Segregation distortion in doubled haploid lines of barley (Hordeum vulgare L.) detected by simple sequence repeat (SSR) markers

A total of 147 simple sequence repeat (SSR) markers (including86 barley and 61 wheat microsatellite markers) were tested for their segregation in a doubled haploid (DH) and an F₂ population of barley. The DH population consisted of 71 doubled haploid lines, developed from F₁ plants of a cross between Tadmor and WI2291using isolated microspore culture technique. A genetic linkage map consisting of 43 microsatellite markers was constructed using the DH population. Particularly on chromosome 4H microsatellite markers showed distorted segregation ratios. Segregation of DH lines based on molecular markers were compared with segregation of 92 F₂ lines from the same cross. The proportion of loci deviating from the expected monogenic segregation ratios in the DH population was significantly higher (19/43loci, 44%) than in the F₂ population (7/43 loci, 16%). The deviation was biased towards the WI2291 parent alleles. In line with this observation, WI2291 was found to perform better than Tadmor in regenerating green plantlets with the isolated microspore-culture technique. This revised version was published online in July 2006 with corrections to the Cover Date.     

Two genes and linked RAPD markers involved in resistance to Fusarium oxysporum f. sp. Ciceris race 0 in chickpea

The inheritance of resistance to fusarium wilt race 0 of chickpea and linked random amplified polymorphic DNA (RAPD) markers were studied in two F₆:₇ recombinant inbred line (RIL) populations. These RILs were developed from the crosses CA2156 × JG62 (susceptible × resistant) and CA2139 × JG62 (resistant × resistant), and were sown in a field infected with fusarium wilt race 0 in Beja (Tunisia) over 2 years. A1:1 resistant to susceptible ratio was found in the RIL population from the CA2156 × JG62 cross, indicating that a single gene with two alleles controlled resistance. In the second RIL population (CA2139 × JG62) a 3:1 resistant to susceptible ratio indicated that two genes were present and that either gene was sufficient to confer resistance. Linkage analysis showed a RAPD marker, OPJ20600, linked to resistance in both RIL populations, which is present in the resistant parent JG62.     

Distorted Segregation for Mildew Resistance in Doubled Haploid Lines of Spring Barley

The mildew reactions of the second generation of doubled haploid (DH) plants, derived from anther culture of crosses among three spring barley lines carrying different Mla mildew resistance alleles and the cv. ‘Pallas’, were analyzed by using a set of three European and one Israeli mildew isolate. The results indicated, (1) a significant level of distortion segregation in favour of resistant DH genotypes, which was possibly due to linkage of mildew resistance genes on chromosome 5 with genes for plant regeneration and (2) various degrees of dominance for the different resistance genes studied as well as the possible action of modifier genes.     

节瓜抗镰刀菌酸突变体的筛选和特性研究

枯萎病是节瓜的主要病害之一。为了创新节瓜抗枯萎病资源,利用枯萎病毒素镰刀菌酸（FA）为胁迫剂,以节瓜不定芽为筛选材料,进行了节瓜抗镰刀菌酸变异体离体筛选研究。试验结果表明,FA胁迫的适宜浓度为60~80mg•L-1。筛选出的抗性细胞系继代培养后仍保持对FA抗性,其再生植株的R1、R3代株系对节瓜枯萎病的抗性可以达到中抗至抗病级;与供体材料相比,再生系后代株系除枯萎病抗性增强外,其他主要农艺性状没有明显变化。表明利用抗FA细胞系离体筛选技术改良和创新节瓜抗枯萎病材料是可行的。     

Segregation of genetic markers among wheat doubled haploid lines derived from wheat x maize crosses

Summary Wheat doubled haploid (DH) lines were produced from the F₁ hybrid, Fukudo-komugi x Oligo Culm, through intergeneric crosses between wheat and maize. F₂ plants and 203 DH lines were analyzed for the segregation of the eight genetic markers, namely, grain proteins, grain esterases, GA-insensitivity and glume traits. The segregation in the F₂ plants fitted to the expected ratios. No deviation was observed among the DH lines, either, except for the glume pubescence. The result indicates the absence of correlation between the markers investigated and the efficiency of embryo formation in the DH lines.     

Segregation of 12 isozyme genes among doubled haploid lines derived from a japonica x indica cross of rice (Oryza sativa L.)

