Recombinant Substitution Lines Research Articles

Substitutions and Deletions of Genes Related to Grain Hardness in Wheat and Their Effect on Grain Texture

ABSTRACTThe Hardness (Ha) locus on chromosome 5D is the main determinant of grain texture in hexaploid wheat (Triticum aestivum L.). Puroindoline (Pina‐D1, Pinb‐D1) and Grain Softness Protein (Gsp‐D1) genes are tightly linked at this locus. Additional copies of the Gsp‐1 gene are present on chromosomes 5A and 5B. Mutations in the Pina‐D1 and Pinb‐D1 genes have been individually associated with grain hardness, but it is not known if mutations at both loci may further increase hardness or if additional copies may reduce it. In addition, there is no clear evidence of the effect of the Gsp‐1 genes on grain texture. To answer these questions, we compared the effect of different dosages of puroindoline and Gsp‐1 genes on grain texture. Isogenic substitution and deletion lines for homoeologous group 5 in ‘Chinese Spring’ (CS) were evaluated in two replicated field trials with 13 blocks each. Deletions or allelic variants of Gsp‐A1 and Gsp‐B1 did not produce significant effects on grain texture, suggesting that these genes do not have a critical role in grain hardness. Simultaneous deletions of Pina‐D1 and Pinb‐D1 in deletion line 5DS‐2 and substitution line CS (Red Egyptian 5D) resulted in significantly higher hardness index values than all other lines including CS (Timstein 5D) carrying a single Pina‐D1 deletion (P = 0.02). The incorporation of additional copies of Pina‐Am1 and Pinb‐Am1 from T monococcum L. in recombinant substitution line 5A/5Am in CS resulted in significantly softer grains than those from the CS control (P < 0.01).

Mapping of genes regulating abiotic stress tolerance in cereals

The location of major QTLs or even genes controlling abiotic stress tolerance is now possible by the application of marker-mediated techniques. This is achieved by exploiting precise genetic stocks, such as doubled haploids (DHs), recombinant substitution lines (RSLs) and recombinant inbred lines (RILs), along with the comprehensive genetic maps now available through the application of molecular marker techniques. These strategies are illustrated here showing how QTLs/genes affecting vernalization response, cold tolerance, osmotic adjustment, osmolite accumulation (free amino acids, polyamines and carbohydrates), salt tolerance and cold-regulated protein accumulation have been identified and located. Also, an example of marker-assisted selection (MAS) for frost tolerance is presented. Major loci and QTLs affecting stress tolerance inTriticeaehave been mapped on the group 5 chromosomes, where the highest concentration of abiotic stress-related QTLs (vernalization response, frost tolerance, salt tolerance and osmolite accumulation) was located. A conserved region with a major role in osmotic adjustment has been located on the group 7 chromosomes.

Effect of Triticum monococcum glutenin loci on cookie making quality and on predictive tests for bread making quality

The effects of Triticum monococcum glutenin loci on cookie making quality and predictive tests for bread making quality were evaluated in recombinant substitution lines (RSLs) between chromosome 1Am from T. monococcum and chromosome 1A from Chinese Spring. All four combinations of high molecular weight (H Mr-GS) and low molecular weight glutenin alleles (L Mr-GS) were studied in a factorial design to evaluate their interactions. Grain protein content was used as a covariable to evaluate the effect of these loci independently of the variation in protein content among lines. No significant interactions were detected indicating an additive effect. RSLs carrying the HMr -GS from T. monococcum showed a 13·6% increase in SDS sedimentation volume (p=0·004) and a significant reduction in cookie diameter (−5·2%,p =0·02), and cookie quality (−6·8%, p=0·02). RSLs carrying the LMr -GS from T. monococcum showed a significant decrease in the proportion of polymeric protein (−2·8%, p<0·0001), SDS sedimentation volume (−8·1%,p =0·03) and gluten strength (−16·5%, p=0·01), and a significant increase in cookie quality (5·9%, p=0·05). The T. monococcum LMr. -GS allele has potential value to be used in soft wheat breeding programs. These results suggest that diploid T. monococcum could be a valuable source for new allelic variation for storage proteins loci and new quality characteristics.

