Bulk pollen pollination in maize for efficient construction of introgression populations with high genome coverage

Introgression populations consist of a set of introgression lines or families, constructed by continuous backcrossing to the recurrent parent, while carrying a limited number of chromosome segments from a donor parent in their genomes. Increasing the genome coverage is an important aim when constructing introgression population. In this study, we proposed bulk pollen pollination (BPP) method and used it to increase the genome coverage of a maize introgression population. The results showed that the genome coverage of the introgression population constructed using BPP method reached 100% at BC₃ generation, which accorded with the simulation result. The BPP‐based BC₃F_1:2 population could identify most quantitative trait loci (QTL) detected using the F_2:3 population, especially major QTL. Simulation analysis showed that the genome coverage of introgression population increased with the increase of population size and the number of bulked plants, and decreased with the increase of backcross generation. Our results proved the reliability of the BPP‐based introgression population in increasing genome coverage and detecting QTL, and provided references for constructing high‐coverage introgression populations.     

Genetic dissection of seed storability using two different populations with a same parent rice cultivar N22

Seed storability in rice (Oryza sativa L.) is an important agronomic trait. Two segregating populations with N22 (indica) as a common parent, viz. a set of 122 backcross-inbred lines (BILs) derived from the backcross Nanjing35 (japonica)/N22//Nanjing35 and another population comprising 189 recombinant inbred lines (RILs) from the cross of USSR5 (japonica) and N22, were studied to detect quantitative trait loci (QTL) controlling seed storability. Germination percentage (GP) was used to evaluate seed storability after aging treated under three different conditions, viz. natural, artificial and combined aging treatments. A total of seven QTLs were identified on chromosomes 1, 2, 5, 6 and 9. Among them, a major QTL, qSSn-9, was common in the two populations. In contrast, four QTLs (qSSnj-2-1, qSSn-2-2, qSSn-5 and qSSn-6) were detected in BILs and the QTL qSSn-1 was identified in RILs, which was a new QTL for seed storability. The N22-derived alleles increased the seed storability at all the loci except qSSnj-2-1. We also investigated the effect of QTLs using five selected lines with high storability from BILs and verified qSSn-5 with a near-isogenic line (NIL). These results provide an opportunity for pyramiding or map-based cloning major QTLs for seed storability in rice.     

Heritability of early blight resistance in a Lycopersicon esculentum×Lycopersicon hirsutum cross estimated by correlation between parent and progeny

Sixteen‐hundred BC₁ plants of a cross between an early blight (EB) susceptible tomato (Lycopersicon esculentum Mill.) breeding line (‘NC84173’ maternal and recurrent parent) and a resistant accession (‘PI126445’) of the tomato wild species Lycopersicon hirsutum Humb. and Bonpl. were grown in a field in 1998. This population was segregating (among other traits) for growth habit, self‐incompatibility and earliness in maturity. To eliminate confounding effects of these factors on disease evaluation and h² estimation, plants that were self‐incompatible, indeterminate and/or late‐maturing were eliminated. The remaining plants (146), which were self‐compatible and determinate (sp./sp.) in growth habit, with early‐ to mid‐season maturity, were evaluated for EB resistance and self‐pollinated to produce BC₁S₁ seed. The 146 BC₁S₁ progeny families, consisting of 30 plants per family, were grown in a replicated field trial in 1999 and evaluated for EB resistance and plant maturity. For each of the 146 BC₁ plants and corresponding BC₁ families, the area under the disease progress curve (AUDPC) and final disease severity (final percentage defoliation) were determined and used to measure disease resistance. The distributions of the AUDPC and final percentage defoliation values in the BC₁ and BC₁S₁ generations indicated that resistance from ‘PI126445’ was quantitative in nature. Estimates of h² for EB resistance, computed by correlation between BC₁S₁ progeny family means and BC₁ individual plant values, ranged from 0.69 to 0.70, indicating that EB resistance of ‘P1126445’ was heritable. Across BC₁S₁ families, a small, but significant, negative correlation (r = ‐0.26, P < 0.01) was observed between disease resistance and earliness in maturity. However, several BC₁S₁ families were identified with considerable EB resistance and reasonably early maturity. These families should be useful for the development of commercially acceptable EB‐resistant tomato lines.     

