mapped using an RIL population derived from the cross of "Wangshuibai" with "Nanda2419" which has a good TCR. By whole genome scanning we identified five, four and four chromosome regions conditioning, respectively, percent embryos forming a callus (PEFC), percent calli regenerating plantlets (PCRP), and number of plantlets per regenerating callus (NPRC). The major QTLs QPefc.nau-2A and QPcrp.nau-2A were mapped to the long arm of chromosome 2A, explaining up to 22.8% and 17.6% of the respective phenotypic variance. Moreover, two major QTLs for NPRC were detected on chromosomes 2D and 5D; these together explained 51.6% of the phenotypic variance. We found that chromosomes 2A, 2D, 5A, 5B and 5D were associated via different intervals with at least two of the three TCR indexes used. Based on this study and other reports, the TCRs of different explant types of wheat may be under the control of shared or tightly linked genes, while different genes or gene combinations may govern the stages from callus induction to plantlet regeneration. The importance of group 2 and 5 chromosomes in controlling the TCRs of Triticeae crops and the likely conservation of the corresponding genes in cereals are discussed.     

Seed quality QTLs identified in a molecular map of early maturing soybean

This study identified QTLs influencing seed quality characters in a cross of two early maturing soybean (Glycine max [L.] Merr.) cultivars (Ma.Belle and Proto) adapted to the short growing seasons of Central Europe. A molecular linkage map was constructed by using 113 SSR, 6 RAPD and 1 RFLP markers segregating in 82 individuals of an F₂ population. The map consists of 23 linkage groups and corresponds wellto previously published soybean maps. Using phenotypic data of the F₂-derived lines grown in five environments, four markers for protein content, three for oil content and eight for seed weight were identified. Four from fifteen seed quality QTL-regions identified in the present study were also found by other authors. Markers associated with seed weight QTLs were consistent across all environments and proved to have effects large enough to be useful in a marker-assisted breeding program, whereas protein and oil QTLs showed environmental interactions. Received: 9 October 2000 / Accepted: 26 February 2001     

Mapping QTLs on chicken chromosome 1 for performance and carcass traits in a broiler x layer cross

With the objective of mapping quantitative trait loci (QTLs) for performance and carcass traits, an F2 chicken population was developed by crossing broiler and layer lines. A total of 2063 F2 chicks in 21 full-sib families were reared as broilers and slaughtered at 42 days of age. Seventeen performance and carcass traits were measured. Parental F(0) and F1 individuals were genotyped with 80 microsatellites from chicken chromosome 1 to select informative markers. Thirty-three informative markers were used for selective genotyping of F2 individuals with extreme phenotypes for body weight at 42 days of age (BW42). Based on the regions identified by selective genotyping, seven full-sib families (649 F2 chicks) were genotyped with 26 markers. Quantitative trait loci affecting body weight, feed intake, carcass weight, drums and thighs weight and abdominal fat weight were mapped to regions already identified in other populations. Quantitative trait loci for weights of gizzard, liver, lungs, heart and feet, as well as length of intestine, not previously described in the literature were mapped on chromosome 1. This F2 population can be used to identify novel QTLs and constitutes a new resource for studies of genes related to growth and carcass traits in poultry.     

Integration genetic linkage map construction and several potential QTLs mapping of Chinese shrimp (Fenneropenaeus chinensis) based on three types of molecular markers

In this study, totally 54 selected polymorphic SSR loci of Chinese shrimp (Fenneropenaeus chinensis), in addition with the previous linkage map of AFLP and RAPD markers, were used in consolidated linkage maps that composed of SSR, AFLP and RAPD markers of female and male construction, respectively. The female linkage map contained 236 segregating markers, which were linked in 44 linkage groups, and the genome coverage was 63.98%. The male linkage map contained 255 segregating markers, which were linked in 50 linkage groups, covering 63.40% of F. chinensis genome. There were nine economically important traits and phenotype characters of F. chinensis were involved in QTL mapping using multiple-QTL mapping strategy. Five potential QTLs associated with standard length (q-standardl-01), with cephalothorax length (q-cephal-01), with cephaloghorax width (q-cephaw-01), with the first segment length (q-firsel-01) and with anti-WSSV (q-antiWSSV-01) were detected on female LG1 and male LG44 respectively with LOD> 2.5. The QTL q-firsel-01 was at 73.603 cM of female LG1. Q-antiWSSV-01 was at 0 cM of male LG44. The variance explained of these five QTLs was from 19.7-33.5% and additive value was from -15.9175 to 7.3675. The closest markers to these QTL were all SSR, which suggested SSR marker was superior to AFLP and RAPD in the QTL mapping.     

A gene controlling sex in grapevines placed on a molecular marker-based genetic map.