More QTL for flowering time revealed by substitution lines in Brassica oleracea

Seventy-nine recombinant backcross substitution lines from a cross between Brassica oleracea var. italica and Brassica oleracea var. alboglabra were grown in field trials over five years along with the alboglabra recurrent parent. Plants were scored for the days from sowing to the opening of the first flower, and lines that flowered significantly earlier or later than the recurrent parent were identified. Based on the lengths of the substitutions, evidence for 11 QTL on chromosomes O1, O2, O3, O5 and O9 was found, five of which mapped to similar regions to five of the six found in a previous analysis of doubled haploid lines from the same cross. Several of the QTL were linked closely in repulsion.

Differential effects of the null alleles at the three Wx loci on the starch-pasting properties of wheat

The amylose/amylopectin ratio and the pasting properties of wheat starch are important in producing marketable flour products, especially Japanese noodles. To determine if null mutations at the three Wx loci confer differences in starch-pasting viscosity, we analyzed the variation associated with the null mutations in three separate sets of recombinant substitution lines of chromosomes 7A, 4A and 7D produced from crosses between Chinese Spring and three single-chromosome substitution lines carrying the null Wx alleles. Differential effects of null alleles at the three Wx loci on starch-pasting properties were revealed. With respect to chromosome 4A, the effect of the Wx-B1b allele, giving a higher peak and breakdown viscosity, was unambiguous. In addition, a QTL of minor effect was identified near the centromere on the short arm. The presence or absence of the Wx-A1 protein gave some variation in peak and breakdown viscosity, but the effects of Wx-Alb were much smaller than those of the Wx-Blb allele. Associated effects of the Wx-D1 locus were detected for the breakdown viscosity as the null Wx-D1b allele produced a higher viscosity than the wild-type Wx-D1a. While negative correlations between amylose content and breakdown viscosity were common in the three populations, the null mutations at the Wx loci produced some variation independent of amylose content. The genetic variation detected for breakdown viscosity was more evident than that for peak viscosity in all three recombinant populations.

A new powdery mildew resistance gene: Introgression from wild emmer into common wheat and RFLP-based mapping

An Israeli accession (TTD140) of wild emmer, Triticum turgidum var. dicoccoides, was found resistant to several races of powdery mildew. Inoculation of the chromosome-arm substitution lines (CASLs) of TTD140, in the background of the Israeli common wheat cultivar ‘Bethlehem’ (BL), with five isolates of powdery mildew revealed that only the line carrying the short arm of chromosome 2B of wild emmer (CASL 2BS) exhibited complete resistance to four of the five isolates. To map and tag the powdery mildew resistance gene, 41 recombinant substitution lines, derived from a cross between BL and CASL 2BS, were used to construct a linkage map at the gene region. The map, which encompasses 69.5 cM of the distal region of chromosome arm 2BS, contains six RFLP markers, a morphological marker (glaucousness inhibitor, W1I), and the powdery mildew resistance gene. Segregation ratios for resistance in F2 of BL × CASL 2BS and in the recombinant lines, combined with the susceptability of F1 progeny to all tested isolates, indicate that resistance is controlled by a single recessive allele. This alleleco-segregated with a polymorphic locus detected by the DNA marker Xwg516, 49.4 cM from the terminal marker Xcdo456. The new powdery mildew resistance gene was designated Pm26.

Identification of genetic loci affecting amylose content and agronomic traits on chromosome 4A of wheat

Chromosome 4A of wheat carries the Wx-B1 gene encoding the granule-bound starch synthase involved in amylose synthesis in the endosperm. To determine the pleiotropic effects of this locus and effects of independent QTLs on agronomic traits, genetical analysis of chromosome 4A was conducted using 98 single-chromosome recombinant substitution lines derived from a cross of Chinese Spring and Chinese Spring (Kanto107 4A) with a low amylose content due to the null Wx-B1b allele. For amylose content, most of the genetic variation was explained by the allelic difference at the Wx-B1 locus. An additional QTL of minor effect was mapped in the 6.2-cM Xbcd1738/Xcdo1387 interval on the short arm, where the allele from Kanto107 led to an increase in amylose content. Field trials over two seasons revealed a pleiotropic effect of Wx-B1, or else the effect of a closely linked QTL, on ear emergence time. A QTL linked to Wx-B1 was detected for plant height. For plant yield and its components, there was no evidence for significant main effects associated with Wx-B1 or adjacent regions. One plant-yield QTL was identified by RFLP markers on the short arm and this was identical to QTLs controlling spikelet number/ear and grain weight/ear. At these QTLs for agronomic traits, alleles from Kanto107 contributed to an earlier emergence time, a height reduction and an yield increase.

