The Journal of Heredity 2001:92(3)
© 2001 The American Genetic Association 92:254-259
Production of Near-Isogenic Lines and Marked Monosomic Lines in Common Wheat (Triticum aestivum) cv. Chinese Spring
From the Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama 244-0813, Japan.
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Abstract |
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Sixteen near-isogenic lines (NILs) carrying a marker gene were produced by the recurrent backcrossing method in the genetic background of common wheat (Triticum aestivum) cv. Chinese Spring (CS). Three genes from alien species showed segregation distortion. In NILs carrying a marker gene of rye (Secale cereale) or Aegilops caudata, the alien chromosome segments were detected by fluorescence in situ hybridization (FISH). The NILs were grown with replications and the effect of marker genes on plant morphology in the genetic background of CS was investigated. These NILs were further crossed with the corresponding monosomics of CS and 13 monosomic lines whose monosome carries a respective marker gene were established and named "marked monosomics." Many of the marked monosomics were distinguishable from the disomic NILs because of the different dosage effect of the genes. The NILs are utilized for studies on gene isolation or gene regulation. Marked monosomics are useful not only for monosomic analysis but also for production of homologous chromosome substitution lines because chromosome observation is not required.
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Introduction |
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Near-isogenic lines (NILs) are important genetic stocks for investigating the function and regulation of single genes. These are also useful for isolating genes (Kojima et al. 1998). In common wheat (Triticum aestivum L.), several NILs have been produced (see genetic catalogue by McIntosh et al. 1997); for example, Tsunewaki and others produced a series of NILs using cultivar S-615 as their common genetic background (Tsunewaki 1998; Tsunewaki and Ebana 1999; Tsunewaki and Koba 1979), and Koval (1997) used cv. Novosibirskaya-67 as the recipient for his NILs.
I report herein 16 NILs produced in the genetic background of the cultivar Chinese Spring (CS). The introduced genes manifest obvious morphologic change even in the hexaploid genetic background of common wheat. Indeed, some of the phenotypes are key characters for wheat taxonomy. The NILs were developed to elucidate the effects of these genes on the development and physiology of wheat. Since CS is regarded as the standard genetic background in wheat genetics after Sears (1954) first produced a monosomic series in this cultivar, a detailed description of gene function in this genetic background is valuable. I crossed the NILs to the monosomic lines of CS and produced novel monosomic lines whose monosome carries a marker gene. These lines, called "marked monosomics," allow isolation of monosomic plants without microscopic observation. I discuss the usefulness of these lines in the genetic analysis of common wheat.
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Materials and Methods |
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Production of NILs
NILs were produced by the recurrent backcross method using common wheat (Triticum aestivum L.) cv. Chinese Spring (CS) as the recurrent parent. The original sources of the genes are listed in Table 1. The gene nomenclature follows the rules for gene symbols in wheat (McIntosh et al. 1998). In the Hg and q loci, the alleles from T. dicoccoides Körn. (2n = 4x = 28, genome AABB) were tentatively designated here as Hgb and qb to distinguish them from the other sources. The gene for black glume was designated as Bg-C1 because it was derived from the C genome of Aegilops caudata L. (2n = 2x = 14, CC). The suppressor genes for glaucousness, Iw1 and Iw2, were recently renamed from WI1 and WI2, respectively, because their loci were found to be different from the genes for glaucousness, W1 and W2 (Tsunewaki and Ebana 1999).
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To produce nine NILs of C, Hg, Rht-B1c, Rht-D1c, Iw1, Iw2, Hp1, Hp2, and Hp3, the NILs of S-615 were used as the secondary sources for backcrosses with CS (Tsunewaki 1998; Tsunewaki and Ebana 1999; Tsunewaki and Koba 1979). The genes for hairy neck—Hp1, Hp2, and Hp3—originated from a single gene located on the chromosome 5R of rye (Secale cereale L.) (2n = 2x = 14, RR; Driscoll and Sears 1965; Sears 1967). For Bg-C1, common wheat strain P168 (Kihara 1951), a derivative of a hybrid between common wheat and Ae. caudata, was used, where chromosome 1D is substituted by chromosome 1C of Ae. caudata (Muramatsu 1959).
CS carries a dominant awn inhibitor and modifier, B2 and Hd, on the long arm of chromosome 6B and the short arm of chromosome 4A, respectively (Sears 1954). To accurately introduce each recessive allele from an awned cultivar, Norin 26 (hdhd b2b2), to CS, the pollen of Norin 26 was first crossed to the monosomics 4A and 6B of CS. The hybrids with a short awn, that were expected to be monosomics, were crossed with CS, and the F1s were self-pollinated to obtain recessive homozygotes in F2. Because hd and b2 are completely recessive, self-pollination was carried out after every backcross to obtain recessive homozygotes. The sphaerococcum gene (s1) is also recessive. However, after several backcrosses, its heterozygotes could be distinguished from the dominant homozygotes.