Summary The segregation of 12 heterozygous isozyme markers was analyzed among F₂ plants and 51 anther culture (AC)-derived lines obtained from the japonica × indica cross of rice, IRAT 177 × Apura. All the lines except two were homozygous products of recombination of the two parental phenotypes. Doubled haploid (DH) lines derived from plants regenerated from the same callus were identical, confirming previously obtained results in rice. Surprisingly, some lines derived from different calli were also identical, suggesting a phenomenon of early callus fragmentation. All these observations at the isozyme level were confirmed by field evaluation. Deviations of segregations from the expected 1 : 1 ratio were observed at 4 loci among the DH lines. Among these, two were also noted among the F₂ plants. The two other distortions, both in favor of the japonica allele, were observed specifically in the AC-derived materials.Although this concerns a small proportion of the genes under study, it suggests that the embryogenic microsporal population does not represent a random gametic array. On the other hand, evaluation of recombination between isozyme genes located on chromosome 6 appears consistent with F₂ data and data previously recorded on the other japonica × indica crosses. The potential use of isozymes in breeding doubled haploids derived from remote crosses in rice is discussed.Abbreviations MCPA = 2-methyl-4-chlorophenoxyacetic acid - IAA = indolacetic acid - AC plant or line = anther culture-derived plant or line - DH line = doubled haploid line     

Detecting and estimating segregation distortion and linkage between glufosinate tolerance and blackleg resistance in Brassica napus L.

Summary The segregation and linkage between glufosinate (transgenes ‘Rf3’ and ‘T177’) and blackleg resistance genes in canola (Brassica napus L.) were assessed using F₁ microspore-derived doubled haploid (DH) populations from four crosses including reciprocals, two involving the transgene ‘Rf3’ and the other two involving the transgene ‘T177’. To relax the assumption of no segregation distortion required for the conventional analysis of segregation and linkage, we employed Bailey's analysis that allows detecting segregation distortion at linked loci. The significant departures from the 1:1 segregation were detected in the crosses involving the transgene ‘T177’ but not in the crosses involving the transgene ‘Rf3’. The apparent deficit of the herbicide tolerant DH lines in the crosses with the transgene ‘T177’ is likely due to differential selection against the gametes carrying ‘T177’ during microspore culture. The linkage was strong between blackleg resistance and the transgene ‘Rf3’ but weak or absent between blackleg resistance and the transgene ‘T177’, suggesting that the two transgenes are probably inserted into distant regions of the genome. The observed linkage offers an opportunity to develop new canola cultivars with both glufosinate tolerance conferred by transgene ‘Rf3’ and blackleg resistance.     

Inheritance of resistance to Fusarium wilt in Indian lentil germplasm (Lens culinaris Medik.)

Summary Three lentil genotypes resistant to Fusarium oxysporum f.sp. lentis viz. Pant L 234, JL 446 and LP 286 were crossed with two susceptible ones. The hybrid plants were all resistant in the eight crosses evaluated. Segregation pattern for wilt reaction in F₂, BC(P₁), BC(P₂) and F₃ generations in field and glasshouse conditions indicated that resistance to Fusarium wilt is under the control of two dominant duplicate genes in Pant L 234 and two independent dominant genes with complementary effects in JL 446 and LP 286. A third dominant gene complementary to the dominant genes in JL 446 and LP 286 is present in two susceptible lines. Allelic tests suggest the presence of five independently segregating genes for resistance. Duplicate dominant genes in Pant L 234 are non-allelic to two dominant genes with complementary effects in LP 286 and JL 446 and the third gene complementary to the two genes in JL 446 and LP 286 in susceptible lines JL 641 and L 9–12. Gene symbols among parental genotypes have been designated.     

RAPD markers linked to a clubroot-resistance locus in Brassica rapa L.

Linkage of random amplified polymorphic DNA (RAPD) markers with resistance genes to clubroot (Plasmodiophora brassicae Wor.) in Brassica rapa L. was studied in a doubled haploid (DH population obtained by microspore culture. Thirty-six DH lines were obtained from F₁ plants from a cross between susceptible ‘Homei P09’ and resistant ‘Siloga S2’ plants. ‘Homei P09’ was a DH line obtained by microspore culture of the Chinese cabbage variety ‘Homei’, which is highly responsive in microspore culture. The resistant line ‘Siloga S2’ was obtained by two rounds of selfing of the fodder turnip ‘Siloga’. Three RAPD markers, RA12-75A, WE22B and WE49B, were found to be linked to a clubroot-resistance locus. These three markers were linked in the DH lines and an F₂ population and should be useful for marker-assisted selection in breeding programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

Segregation for isozymes and HMW glutenins in a doubled-haploid cross of winter wheat carrying 1BL.1RS and 5DL.5RS translocations from rye