Evaluation of maternal parent and puroindoline allele on kernel texture in a reciprocal cross between two hard spring wheat cultivars

Kernel texture is an important characteristic for both the milling and the end-use quality of wheat (Triticum aestivum L.). Gene sequence variation and mutations to the two puroindoline genes (Pina and Pinb), located at the Ha locus on chromosome 5DS, account for the majority of variation in wheat kernel texture. Other factors also influence kernel texture, including effects associated with different maternal parent backgrounds. To investigate the effect of two hard puroindoline alleles in different maternal backgrounds, a population of 228 recombinant inbred lines (RILs) derived from a reciprocal cross between two wheat cultivars ID377s (Pina-D1b/Pinb-D1a) and Klasic (Pina-D1a/Pinb-D1b) were examined in two succeeding generations (F₇ & F₈). Kernel texture was determined using the Single Kernel Characterization System (SKCS) and the RIL puroindoline haplotype was identified by the sequence-specific PCR amplification of each gene. Analysis of variance identified a significant (P 0.001) effect of the maternal parent and puroindoline mutation on kernel texture. RILs containing the Pina-D1b mutation were significantly harder than lines containing the Pinb-D1b mutation. RILs which had Klasic as the maternal parent were significantly harder than those which had ID377s as the maternal parent. When the maternal parent and puroindoline allele were analyzed in combination, RILs derived from Klasic as the maternal parent and the Pina-D1b allele were significantly harder (P 0.001) than those containing the same allele but ID377s as the maternal parent. The same occurred for RILs containing the Pinb-D1b allele, lines with Klasic as the maternal parent were harder than lines with ID377s as the maternal parent. These results corroborate the harder phenotype of the Pina-D1b allele and indicate a significant maternally-inherited contribution to kernel texture variation.     

Unique meiotic behaviourin F1 plants from a cross between a non-tuberous and a tuberous Solanum species in section Petota

Uniformly abnormal meiotic behaviour was observed in 12 F₁-plants from a cross between Solanum etuberosum (non-tuberous) and S. pinnatisectum (tuberous). Per pollen mother cell at MI an average was found of 3.64 bivalents (all rodshaped, 1–2 per cell, heteromorphic), 16.64 univalents (scattered haphazardly on a continuous bipolar spindle) and 0.03 trivalents (all Y-shaped). Lagging chromosomes and precocious division of univalents very frequently occurs, leading to unequal distribution of chromosomes, aneuploid gametes and male sterility. Heteromorphic bivalents at MI, loops in bivalents at pachytene and non-disjunction in one hybrid plant, point to a highly abnormal meiotic behaviour. The occurrence of few trivalents is discussed.Considering that according to the literature nearly normal pairing was observed in the intergeneric F₁-hybrids Lycopersicon esculentum × Solanum lycopersicoides and L. esculentum x S. pennellii, the lack of chromosome pairing in an interspecific F₁-hybrid, of which both Solanum parents belong to the same section, is paradoxical to a plant breeder and might even be conspicuous to a taxonomist.     

Heritability and predicted gain from selection in components of crop duration in divergent chickpea cross populations

Genetic analysis of ten quantitative traits related to crop duration in chickpea was carried out using three F ₂ sib-populations; 272-2 × CDC Anna, 298T-9 × CDC Anna and 298T-9 × CDC Frontier. F ₃ and F ₄ families from these populations were further evaluated for traits found important in the initial study. Also, 112 recombinant inbred lines (RILs) of chickpea cross ICCV 2 × JG 62 were evaluated for days to flowering, days to maturity and reproductive period. An analysis of the F ₂ population data using the mixed model approach revealed that the additive component of variance was significant for days to flowering, days to first podding and days to first pod maturity, while dominance genetic variance was significant for morphological components of crop duration such as height to first pod and height at flowering. Comparatively high heritability estimates (39–48%) were obtained for days to flowering, days to first pod maturity, percent pod maturity at four months after planting and days to maturity based on offspring-parent (F ₄ and F ₃ generations) regression and/or analysis of variance for the RIL population. The predicted gain from selection as a percentage of the population mean was low (5% or less) for these key components of crop duration owing to the low variability detected within the populations, the exception being percent pod maturity. To maximize gain from selection in these traits, it is therefore, essential to increase genetic variability among the progenies, potentially through multi-parent crosses that may involve gene introgression from across desi and kabuli types of chickpea and from wild progenitors.     