In addition to these lines with a single marker gene, a line with C, Hg, and Hp1 was produced as a multiple marker line of CS. For this line, a multiple marker line of S-615 carrying C, Hg, Hp1, and B1 (Tsunewaki K, unpublished data) was used as the secondary parent. Because CS is an awnless wheat by B2 and Hd, B1 for awn inhibition in the multiple marker line of S-615 could not be introduced to the genetic background of CS.
For dominant genes, the B8F1 or B9F1 plants were self-pollinated and the homozygotes for the genes were selected in the F3 in which no recessive homozygotes segregated. For recessive genes, the homozygotes were selected in B7F2 or B8F2 and used in the experiment (Table 2).
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Field Experiment
All of the NILs except that of qb were grown in nursery boxes in autumn 1997 and transplanted to the experimental field of Kihara Institute for Biological Research in winter with four replications. Each replication included three plants, each 20 cm apart in a row. Two plants were randomly selected and the following characters were measured: plant height from ground base to ear neck (PH; cm), length of the first (peduncle), second, and third internodes from spike (1I, 2I, and 3I; cm), culm diameter 10 cm below the spike neck (CD; mm), flag leaf length and width (FL and FW; mm), spike length from the neck to the tip (SL; mm), number of spikelets per spike (NS), number of grains per spike (NG), 100 grain weight (GW; g), awn length at the middle of the spike (AL; mm), and heading date when spike neck emerged from leaf sheath counting from May 1 (HD).
Production of Marked Monosomic Lines
All of the NILs except Hgb and qb were crossed with their appropriate monosomics. F1 monosomics were selected by chromosome observation using the ordinary acetocarmine-squash method. These plants were self-pollinated and the relation between chromosome numbers and morphology were examined.
Detection of Alien Chromosome Segments by Fluorescence in Situ Hybridization
The alien chromosomal fragments in the NILs of Bg-C1, Hp1, Hp2, Hp3, and marked monosomic lines were detected by fluorescence in situ hybridization (FISH) probed with genomic DNAs of Ae. caudata (Bg-C1) and rye (Hp genes). The method of FISH was described in Mukai (1996). The hybridization solution included sonicated CS DNA as a blocker to mask the homologous sequences between common wheat and the alien species.
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Results |
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Segregation of Genes
In the backcross progeny, all of the dominant marker genes segregated as expected in the ratio of 1:1 (Table 2). However, in selfed progeny, three genes—Hgb, Bg-C1, and Hp2—that derived from alien species showed significant segregation distortion. In Hgb and Bg-C1, all plants in selfed progeny carried the dominant genes, indicating that the pollen carrying the marker genes were preferentially transmitted to the next generation. On the other hand, in Hp2 the noncarrier appeared more than the carriers, and the segregation ratio fit to a 1:1 ratio, suggesting that the gene was not transferred by the pollen.
Effect of Marker Genes on Morphology
All of the marker genes clearly demonstrated their phenotype in the genetic background of CS (Figures 1 and 2). In addition, the genes also affected other characters (Table 3).
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Gene C shortened the spike (Figure 2A) and thus decreased the spikelet number. However, it increased the number of grains per spike because the number of grains per spikelet increased from 2.6 to 3.7. This gene did not affect plant height, indicating that the length of rachis and culm are controlled by independent genetic systems.
The genes Hg and Hgb made the glume hairy (Figures 1B,C and 2B). They did not show a clear pleiotropic effect except for a slight reduction in the number of spikelets in Hg and culm diameter in Hgb.
Gene q elongated spikes (Figure 1D), increased the number of spikelets, and reduced the culm diameter. The function of qb was similar to that of q (Figure 1E). The glume morphology was similar to spelt wheat.
The effect of Rht-B1c and Rht-D1c on the morphologic characters was very large (Figure 2C,D). They shortened the first, second, and third internode lengths and, as a result, reduced the plant height. The action of Rht-D1c was stronger than that of Rht-B1c. These genes also made shorter and wider flag leaves. Spikes were, however, rather longer than those of the normal line (Figure 1F,G). The flowers opened before the spikes fully emerged, reflecting the delay of heading.
The s1 gene also had a large effect on the morphology (Figure 2E). This gene shortened plant height and spike length, and increased the number of spikelets per spike. There are significantly fewer grains per spike because of the reduction in the grain numbers per spikelet. The seeds were small and round. The lines without awn suppressors, Hd and B2, showed a short awn (Figure 2F,G) and a slight increase in spike length and number of grains per spikelet.
Both Iw1 and Iw2 suppressed glaucousness of leaves, culms, and spikes, and its expression in nodes was the most marked (Figure 2H). When dried the spikes of both NILs became more yellowish (Figure 1H,I). One of the genes, Iw1, did not show any remarkable effect on morphology, whereas another gene, Iw2, elongated the spike and increased the number of spikelets.
