Plants were harvested 45 days after germination when the differences in root characters could be efficiently measured among different recombinant lines. The data for different shoot characters were recorded and the tubes containing roots were stored at 4°C until processing. The roots were washed and recovered without damage using a Xotation technique . The shoot characters measured were longest leaf length , maximum width of the longest leaf , leaf area , plant height , number of tillers , and dry shoot biomass . The root characters measured were number of roots greater than 30 cm , longest root length , total length of roots greater than 30 cm , shallow root weight , deep root weight , dry root biomass , and root biomass to shoot biomass ratio .The shoot and root data were subjected to the analysis of variance for each year .The overall rooting ability of each genotype was calculated by ranking each genotype for individual root traits in each replication in each of the 3 years. Genotypes with the highest values were ranked 5 and those with the lowest values were ranked 1. Subsequently, all the ranks of root characters for each genotype were summed providing a measure of the rooting ability index for each genotype at different replications . The genotypic rank sums averaged across the years were subjected to the non-parametric Quade analysis developed for randomized complete block designs to differentiate genotypes for overall rooting ability.Phenotyping of recombinants was necessary to test the general applicability of the consensus 1RS-1BS map in locating a 1RS region showing better rooting ability. Various shoot and root traits were studied using two parents and three recombinants covering the whole 1RS-1BS map. There were significant differences among years for all shoot characters measured, except for the maximum width of the longest leaf .
Significant differences were found among the genotypes for shoot characters, except for maximum width of the longest leaf and leaf area. Genotype £ year interaction was significant only for the number of tillers per plant . This interaction was due to changes in the magnitude of the genotypic means across different years rather than changes in ranking of the means. Therefore, shoot characters in Table 2 are represented by means averaged across years. Pavon 1RS.1BL was taller,growing tomatoes hydroponically had longer leaf length and a greater root-to-shoot ratio than Pavon 76. Since Pavon 1RS.1BL and Pavon 76 had similar shoot biomass , greater root-to-shoot biomass ratio in the former genotype indicated greater root biomass in 1RS.1BL than Pavon 76 . Leaf area in 1B + 2 was the highest followed by Pavon 1RS.1BL . Despite significant differences observed among the genotypes for shoot characters, the differences were relatively small, except for the shoot biomass in 1B + 2 in the third year which was due to greater number of tillers per plant and plant height . Otherwise, the rest of the genotypes did not show large differences for combined as well as for individual years for most of the shoot traits. There were significant differences among years for all root characters measured . Significant differences were found among the genotypes for all the root characters measured, except for longest root length. The genotype £ year interaction was significant only for the number of roots greater than 30 cm. Therefore, means for root characters were averaged across 3 years . The number of roots greater than 30 cm and root biomass in Pavon 1RS.1BL was greater than those in Pavon 76, which confirmed the results reported earlier . The number of roots greater than 30 cm in Pavon 1RS.1BL, 1B + 2 and 1B + 38 was similar, but greater than those in Pavon 76 and T-14 . A similar trend was observed for the total length of roots greater than 30 cm. Shallow root weight was highest in Pavon 1RS.1BL followed by 1B + 2 , and 1B + 38 . The lowest shallow root weight belonged to T-14 . Deep root weight in Pavon 1RS.1BL, 1B + 2, and 1B + 38 were similar, but greater than those in Pavon 76 and T-14.
The greater dry root biomass observed in Pavon 1RS.1BL as compared to Pavon 76 was because of a combination of greater shallow and deep root weight in the former than in the latter genotype .Genetic mapping in wide hybrids has been performed for many plant species, particularly in diploids including barley , chickpea , lentils , onion , Nicotiana species , and tomato . In the early days of genetic mapping with molecular markers, wide hybrids were the approach of choice, for it guaranteed much higher levels of polymorphism than in intra-specific hybrids. Despite notable instances of non-Mendelian segregation and skewed distribution of recombination, wide hybrids produced useful genetic maps with higher marker saturation at considerably less cost and eVort . The use of wide hybrids in allopolyploids is more complicated than in diploids as allopolyploids tend to have some kind of chromosome pairing control system in place that limits crossing over to homologues. Hence, homoeologous pairing may be low or even non-existent. In this study, recombinants produced by crossing over were used. These recombinants were selected from a population of 103 such recombinants developed by Lukaszewski . The entire recombinant population was selected from a population of ca. 17,000 progeny with the Ph1 system disabled. If the assumption is made that crossing over in the Ph1+ and Ph1¡ wheats is the same, and they appear to be, except for the absence of multiple crossovers per arm , then the sample analyzed here would be equivalent to a population of 136 back cross progeny, a sensible number giving the maximum resolution level of 0.7 cM. The physical distribution of 68 recombinant breakpoints used in the present study is shown in Fig. 3. With a total of 20 physical and molecular markers, we constructed the combined genetic map of 1RS-1BS recombinant breakpoints in Pavon 76 background. The genetic map produced here has an average density of 2.5 cM. The maps shown in Figs. 2 and 4 represent only the physical 40% of the distal ends of the 1S arms as no recombination took place in the proximal 60% of the arm. Any loci in this region would show complete linkage with the centromere.