QTL mapping of genes controlling ear emergence time and plant height on chromosome 5A of wheat

Chromosome 5A of wheat carries major gene loci for agronomic traits including the vernalization requirement (Vrn-A1) and ear morphology (Q). To determine whether the genetic variation for ear emergence time and plant height is attributable to either of these major genes as pleiotropic effects or independent QTL, we combined a RFLP map constructed from 120 recombinant substitution lines derived from a cross between ‘Chinese Spring’ (Cappelle-Desprez 5A) and CS(Triticum spelta 5A) with data collected from field trials over 3 years. For ear emergence time the main effects on flowering time were by Vrn-A1 and QEet.ocs-5A.1, the latter a QTL in the 28.6-cM Xcdo584/Q interval linked to Q by less than 10 cM. The CS(T. spelta 5A) allele at QEet.ocs-5A.1 contributed to an earlier ear emergence time by 2.7–6.0 days, which was approximately equal to the effects of Vrn-A1. For plant height, three QTLs were identified on the long arm and linked in repulsion. The CS(T. spelta 5A) allele at Vrn-A1 or closely linked to Xfba068 contributed to a height reduction of 3.5–6.1 cm, whereas both the Q allele and Qt.ocs-5A.1 allele within the Xcdo1088/Xbcd9 interval from CS(Cappelle-Desprez 5A) produced a shorter plant. When plant height was partitioned into culm length and ear length, the Vrn-A1 allele and CS(Cappelle-Desprez 5A) allele at QCl.ocs-5A.1 within the Xcd1088/Xbcd9 interval were found to contribute to a shorter culm. CS(T. spelta 5A) allele at q was a major determinant of a long ear, together with minor effects at QEl.ocs-5A.1 within the Xcdo1088/Xbcd9 interval.

Control of ear emergence time by chromosome 3A of wheat

AbstractChromosome 3A of wheat is known to carry earliness per se genes. To determine the number of genes and their arm location, ear emergence time under a controlled environment was nvestigated using ditelosomic lines and homozygous recombinant substitution lines developed between chromosome 3A homologues from (Timstein) and (Chinese Spring) (CS) in a CS genetic background. Because the ear emergence distribution was discontinuous and two separate modes were produced, the 86 recombinant lines could be divided into 21 lines as the early ‘Timstein’3A type and 65 lines as the late CS type. This agrees with the 1:3 independent segregation of two genes both located on chromosome 3A. Therefore, two hypotheses can be proposed, either CS(‘Timstein’ 3A) carries two genes and both are necessary to give early ear emergence, or one gene for early ear emergence is present on (Timstein) 3A, but a suppressor is on CS 3A. The behaviour of ditelosomic 3AL and 3AS lines, with an ear emergence time identical to that of CS, suggested that one gene is located on the long arm and the other is on the short arm.

Wheat Grain Hardness Among Chromosome 5D Homozygous Recombinant Substitution Lines Using Different Methods of Measurement

ABSTRACTThe level of grain hardness of wheat (Triticum aestivum) cultivars profoundly affects milling properties and end‐use. We examined grain hardness among a genetically defined set of 83 chromosome 5D homozygous recombinant substitution lines derived from soft wheat cv. Chinese Spring and hard wheat cv. Cheyenne and compared four common methods of measuring wheat grain hardness. Measures of grain hardness included a modified particle size index, Brabender Quadrumat flour milling, near‐infrared reflectance (NIR) spectroscopy, and the single‐kernel characterization system (SKCS). Duncan's multiple range test was used to group recombinant lines according to parental classes. Quadrumat milling fractions, percent bran and middlings, were well correlated to NIR and SKCS grain hardness, whereas break flour, a traditional measure of grain hardness, was poorly correlated to other hardness measures. NIR and SKCS grain hardness measures provided the greatest and similar mean separations. Both methods identified recombinant lines as being significantly outside either parental class and significantly different from and in between the two parental classes. Between two divergent environments, correlations (r) for Quadrumat bran and middlings percents and NIR and SKCS hardness ranged from 0.83 to 0.94. Analysis of variance indicated that lines differed substantially for hardness, and hardness was highly influenced by environment, albeit consistently, as indicated by low line‐location model interaction terms. The results confirmed the presence of major allelic differences assignable to chromosome 5D and suggested the action of minor gene(s). Break flour, in particular, showed strong indications of transgressive segregation independent of the Hardness (Ha) locus. The Perten 4100 SKCS provided the best (most discriminating) measure of the material properties of the wheat endosperm manifested by the action of the Ha locus.