Recurrent selection of cocoa populations in Côte d'Ivoire: comparative genetic diversity between the first and second cycles

In Côte d'Ivoire, the cocoa breeding programme has been based on the creation of hybrids between different genetic groups. From 1990 onward, a reciprocal recurrent selection programme has been set up with the purpose of improving simultaneously the characteristics of the two main genetic groups: Upper Amazon Forastero (UA) and a mixture of Lower Amazon Forastero (LA) and Trinitario (T). Based on data obtained from 12 microsatellite primers, the genetic diversity and genetic distances of the parental populations used in the first and second selection cycles are presented. The results revealed that the diversity of populations UA0 and UA1 on the one hand and (LA+T)0 and (LA+T)1 on the other is similar. The genetic distances were small between the parental populations used for the first and second cycles. Genetic diversity was greater in the UA group than in the LA+T group. The number of rare and of private alleles was reduced for both genetic groups, as well as the number of the frequent alleles in the LA+T group.     

An evaluation of F2 populations from a cross between Cucurbita pepo L. and C. moschata Dutch. for resistance to cucumber mosaic virus

Summary Several varieties of Cucurbita moschata were found resistant to cucumber mosaic virus (CMV). Inter-specific hybridization between C. pepo and a CMV resistant variety of C. moschata resulted in partial fertile hybrids which set F₂ seeds. Cotyledonary inoculation of F₂ seedlings with CMV brought various results, depending on seasonal differences and developmental stages at which observations were made. Resistance to CMV generally declined under conditions of low temperature, short days and low light intensity.     

Recombination of gliadin genes of chromosome 1D in the common wheat hybrid carrying the introgression from Aegilops cylindrica

Recombination of co‐gliadin components of the allele ‘_/’ at the Gli‐D1 locus and the allele at the respective locus from Aegilops cylindrica was revealed in the common wheat hybrid carrying the introgression from Ae. cylindrica. Gliadins of single seeds from F₂ plants of the cross between the winter common wheat variety ‘Albatros Odesskii’ and the common wheat line carrying the introgression from Ae. cylindrica were analyzed by acid polyacrylamide gel electrophor‐esis. The average frequency of recombination detected between the gliadin components was 0.35%.     

不同生境Bt分离株晶体含量与靶标昆虫生物活性关系分析

通过醋酸钠-抗生素筛选法从6个不同生境中筛选得到了苏云金芽孢杆菌新菌株W14、ZY1、3346、TMQ2、B31和P91,镜检可观察到它们的伴孢晶体呈小菱形和不规则形状。SDS-PAGE分析结果表明6个菌株表达的晶体蛋白分子量为130 kDa,对其杀虫晶体蛋白的测定,结果发现这些菌株晶体蛋白含量均不高,仅为0.20%-0.29%,而B31则无晶体含量。同时对这6个Bt菌株进行了生物测定,发现所有菌株对棉铃虫杀虫活性均较低。由此可见,特殊生境获得的Bt菌株与常规菌株晶体含量不同,且晶体含量过低是其杀虫活性不高的根本原因。     

Sexual hybridisation in crosses of cultivated Brassica species with the crucifers Erucastrum gallicum and Raphanus raphanistrum: Potential for gene introgression