The advantage of using these recombinant breakpoints in developing a genetic map is in their physical differences from one another. Here, we used two methods to map. Firstly, we used the recombinant breakpoints to develop a genetic map of the arm, and secondly, we used the map to classify the breakpoints. Each interval or genetic distance between two markers is represented by one or more recombinant breakpoints that most often include both reciprocal configurations of the chromosomes. This physical separation of the breakpoints further refines the genetic map to 0.7 cM level. The reciprocal nature of chromosomes with breakpoints in any given interval will permit allocation of identified genetic loci to very narrow physical segments of rye chromatin. This may be a useful tool in dissecting the genetic components of a particular gene of interest or an important agronomic trait present on 1RS. Somers et al. detected an average of one single nucleotide polymorphism per 540 bp ESTs in wheat and demonstrated the reliability of designing PCR primers for locus-specific amplicon production. In this study, we targeted a single chromosome arm, 1BS,hydroponic growing supplies and expected that most of the 91 EST loci allocated to this arm could be converted to 1BS locus-specific amplicon markers. Given that the recombination was intergeneric, we expected that most of these markers would be polymorphic between wheat and rye, and so would produce a highly saturated map. However, only four PCR-based markers showed reliable polymorphism, though we cannot rule out sequence-based polymorphisms that we could not detect. The sequences of the 91 EST loci on wheat 1S arms were used to search rice orthologs using TIGR rice version 4.0 gene models, and 54 of these sequences had rice blastx hits of e-20 or better, comprising 52 rice gene models. Of the 52 models, were clustered on chromosome 5 of rice, which is known to besyntenic to chromosomes 1S of Triticeae . In the present study, two gene models with genebank accessions viz; BE497177 and BF483588 were found to be represented as markers, Xucr_4 and Xucr_8, respectively . The rice annotation of Xucr_8 is described as a drought induced 19 protein which can be helpful in studying drought. However, a high frequency of insertions and deletions in rice and maize make these genomes more Xuid at the DNA sequence level than indicated by Southern analyses . Testing a total of 16 SSR and eSSR markers yielded four polymorphic amplified products, which were comparable to the previous studies . Röder et al. found different band sizes for cultivar and synthetic wheat, though the differences were comparable. Only 1 out of 9 eSSR markers could be mapped on 1BS-1RS. This low success rate may be due to low polymorphism between wheat and rye 1S arms. Whether this indicates high conservation of genic regions across species we cannot tell at this point. Peng and Lapitan showed amplification of 15 wheat eSSR markers in rye as well as barley. They detected polymorphism between wheat and barley but did not mention polymorphism between wheat and rye. A comparison of the current 1RS- 1BS map with the SSR and eSSR maps of the 1B chromosome shows a good agreement in marker order, location, and relative positions in its distal part, regardless of the projection mode used. A similar approach was used to generate a genetic map of ph1b-induced 2R-2B intergeneric recombinants, with a similar success , validating the wide-hybrid approach to mapping. The integration of physical and molecular markers in the present 1RS-1BS map also provided the alignment of recombinant breakpoints over each map interval.
This information offers great potential to study agronomic traits affected by the introduction of alien 1RS chromatin into wheat. Our working hypothesis was if a 1B + line shows some specific trait then this trait should be absent in its complementary T-line, and vice versa. To check the applicability of this concept, we looked at different root characters in wheat. Studies during the past few years showed an increased root biomass in wheat with the alien 1RS chromosome arm over standard spring wheat Pavon 76 and found a positive correlation of increased root biomass with grain yield . Recent studies in rice and maize have also correlated the QTLs for root traits with QTLs for yield under Weld conditions . A major limitation to study roots is the difficulty in making observations as the process is very laborious and time consuming. In the present study, we tested only three recombinant chromosomes with breakpoints selected to divide the recombining portion of the arm into three segments of roughly similar lengths. The experiments were conducted in 3 years at the same months of the year, in replicated trials, to determine the magnitude of the genotype £ year interaction and the repeatability of results from the sand-tube technique. Significant variation was observed from year to year , with considerably higher means in the third year. This might have been due to relatively lower temperatures in the third year providing better conditions for vegetative growth. The significant genotype £ year interaction observed for number of roots greater than 30 cm was due to only one genotype, T-14, producing more roots only in year 2. Otherwise, other genotypes showed similar trends for this character across the 3 years. The lack of signiWcant genotype £ year interaction for most of the root characters examined indicated high repeatability for these root traits. Quade analysis used on rank sums separated the five genotypes in two groups viz., Pavon 1RS.1BL, 1B + 2 and 1B + 38 containing distal rye chromatin with higher rooting ability and Pavon 76 and T-14 lacking the distal rye segment with lower rooting ability . Overall, the wave genotypes examined showed relatively small variation for shoot characters but they differed in root characteristics including root biomass.