Triticum turgidum L. 6A and 6B recombinant substitution lines: extended linkage maps and characterization of residual background alien genetic variation

Triticum turgidum L. var ‘durum’ cv ‘Langdon’-T. t. var ‘dicoccoides’ chromosome 6A and 6B recombinant substitution lines (RSLs) and a F2 population derived from a ‘Langdon’-T. t. var ‘dicoccoides’ disomic chromosome 6A substitution lineבLangdon’ cross were analyzed with the objective of markedly increasing the number of markers assigned to and the resolution of previously constructed 6A and 6B linkage maps. Fifty-seven markers were added to the 6A RSL-population map, which now consists of 73 markers that span 111 cM, and 40 markers were added to the 6B RSL-population map, which now consists of 56 markers that span 123 cM. With the exception of 2 6B loci, all of the loci on the two RSL-population maps were ordered at a LOD score ≥3.0. Thirty-seven orthologous markers were mapped in the two chromosomes and colinearity between them is strongly indicated. The 6A RSL-population map and the F2-population map are highly similar, indicating that the former population, which consists of 66 lines, can be reliably used for mapping, as was previously demonstrated for the 6B RSL population. In the absence of selection and genetic drift, the lines in a RSL population, except at loci in the substituted/recombined chromosome, should be near-isogenic. An unexpected finding was that at least 26 and possibly 29 of the RFLPs detected in the RSL populations (18% of the markers analyzed) are not located in the substituted/recombined chromosomes. Linkage analysis of the markers disclosed that at least 19 of them are located in six or seven segments that span approximately 10 cM and 17 cM of the genetic lengths of 6B and 6A, respectively, in the 6A and 6B RSL populations, respectively, a finding that suggests that 40 or more alien segments spanning 8–15% of the genetic length of the 13 unsubstituted chromosomes are present in both of the RSL populations. Alien alleles are fixed in many RSLs for most of the markers, in most cases at a frequency consistent with theoretical expectations. Highly distorted segregation favoring the alien allele was detected for all of the markers in 2 of the segments, however. Nine of the markers were among those mapped in the substituted/recombined chromosomes; the linkage data obtained for the other 10 was sufficient to assign them to approximate map positions.

Identification of an RFLP interval containingPch2on chromosome 7AL in wheat

The gene Pch2 in 'Cappelle Desprez' is one of two genes found in hexaploid wheat known to confer resistance to eyespot disease. This study was conducted to develop an RFLP linkage map of the distal portion of wheat chromosome 7AL, and to locate and identify markers closely associated with Pch2 for use in marker-assisted selection. Ten loci in addition to Pch2 were mapped on chromosome 7AL, using segregation data from 102 homozygous chromosome 7A recombinant substitution lines derived from 'Chinese Spring' x 'Chinese Spring' ('Cappelle Desprez' 7A). The Pch2 locus was bracketed by two RFLP markers, one 11.0 cM distal to Xcdo347 and the other 18.8 cM proximal to Xwg380. The position of Pch2 on chromosome 7AL is similar to that of Pch1 on chromosome 7DL, suggesting that these resistance genes are homoeoloci. Although no single marker was closely linked to Pch2, simultaneous selection of the flanking RFLP markers Xcdo347 and Xwg380 could be used for selecting Pch2, since double recombination occurred in only 3% of the recombinant population. The use of the flanking RFLP markers to select for Pch2, in combination with previously identified Pch1-linked markers, would facilitate the development of cultivars carrying two genes for resistance to eyespot.

Linkage relations among eyespot resistance gene Pch2, endopeptidase Ep‐A1b, and RFLP marker Xpsr121 on chromosome 7A of wheat

AbstractMarker‐based selection of Ep‐D1b has been used successfully to incorporate Pch1, the gene for eyespot resistance on chromosome 7D, into commercial wheat. However, attempts to transfer resistance conferred by Pch1 (on chromosome 7A) through selection for Ep‐A1b have not always been successful. Linkage relations among eyespot resistance gene Pch2, a gene encoding for an isozyme of endopeptidase, Ep‐A1b, and RFLP marker Xpsr121 on chromosome 7A were determined using 80 homozygous recombinant substitution lines. The recombinant lines were derived from eyespot susceptible ‘Chinese Spring’ hybridized with a resistant disomic substitution line of ‘Cappelle Desprez’ that has chromosome 7A substituted into ‘Chinese Spring’. Segregations of Pch2, Ep‐A1b and Xpsr121 fit an expected 1:1 single‐locus ratios based on χ2 tests. Linkage analysis revealed that Pch2 was not tightly linked to Ep‐Alb (15% recombination). However, close linkage (3.8% recombination) existed between Ep‐A1b and Xpsr121. The order of these loci is Pch2‐Xpsr121‐Ep‐A1b. Unlike Pch1 and Ep‐D1b, where little or no recombination is found, Pch1 and Ep‐A1b showed considerable recombination and therefore linkage cannot be utilized efficiently in marker‐based selection.