The doubled haploid (DH) wheat line ‘dh 5841’ carrying two translocations from rye, 5DL.5RS and 1BL.1RS, has been crossed to the subline of wheat cultivar ‘Amadeus 7143’ with a 1BL.1RS translocation. The resulting F₁ hybrid IJ 98 with a heterozygous 5DL.5DS‐5DL.5RS chromosome pair has been used to produce doubled haploids. A total of 57 DH lines were obtained from plantlets regenerated in anther culture after successful colchicine treatment and seed set. These lines were identified regarding the constitution of chromosome 5D (5DL.5DS or 5DL.5RS) by means of isoenzyme marker analysis. Thirty DH lines possessed the 5DL.5DS chromosome, while the remaining 27 lines carried the 5DL.5RS translocation. For some of these lines, the 5DL.5RS chromosome was cytologically confirmed by C‐banding. Furthermore, the DH lines were evaluated for their high molecular weight glutenin subunit composition. All possible combinations for the four independent loci —Skdh, Glu‐Al, Glu‐B1 and Glu‐D1— were detected in only 57 DH lines and no segregation distortion was observed.     

Expression of dormancy in a spring wheat cross grown in field and controlled environment conditions

Pre-harvest sprouting resistance in white-seeded wheat, Triticum aestivum L. is a genetically complex trait that varies with environmental conditions. Such variation causes difficulty in phenotypic characterization of populations to study inheritance or develop suitable DNA markers. To minimize random environmental effects, we evaluated controlled environments to measure dormancy. A population of 380 doubled haploid lines, AC Karma/SC8021V2, was evaluated in the glasshouse where the developing grains would not be exposed to moisture and greater consistency in temperature could be achieved. AC Karma is sprouting susceptible and SC8021-V2 is sprouting resistant. The population plus eight checks were seeded in early spring so the plants would reach physiological maturity under long days, requiring less supplemental light, and when the external temperature would be low enough that it would not cause difficulty in cooling the glasshouse. An alpha-lattice in a randomized complete block design with three replications was used. The blocks were arranged to minimize the environmental sources of variability in the glasshouse within each block. A sub-set of this population was tested in six field environments. Dormancy was characterized by germination of seed harvested near physiological maturity, from which a germination resistance index was calculated. The dormancy expressed in the glasshouse was significantly correlated with five of six field environments and highly significant in two of these. There was significant bidirectional transgressive segregation in both field and glasshouse environments. We are currently repeating this glasshouse experiment to confirm the results.     

甘蓝型油菜DH系在不同生态区SPAD值的差异分析

以甘蓝型油菜纯合双单倍体(DH)为材料,研究不同生态环境下油菜叶绿素SPAD值的变化状况,揭示叶绿素含量遗传表现规律,创制更高叶绿素含量种质资源,以期为油菜高光效育种奠定方法和理论基础。选取了170份甘蓝型油菜DH系,连续3年分别种植在冬油菜区(陕西大荔)和春油菜区(甘肃张掖)。结果表明:无论是不同遗传背景的DH系、还是同一亲本下的不同DH系材料,油菜DH系叶绿素含量在田间既表现出遗传稳定性,又容易受到外界环境影响,即在相似的生态条件下,同一区域即便不同年份的各DH系材料,叶绿素SPAD值的表现基本一致,但在不同生态区,油菜DH系的SPAD值差异很大。通过对油菜DH系亲本和DH系叶绿素SPAD值频数分布分析,叶绿素含量连续3年在2个生态环境具有广泛的连续分布,并且服从正态或者近似于正态分布,以及超亲分离的特点。这些都表明油菜叶绿素含量是一个典型的数量性状,受到多对基因的控制。     

The detection of QTLs controlling bacterial wilt resistance in tobacco (N. tabacum L.)

The bacterial tobacco wilt caused by Ralstonia solanacearum is one of the most destructive soil-borne diseases worldwide. One strategy to improve the resistance to bacterial wilt is to make use of plant varieties expressing wilt resistance genes. To characterize the genetics of wilt resistance and to identify relevant molecular markers for use in plant breeding, quantitative trait loci (QTLs) affecting tobacco bacterial wilt resistance were mapped in the F_2:3 and F_2:4 progeny produced from two crosses between the wilt-resistant breeding lines Enshu and Yanyan97 and the susceptible cultivar TI448A. A linkage map containing 118 loci in 24 linkage groups was constructed for 236 lines from the Enshu×TI448A cross, and a linkage map containing 96 loci in 24 linkage groups was constructed for 264 lines from the Yanyan97×TI448A cross. The wilt resistance of the progeny was examined in field trials conducted in Xuancheng, China, in 2010, 2011, and 2012. The disease severity was assessed on stems using separate rating scales. Mapmaker/EXP 3.0 and Mapmaker/QTL 1.1 were used to identify the qBWR-3a, qBWR-3b, qBWR-5a and qBWR-5b QTLs in linkage group 3 and 5; these four loci were strongly associated with resistance and explain 9.00, 19.70, 17.30, and 17.40 % of the variance in resistance, respectively. The close linkage of the markers PT20275 and PT30229 was detected in both the TI448A×Enshu and TI448A×Yanyan97 crosses, and this linkage group could be used to select individual resistant plants. These findings suggest that one strategy to combat bacterial wilt could be to exploit the resistance genes of the Enshu and Yanyan97 strains.     