Genetic maps of Vitis (2n = 38) have been constructed from an interspecific hybrid population of 58 seedlings of the cross 'Horizon' ('Seyval' x 'Schuyler') x Illinois 547-1 (V. cinerea B9 x V. rupestris B38). The maps were initially constructed based on 277 RAPD (random amplified polymorphic DNA) markers using a double-pseudotestcross strategy. Subsequently, 25 microsatellites, 4 CAPS (cleaved amplified polymorphic sequence), and 12 AFLP (amplified fragment length polymorphism) markers were added to the maps. Another 120 markers, mostly those segregating 3:1, were also assigned but not positioned on the linkage groups in the two maps. The 'Horizon' map consisted of 153 markers covering 1199 cM, with an average map distance of 7.6 cM between markers. The Illinois 547-1 map had 179 markers covering 1470 cM, with an average map distance of 8.1 cM. There were 20 linkage groups in each map, one more than the basic number of chromosomes in grapes. Ten linkage groups in each map were identified as homologous using 16 microsatellite and 2 CAPS markers polymorphic in both parents. A single locus controlling sex in grapes mapped close to a microsatellite marker. These maps provide enough coverage of the genome for QTL (quantitative trait loci) analysis and as a starting point for positional gene cloning in grapes.     

Diallel analysis of androgenetic plant production in hexaploid Triticale (X. triticosecale,Wittmack)

Summary Studies were made on the genetic determination of androgenetic plant yield and its two components: embryo production and green plant regeneration. This involved the analysis of a complete 7×7 diallel cross of 4 androgenetic lines and 3 lines obtained by pedigree selection, one of them having the Triticum timopheevi cytoplasm. The three traits analysed are both heritable and environmentally influenced (by season and culture medium composition). The analysis of embryo production shows a mainly nuclear inheritance, with predominantly additive gene action, but also a favourable effect of Triticum timopheevi cytoplasm. Green plant regeneration has a more complex genetic determination, with additive as well as non-additive gene action and cytoplasmic influences. Hybrids appear superior to inbred lines for embryogenesis and green plant yield, but not for green plant regeneration. Androgenetic lines used as parents did not show superiority over other parents either in their own value or in the transmission of androgenetic abilities. Genetic improvement seems to be possible by recombination in progenies of hybrids between lines having complementary abilities.     

Mapping QTLs for root morphological traits inBrassica rapa L. based on AFLP and RAPD markers

Root growth and thickening plays a key role in the final productivity and even the quality of storage roots in root crops. This study was conducted to identify and map quantitative trait loci (QTLs) affecting root morphological traits in Brassica rapa by using molecular markers. An F2 population was developed from a cross between Chinese cabbage (Brassica rapa ssp. chinensis) and turnip (B. rapa ssp. rapifera), which differed greatly in root characters. A genetic map covering 1837.1 cM, with 192 marker loci and 11 linkage groups, was constructed by using this F2 population. The F3 families derived from F2 plants were grown in the field and evaluated for taproot traits (thickness, length, and weight). QTL analysis via simple interval mapping detected 18 QTLs for the 3 root traits, including 7 QTLs for taproot thickness, 5 QTLs for taproot length, and 6 QTLs for taproot weight. Individually, the QTLs accounted for 8.4-27.4% of the phenotypic variation. The 2 major QTLs, qTRT4b for taproot thickness and qTRW4 for taproot weight, explained 27.4% and 24.8% of the total phenotypic variance, respectively. The QTLs for root traits, firstly detected in Brassica crops, may provide a basis for marker-assisted selection to improve productivity in root-crop breeding.     

Development of a genetic linkage map for Pinus radiata and detection of pitch canker disease resistance associated QTLs

Key message

The heritability of genetic resistance of radiata pine against Fusarium circinatum was not clear. We demonstrated that there are at least 3 QTLs that could be involved in this resistance/susceptibility.

Abstract

A genetic linkage map was developed for Pinus radiata, using Amplified Fragment Length Polymorphism (AFLP), Inter-Simple Sequence Repeat (ISSR), Selective Amplification of Microsatellite Polymorphic Loci (SAMPL), and Simple Sequence Repeat (SSR) molecular markers, based on a two-way pseudo-testcross strategy, using 86 individuals of a F1 full-sib family and 787 molecular markers for genotyping. Linkage analysis generated a map of medium to high density for each parent, with 1,060 and 1,258 cM for parents XO and XP, respectively. A total of 458 markers were mapped on 12 linkage groups (LG) in XO and XP, which equals the number of haploid chromosomes present in P. radiata. Analysis of quantitative trait loci (QTL) for resistance against pitch canker disease caused by Fusarium circinatum was made using Bayesian Information Criterion (BIC). In the XO parental map, two groups (LG-1 and LG-9) showed high probabilities for one or more QTLs. Only one group (LG-9) in the XP parental map showed probability for one or more QTLs. The results indicate that resistance to pitch canker is inherited from both parents. These results provide the basis for further studies focused on structure, evolution, and function of the P. radiata genome.     