Use of recombinant substitution lines in the construction of RFLP-based genetic maps of chromosomes 6A and 6B of tetraploid wheat (Triticum turgidum L.)

RFLP-based genetic maps of chromosomes 6A and 6B of Triticum turgidum have been constructed using data obtained by the study of Triticum turgidum var 'durum' cv 'Langdon'-T. t. var 'dicoccoides' recombinant substitution lines (RSLs) supplemented with data obtained from F3 families derived from 'Langdon' dicoccoides 6A and 6B disomic substitution lines. The average RFLP frequencies detected for the two chromosomes in a test of 45 DNA clones with six restriction enzymes were 56% and 53%, respectively, and a subset of 32 clones gave frequencies of 75% and 72%, respectively. Seventeen loci were mapped in 6A and 18 in 6B. With the possible exception of 5 loci in the centromeric region of 6A, all of the mapped 6A and 6B loci are located in the same arm as are homologous loci in hexaploid wheat, and the linear order of the loci is the same in the two chromosomes, except possibly close to the centromere. Major differences in genetic distances exist between homologous loci located in the proximal regions of the 6AL and 6BL linkage groups, however, the distances being much larger in the former than in the latter. The 6B maps that were constructed using data from both the RSL and the F2 populations and using data from the RSL population alone closely resemble one another, indicating that the 6B RSL population, composed of 85 lines, can be reliably used for genetic mapping. Additional studies must be conducted before the utility of the 6A RSL population, composed of 66 lines, can be adequately assessed.

Use of double-ditelosomic and normal chromosome 1D recombinant substitution lines to map Sr33 on chromosome arm 1DS in wheat

Chromosome 1D homozygous recombinant substitution lines derived from Triticum aestivum 'Chinese Spring' (cross 1) or 'Chinese Spring' double-ditelosomic 1D (cross 2) hybridized with a disomic substitution line of Triticum tauschii chromosome 1D in 'Chinese Spring' were used to investigate the linkage relationships among Glu-D1, encoding subunits of high molecular weight glutenin storage proteins; Gli-D1, encoding gliadin storage proteins; Sr33, conferring stem rust resistance; and the centromere. Based on analysis of 88 and 91 recombinant substitution lines of crosses 1 and 2, respectively, Sr33 is tightly linked to Gli-D1 on chromosome arm 1DS (5.6 and 7.6% recombination) and less tightly to the centromere (29.6%, cross 2) and to Glu-D1 (40.9 and 39.5%). The order of the loci is Glu-D1 – centromere – Sr33 – Gli-D1.Key words: stem rust resistance, seed storage protein, glutenin, gliadin, Triticum aestivum, Triticum tauschii.

Linkage relations of Gli-D1, Rg2, and Lr21 on the short arm of chromosome 1D in wheat

Homozygous recombinant substitution lines, derived from a cross of Triticum aestivum 'Chinese Spring' with a disomic substitution line of Triticum tauschii chromosome 1D in 'Chinese Spring', were used to investigate the linkage relationships among the loci Glu-D1, encoding high molecular weight glutenin storage proteins, Gli-D1, encoding gliadin storage proteins, Rg2, controlling glume color, and Lr21, conferring leaf-rust resistance. Gli-D1, on chromosome arm 1DS, is tightly linked to Rg2 and Lr21 (1.4 and 5.6% recombination, respectively). The order of the loci is Gli-D1–Rg2–Lr21. Glu-D1, on chromosome arm 1DL, segregates independently (P = 0.43) of Gli-D1, Rg2, and Lr21. The position of Glu-D1, Gli-D1, Rg2, and Lr21 in the genetic linkage map of chromosome 1D agrees with the position of storage protein, glume color, and rust-resistance loci on chromosomes 1A and 1B.Key words: leaf-rust resistance, seed storage protein, glutenin, gliadin, glume color, Triticum aestivum, Triticum tauschii.