海岛棉枯萎病抗性与类黄酮代谢相关基因表达量的相关

枯萎病是危害海岛棉生产的重要因素之一, 研究枯萎病抗性分子机制将为培育抗病海岛棉品种、解决枯萎病对海岛棉的危害问题提供坚实的基础。本研究在前期转录组测序的基础上, 对海岛棉枯萎病抗性差异表达基因进行分析(DEG, Differentially Expressed Gene); 以7个抗病性表现不同的海岛棉品种为材料, 利用qRT-PCR的方法研究抗病差异表达基因在不同接菌时间点的表达量差异, 并分析基因表达量与病情指数的相关性。结果表明, DEG分析得出类黄酮代谢通路相关基因与海岛棉枯萎病抗性有关。qRT-PCR分析显示抗病材料中类黄酮代谢通路关键基因的表达量显著高于感病材料。在接菌后多个时间点, 类黄酮代谢通路中的关键基因TT7、CHI和DFR在抗病材料中的表达量显著或极显著高于感病材料, 其中CHI和DFR基因的表达量与病情指数呈显著负相关。综上所述, 类黄酮代谢通路相关基因对海岛棉枯萎病抗性均有影响, 且CHI、TT7和DFR基因是关键基因。     

Morphological and chemical characteristics of doubled haploids of flue-cured tobacco combining resistance to Thielaviopsis basicola and TSWV

Thielaviopsis basicola and Tomato spotted wilt virus (TSWV) are the most important problems in a moderate climate zone. Previously obtained doubled haploids (DH) of F₁ hybrids of the flue-cured line WGL3 resistant to Th. basicola and the dark-cured line PW-834 carrying RTSW-al gene provided the research material. Biological tests and SCAR markers linked with TSWV were applied to confirm resistance of DH. Lines combining resistance to TSWV and Th. basicola were evaluated for morphological and chemical characteristics. Most of DH were significantly shorter than parents but two lines, 31/A/2 and 31/B/3, were close to the flue-cured WGL3. Usually DH possessed fewer leaves while one of them 31/B/3, exceeded parental forms. The doubled haploids flowered later than their parents. The most negative effect was reduced area of mid-position leaves of DH. It might be explained by a recombination during microsporogenesis in F₁, however the influence of ‘Polalta’-derived RTSW-al gene cannot be excluded. Extensive line to line variation for nicotine and sugars content was not associated with the genes for TSWV and Th. basicola resistance. Biological tests and field performance of DH revealed potential to overcome the negative effect of coupling between the RSTV-al gene and genes responsible for the morphological deformations.     

Phenotypic distribution of barley SSD lines and doubled haploids derived from F1 and F2 hybrids

Summary The doubled haploid (DH) system and the single seed descent (SSD) technique are frequently applied in breeding of self-pollinated crops to rapidly obtain homozygous lines from heterozygous hybrids. This study presents a comparison of populations of barley DH and SSD lines in terms of traits of stem structure. The SSD and DH lines derived from F₁ and F₂ hybrids Roland x Apex were examined in a field experiment. On the basis of a comparison of means, variances and correlations between traits in the F1DH, F2DH and SSD populations the occurrence/absence of linkage between genes responsible for the analysed traits was inferred. Independent inheritance was found for 1000-grain weight and the length of particular internodes, spike length and stem wall thickness. Moreover, no linkage was found for stem wall thickness and spike length, length of internodes I, II and thickness of stem walls, stem diameter and thickness of stem walls. The results obtained for the other pairs of traits indicate the presence of linkage.     