Construction of a genetic map for Caulobacter crescentus.

RP4-mediated conjugation has been used to transfer large fragments of chromosomal material in Caulobacter crescentus. In this system, conjugation proceeds from multiple origins, and haploid recombinants are recovered at frequencies of 10(-6) and 10(-7) per donor cell. The data from five-factor crosses were subjected to computer-assisted crossover analyses as a rapid method to determine marker order. Using this information and data from additional two- and three-factor crosses mediated by RP4 or the generalized transducing bacteriophage phi Cr30, we constructed the first genetic map for C. crescentus.     

A molecular linkage map of tomato displaying chromosomal locations of resistance gene analogs based on a Lycopersicon esculentum x Lycopersicon hirsutum cross.

A molecular linkage map of tomato was constructed based on a BC1 population (N = 145) of a cross between Lycopersicon esculentum Mill. line NC84173 (maternal and recurrent parent) and Lycopersicon hirsutum Humb. and Bonpl. accession PI126445. NC84173 is an advanced breeding line that is resistant to several tomato diseases, not including early blight (EB) and late blight (LB). PI126445 is a self-incompatible accession that is resistant to many tomato diseases, including EB and LB. The map included 142 restriction fragment length polymorphism (RFLP) markers and 29 resistance gene analogs (RGAs). RGA loci were identified by PCR amplification of genomic DNA from the BC1 population, using ten pairs of degenerate oligonucleotide primers designed based on conserved leucine-rich repeat (LRR), nucleotide binding site (NBS), and serine (threonine) protein kinase (PtoKin) domains of known resistance genes (R genes). The PCR-amplified DNAs were separated by denaturing polyacrylamide gel electrophoresis (PAGE), which allowed separation of heterogeneous products and identification and mapping of individual RGA loci. The map spanned 1469 cM of the 12 tomato chromosomes with an average marker distance of 8.6 cM. The RGA loci were mapped to 9 of the 12 tomato chromosomes. Locations of some RGAs coincided with locations of several known tomato R genes or quantitative resistance loci (QRLs), including Cf-1, Cf-4, Cf-9, Cf-ECP2, rx-1, and Cm1.1 (chromosome 1); Tm-1 (chromosome 2); Asc (chrromosme 3); Pto, Fen, and Prf (chromosome 5); 01-1, Mi, Ty-1, Cm6.1, Cf-2, CF-5, Bw-5, and Bw-1 (chromosome 6); I-1, 1-3, and Ph-1 (chromosome 7); Tm-2a and Fr1 (chromosome 9); and Lv (chromosome 12). These co-localizations indicate that the RGA loci were either linked to or part of the known R genes. Furthermore, similar to that for many R gene families, several RGA loci were found in clusters, suggesting their potential evolutionary relationship with R genes. Comparisons of the present map with other molecular linkage maps of tomato, including the high density L. esculentum x Lycopersicon pennellii map, indicated that the lengths of the maps and linear order of RFLP markers were in good agreement, though certain chromosomal regions were less consistent than others in terms of the frequency of recombination. The present map provides a basis for identification and mapping of genes and QTLs for disease resistance and other desirable traits in PI126445 and other L. hirsutum accessions, and will be useful for marker-assisted selection and map-based gene cloning in tomato.     

Mapping QTLs with epistatic effects and QTL×environment interactions for plant height using a doubled haploid population in cultivated wheat

Quantitative trait loci (QTLs) for plant height in wheat (Triticum aestivum L.) were studied using a set of 168 doubled haploid (DH) lines, which were derived from the cross Huapei 3/Yumai 57. A genetic linkage map was constructed using 283 SSR and 22 EST-SSR markers. The DH population and the parents were evaluated for wheat plant height in 2005 and 2006 in Tai’an and 2006 in Suzhou. QTL analyses were performed using the software of QTLNetwork version 2.0 based on the mixed linear model. Four additive QTLs and five pairs of epistatic effects were detected, which were distributed on chromosomes 3A, 4B, 4D, 5A, 6A, 7B, and 7D. Among them, three additive QTLs and three pairs of epistatic QTLs showed QTL×environment interactions (QEs). Two major QTLs, Qph4B and Qph4D, which accounted for 14.51% and 20.22% of the phenotypic variation, were located similar to the reported locations of the dwarfing genes Rht1 and Rht2, respectively. The Qph3A-2 with additive effect was not reported in previous linkage mapping studies. The total QTL effects detected for the plant height explained 85.04% of the phenotypic variation, with additive effects 46.07%, epistatic effects 19.89%, and QEs 19.09%. The results showed that both additive effects and epistatic effects were important genetic bases of wheat plant height, which were subjected to environmental modifications, and caused dramatic changes in phenotypic effects. The information obtained in this study will be useful for manipulating the QTLs for wheat plant height by molecular marker-assisted selection (MAS).     