Studies were conducted to investigate the crossability of the cultivated Brassica species, Brassica napus (oilseed rape), B. rapa (turnip rape), and B. juncea (brown and oriental mustard), with two related cruciferous weeds that are abundant in certain regions of Canada, Erucastrum gallicum (dog mustard) and Raphanus raphanistrum ssp. raphanistrum (wild radish). Seed was produced without recourse to embryo rescue from all reciprocal crosses except R. raphanistrum × B. juncea. Four hybrid plants were recovered, namely B. napus × E. gallicum, B. napus × R. raphanistrum (two plants), and B. rapa × E. gallicum. The hybrids were characterized by their morphology, RAPD analysis, and cytological examination. The B. rapa × E. gallicum hybrid was extremely vigourous and fertile, and would likely grow in natural habitats. This hybrid produced self-seed and backcrossed readily with the B. rapa parent and, to a lesser extent, with the E. gallicum parent. The B. napus × E. gallicum hybrid was a weak plant, but produced fertile backcross progeny with the E. gallicum parent. The B. napus × R. raphanistrum hybrids were vigourous but mostly sterile. Because of their low vigour and/or sterility, hybrids produced from crosses of B. napus with the cruciferous weeds would not likely be an environmental concern. However, the potential of the B. napus × E. gallicum and B. rapa × E. gallicum hybrids to backcross with E. gallicum may be of concern. This revised version was published online in July 2006 with corrections to the Cover Date.     

Qualitative and quantitative evaluation of cassava bacterial blight resistance in F1 progeny of a cross between elite cassava clones

Deployment of resistant varieties is one major approach to controlling cassava bacterial blight (CBB), caused by Xanthomonas axonopodis pv. manihotis (Xam). To understand the genetic determinism of resistance to CBB, the use of reliable parameters measuring resistance is necessary. In order to test a relevant method for evaluation of quantitative resistance for mapping QTL (quantitative trait loci), the response of 150 F₁ individuals, inoculated with four different Xam strains (CIO-84, CIO-1, CIO-136 and CIO-295), was assessed under controlled conditions. We used two types of evaluations at different intervals after inoculation, one based on a scale of 0 to 5 and the second based on the determination of the bacterial population in the vascular system. Both evaluation types revealed interaction between strains and F₁ genotypes. Population values at 3 and 6 cm from the point of inoculation showed a high level of correlation. By performing an association analysis, at 7 and 15 days after inoculation, a significant positive correlation between both evaluation types was obtained. However, the disease rating at 30 days did not correlate with bacterial populations at either 7 or 15 days after inoculation, except for one strain, CIO-84. Evaluation of the bacterial population in stem tissues is time and labour consuming, consequently, for a rapid and reliable assessment of CBB resistance for QTL analysis, we strongly recommend evaluation based on the use of a symptom scale.     

The integration and insertion site of GbVe1 gene in the genome of transgenic cotton (Gossypium hirsutum)

[Objective] The aim of this study was to obtain the flanking sequences of T-DNA in the transgenic cotton containing a GbVe1 over-expression cassette. [Method] The T-DNA insertion copy number in the transgenic GbVe1 cotton was analyzed by southern blot. Flanking sequences of the transgenic lines with putative single T-DNA insertion copy were obtained using high-efficiency Thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR). The T-DNA insertion sites were further confirmed by PCR with specific primers. [Result] RB-flanking sequences (119-1 018 bp) and LB-flanking sequences (243-516 bp) were obtained from three transgenic lines with low copy number of T-DNA insertion. The AT content was more than 63% in these flanking sequences. A same single insertion site in the intron of Gohir.D01G157600.1 was found in the two transgenic lines 7/100826-152 and 12/100826-393, while two separated insertion sites, one also in the intron of Gohir.-D01G157600.1 and the other in the intergenic region of A12 chromosome, were found in the transgenic line 1/w-ch14. A deletion of 21 bp was found in the insertion site in the intron of Gohir.D01G157600.1. The T-DNA insertion in the intron of Gohir.D01G157600.1 was further confirmed by the specific PCR. [Conclusion] The flanking sequences of T-DNA in the transgenic GbVe1 cotton were obtained and the specific transformation event in the intron of Gohir.D01G157600.1 was further confirmed by PCR.     