棉花抗病核不育两用系的培育及应用研究

１９７８ 年开始选用含棉花核雄性不育基因ｍ ｓ１４的两用系４７３Ａ、抗枯萎病品种陕棉９ 号及抗枯黄萎病品种中棉所１２, 通过杂交转育、病圃与非病圃、可育株与不育株的双重交叉选择, 成功地培育出抗枯萎耐黄萎和抗枯萎抗黄萎的核不育两用系抗Ａ１ 和抗Ａ２。并对其抗性遗传、抗性与产量的配合力及其利用情况进行了研究, 以抗Ａ１ 和抗Ａ２ 作母本已选配成３ 个杂交种应用于生产。     

Mapping resistance genes conferring tolerance to RWA (Diuraphis noxia) in barley (Hordeum vulgare)

The Russian wheat aphid (RWA) is one of the most aggressive pests of barley and wheat. The outbreak of RWA occurred in Argentina in 2008 caused serious damage to barley cultivars. The most effective and sustainable method of RWA control is to identify new resistance genes. The purpose of the current research was to map RWA resistance genes in a set of double haploid (DH) lines of the Oregon-Wolfe Barley (OWB) mapping population derived from the cross between OWB_DOM and OWB_REC. The DH and both parental lines were screened for antixenosis, tolerance and antibiosis to RWA. There was significant variation among the DH lines in most of the traits studied. However, only tolerance resulted in significant quantitative trait loci (QTLs) associated with the molecular markers. Two main QTLs were identified. These explained 90 and 79 % of the variability of foliar area and chlorophyll content, respectively, of infested and control plants. The initial and final foliar area and the variation in foliar area were associated with the same molecular markers on chromosome 2H (BmAc0125, Vrs1, BmAc0144f and BmAg0113e). The positive alleles were provided by OWB_DOM. The content of chlorophyll was associated with the marker loci WMC1E8, MWG912, ABC261, MWG2028 and Blp on chromosome 1H, with the positive alleles provided by OWB_REC. Both parents contributed to different tolerance traits, with foliar area and chlorophyll content remaining as the plant traits most affected by aphid feeding. The QTLs found in this population are new RWA resistance loci. A sequence homology search was performed to derive the putative function of the genes linked to the QTLs.     

Mapping and candidate gene identification of loci determining tolerance to greenbug (Schizaphis graminum, Rondani) in barley

Greenbug is one of the most aggressive pests of barley and wheat. In Argentina, yield losses of wheat, barley, oat and sorghum crops caused by greenbug are chronic and at times severe. Since Marker Assisted selection for greenbug resistance genes in barley is very limited, the purpose of the current study was to map greenbug resistance genes in doubled haploid (DH) lines and to identify candidate genes. A set of DH lines of the Oregon-Wolfe Barley (OWB) mapping population derived from the cross between OWB_DOM and OWB_REC and both parental lines were screened for tolerance to greenbug. There was significant variation among the DH lines in foliar area (FA), dry weight (DW) and chlorophyll contents (Ch) between infested and control DH lines. Three main QTLs were identified. These QTLs explained 82 % of the FA, 80 % of DW and 58 % of Ch variability of infested plants. The initial and final FA and DW of controls and final DW of infested plants were associated with the same molecular markers on chromosome 2H (Vrs1, BmAc0144f, GBR259, GBS705). The final FA of infested plants was significantly linked to molecular markers on chromosome 5H (GBRO986, GBR518, GBM1483, GBR1082). The positive alleles were provided by OWB_DOM. The content of chlorophyll of infested plants was associated with the marker loci Ris44, GBR1608, GBR1637N and GBS0785 on chromosome 7H, with the positive alleles provided by OWB_REC. Both parents contributed to different tolerance traits. The QTLs found in this population are new greenbug resistance loci. A sequence homology search was performed to derive the putative function of the genes linked to the QTLs.     

甘蓝枯萎病抗性基因FOC1序列分析及抗性相关变异位点分析

旨在发掘甘蓝抗枯萎病基因FOC1中与抗性相关的特异性单核苷酸多态性(SNP),为进一步开发FOC1特异分子标记提供理论依据。在对11份甘蓝自交系材料枯萎病抗病表型鉴定基础上,通过基因测序,对每份材料中FOC1等位基因及相应的氨基酸序列变异进行分析。结果表明,FOC1等位基因序列之间共存在92处SNP变异和2处Indel变异,其中包含C381A/G等18个在抗、感材料中表现出特异性差异的SNPs。此18个特异的SNP中转换型比率为86.11%、颠换型比率为13.89%,4种碱基变异率以T/C、G/A最高,分别占47.22%和38.89%,而C/G和A/C变异率共占13.89%。本研究也发现抗、感材料中存在4个特异的SNP,可造成3个氨基酸的变化。