水稻ILP标记遗传图谱的构建

内含子长度多态性(ILP)是一种基于PCR的新型分子标记, 具有许多突出的优点。我们先前利用已公布的籼稻品种93-11和粳稻品种日本晴的基因组序列数据, 已开发了172个水稻ILP标记。为了检验这些ILP标记的可靠性及其在遗传作图中的可用性, 利用一个BC1F1(日本晴/93-11//日本晴)群体, 构建了一张含172个ILP标记座位和13个SSR标记座位的水稻遗传图谱, 总长度为1 905.7 cM。比较显示, 图谱上所有标记的顺序与其物理顺序完全一致, 证明了利用ILP标记进行遗传作图的可行性和有效性。文中还对标记偏分离现象进行了分析, 发现在第6号染色体短臂上存在一个严重偏分离的区域。     

Mapping unexplored genomes: a genetic linkage map of the Hawaiian cricket Laupala

As with many organisms of evolutionary interest, the Hawaiian cricket Laupala genome is not well characterized genetically. Mapping such an unexplored genome therefore presents challenges not often faced in model genetic organisms and not well covered in the literature. We discuss the evolutionary merits of Laupala as a model for speciation studies involving prezygotic change, our choice of marker system for detecting genetic variation, and the initial genetic expectations pertaining to the construction of any unknown genomic map in general and to the Laupala linkage map construction in particular. We used the technique of amplified fragment length polymorphism (AFLP) to develop a linkage map of Laupala. We utilized both EcoRI/MseI- and EcoRI/PstI-digested genomic DNA to generate AFLP bands and identified 309 markers that segregated among F(2) interspecific hybrid individuals. The map is composed of 231 markers distributed over 11 and 7 species-specific autosomal groups together with a number of putative X chromosome linkage groups. The integration of codominant markers enabled the identification of five homologous linkage groups corresponding to five of the seven autosomal chromosomal pairs found in Laupala.     

甘蓝型油菜种胚叶绿素含量的QTL定位

种胚叶绿素含量是影响菜子油加工和销售的重要指标。关于种胚叶绿素的遗传特性还缺乏研究。本文以重庆市油菜工程中心构建的甘蓝型黄子与黑子油菜重组自交系群体为材料,2007年分别在重庆市北碚区和万州区两个实验基地种植,利用本实验室已构建的遗传连锁图谱为基础,采用复合区间作图法(C IM)分析种胚叶绿素的QTL。结果共检测到10个QTL,分别位于第1、2、5、8、21和25连锁群,单个QTL解释表型变异的4.04%～8.50%。研究结果表明胚中叶绿素表现为多基因控制的数量性状,基因表达受环境影响较大。     

Construction of intersubspecific molecular genetic map of lentil based on ISSR, RAPD and SSR markers

Lentil (Lens culinaris ssp. culinaris), is a self-pollinating diploid (2n?=?2x?=?14), cool-season legume crop and is consumed worldwide as a rich source of protein (~24.0%), largely in vegetarian diets. Here we report development of a genetic linkage map of Lens using 114 F₂ plants derived from the intersubspecific cross between L 830 and ILWL 77. RAPD (random amplified polymorphic DNA) primers revealed more polymorphism than ISSR (intersimple sequence repeat) and SSR (simple sequence repeat) markers. The highest proportion (30.72%) of segregation distortion was observed in RAPD markers. Of the 235 markers (34 SSR, 9 ISSR and 192 RAPD) used in the mapping study, 199 (28 SSRs, 9 ISSRs and 162 RAPDs) were mapped into 11 linkage groups (LGs), varying between 17.3 and 433.8 cM and covering 3843.4 cM, with an average marker spacing of 19.3 cM. Linkage analysis revealed nine major groups with 15 or more markers each and two small LGs with two markers each, and 36 unlinked markers. The study reported assigning of 11 new SSRs on the linkage map. Of the 66 markers with aberrant segregation, 14 were unlinked and the remaining 52 were mapped. ISSR and RAPD markers were found to be useful in map construction and saturation. The current map represents maximum coverage of lentil genome and could be used for identification of QTL regions linked to agronomic traits, and for marker-assisted selection in lentil.