Successful backcrosses of somatic hybrids between Sinapis alba and Brassica oleracea with the Brassica oleracea parent

Somatic hybrids between Sinapis alba (2n= 24) and Brassica oleracea (2n= 18) have been backcrossed with the B. oleracea parent. Whereas backcrosses with the diploid B. oleracea parent were unsuccessful, 344 BC₁ seeds could be obtained from inter-valence crosses with tetraploid B. oleracea (2n= 4x= 36). The investigated 96 BC₁ plants segregated for morphological traits and for fertility. They were backcrossed with diploid B. oleracea or self-pollinated, depending on their male fertility. The BC₁F₂ and BC₂ progenies segregated well for the morphological traits. Disturbances were observed especially in the generative phase (flower development and pollen fertility). Both male fertile and male sterile BC₁F₂ and BC₂ plants were obtained and backcrossed or self-pollinated with the B. oleracea parent. The presence of either one of the parental or the cybrid organelle genomes was detected. In the progenies, a stable maternal inheritance of the organelle genome patterns was observed. Isozyme analyses revealed polymorphism for the leucine aminopeptidase (LAP) which was used for the identification of S. alba genes in the progenies. Cytological investigations showed a clear differentiation between the BC₁F₂ and BC₂ plants. Whereas the BC₁F₂ plants possess large numbers of chromosomes ranging from 34 to 40, the BC₂ material was strongly reduced to chromosome numbers ranging from 20 to 22. Preliminary investigation of the meiosis suggests the possibility of introgressions of S. alba-DNA into the B. oleracea genome.     

The inheritance of the naked grain character in oats studied in a cross between the naked variety caesar and the husked variety BO 1/11

Summary The inheritance of the naked grain character in oats was studied in a cross between BO 1/11 (husked) and Caesar (naked), by analysing plants of F₁, F₂, F₃ and both (F₁ × BO 1/11) and (F₁ × Caesar) backcross generations. F₁ and F₂ plants were grown under both glasshouse and field conditions.Results suggest that the naked/husked character is basically governed by one major gene and modifying genes mask the effect of the main gene in the homozygous naked and heterozygous classes.Expression of the naked grain character was higher under glasshouse than under field conditions, indicating the large influence which environment can exert on the expression of this character.     

Quality variations in transgenic rice with a synthetic cry1Ab gene from Bacillus thuringiensis

In order to estimate the potential of transgenic rice, characteristics related to grain quality and starch viscosity were investigated in six japonica lines based on three primary transgenic lines containing a synthetic cry1Ab gene from Bacillus thuringiensis. No significant differences were found between the transgenic lines and the wild type, including negative lines and an untransformed line. All six transgenic lines were comparable in size, milling quality, appearance quality and physicochemical properties to the wild type that were derived from. One exception was that the lines derived from the primary transgenic line TR0‐101 had smaller grains. Crude protein contents were equivalent in all the material tested, but Cry1Ab protein was only detected in grains of transgenic rice and was undetectable in the cooked rice. The viscosity of the starch differed between the transgenic lines, the wild type and other controls, and two transgenic lines had breakdown values (BDV) and setback values (SBV) similar to the wild type. A positional effect of T‐DNA insertion on starch viscosity was found in three primary transgenic lines.     

Segregation patterns of AFLP markers in F1 hybrids of a cross between tetraploid and diploid species in the genus Malus

Malus xiaojinensis, one of the most important wild genotypes in the genus Malus, is resistant to a variety of stresses such as Fe deficiency chlorosis, drought and cold. However, lack of knowledge of its genetic background prevents using genetic analysis to study those agronomic traits and corresponding gene functions. Here, as the first step towards construction of the linkage map of M. xiaojinensis, genetic analysis of the F1 triploid hybrids (M. xiaojinensis × M. baccata) was performed with amplified fragment length polymorphism (AFLP) markers. Using 15 EcoRI‐ MseI primer combinations, 1110 AFLPs were identified, with 31.3% of M. xiaojinensis‐, 12.7% of M. baccata‐specific markers, 54.9% of common markers, and 1.2% of non‐parental markers; 93.3% of the AFLP markers exhibit the expected segregation ratio. Thirty‐two M. xiaojinensis‐specific markers and 47 common markers display a 5 : 1 and 11:1 segregation ratios, respectively, suggesting that M. xiaojinensis is an autotetraploid, or at least an isosyndetic allotetraploid.