Bg-C1 produced black pigmentation in spikes and upper culms in the maturing stage (Figure 1J). No pleiotropic effect of this gene was exhibited.
All three NILs of Hp showed hairs in the spike neck and rachis (Figures 1K–M and 2I). The pleiotropic effect of hairy neck genes was different among the lines. Hp1 decreased plant height and the size of the flag leaf and spikes, while Hp2 decreased plant height and spike length, and Hp3 increased spike length and the number of spikelets. Although multiple marker lines carry C, Hg1, and Hp1, the pleiotropic effect was less than that observed in C and Hp1 (Figure 1N).
Hemizygous Effect of the Marker Genes and Production of Marked Monosomics
In the process of backcrossing to produce NILs, heterozygotes of marker genes in B5F1–B7F1 were crossed as males to the appropriate monosomics of CS to produce marked monosomics (Table 4). In the F1, monosomic plants were selected by chromosome and the morphology observed. In most F1s it was easy to distinguish monosomics with or without the markers (Figure 1). However, it was impossible in the lines of q, b2, and Hp2. Monosomic 5A of CS shows a spelt-like (speltoid) character produced by the decrease of the dosage of gene Q (Sears 1954), and therefore, in this condition, the effect of q on spike morphology was not remarkable. In Hp2, no monosomic plants with Hp character appeared, confirming the above result that Hp2 was hard to transfer from pollen. In b2, none of the monosomics showed the expected half awn, and thus its progeny test was carried out as described later.
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Monosomics carrying the marker gene were self-pollinated, and segregation of homozygote (disomics), hemizygote (monosomics), and noncarrier (nullisomics) was observed (Table 4). The hemizygote of gene C could be easily distinguished from the homozygote by their spike morphology (Figure 2A). The hemizygote showed less compact spikes than the homozygote. Likewise, hemizygotes of genes Hg, Rht-B1c, Rht-D1c, s1, Hp1, and Hp3 could be distinguished from their homozygotes by weaker expression of the marked morphology (Figure 2B–E,I): the hair on the glume or neck of hemizygotes for Hg, Hp1, and Hp3 was obviously shorter and less than their homozygotes (Figure 2B,I). The hemizygotes for Rht-B1c and Rht-D1c were taller than their homozygotes (Figure 2C,D). The spike of hemizygotes for s1 was similar to that of the heterozygotes (Figure 2E). On the other hand, all of the selfed plants of marked monosomic 4A carrying hd were half-awned, and it was difficult but not impossible to distinguish the monosomic hemizygote and disomic homozygote by the awn length (Figure 2F).
The genetic behavior of b2 was complicated. As mentioned above, all of the five monosomic F1s were awnless. Thus the selfed seeds were individually harvested and segregation of awn expression was observed in each population. Two populations showed only awnless plants, indicating that chromosome 6B carried B2. On the other hand, three populations showed segregation of plants with awns (8 plants) and without awns (20 plants). These results indicate that the hemizygote for b2 is awnless though its homozygote is awned in the genetic background of CS. The monosomic F1 of the cross between monosomic 6B of CS and Norin 26 (awned cultivar) was awned, by which I could correctly capture b2. This means that in the genetic background of this F1, the hemizygous state of b2 exhibited awn, whereas in the genetic background of CS, the hemizygotes were awnless. Thus it was found that monosomics and disomics could be obviously separated by the awn expression in the selfed progeny of the marked monosomic 6B line with b2 (Figure 2G).
In Iw1 and Iw2, neither monosomics nor disomics were glaucous, and they were difficult to distinguish by this character (Figure 2H).
Detection of Alien Chromosome Segment in NILs
Of the 16 genes used in this experiment, four were derived from different genomes, that is, Bg-C1 from the C genome of Ae. caudata and Hp1, Hp2, and Hp3 from the R genome of rye (S. cereale). Genomic in situ hybridization using Ae. caudata or rye was carried out to determine the size of alien chromosome segments (Figure 3).
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It was found that the NIL of Bg-C1 carried a translocation chromosome (Figure 3A). Most of the chromosome was derived from the C genome of Ae. caudata. The breakpoint was located on the long arm at about one-third the distance from the centromere. The short arm carried a satellite as described by Muramatsu (1959). Since the same translocated chromosome was also observed in strain P168, the secondary source of Bg-C1, it is not in the backcrossing procedure that the translocation took place (Figure 3A). According to the nomenclature system of chromosome aberration in wheat (Gill et al. 1991a), this translocated chromosome is designated as T1CS.1CL-1DL.
Chromosome 4B of NIL with Hp1 showed a small rye segment at the tip of the long arm and is designated T4BS.4BL-5RL (Figure 3B). This segment was not remarkable by C-banding. The short arm of chromosome 5B of NIL with Hp2 was completely replaced by a rye chromosome segment. This translocated chromosome is designated as T5RL.5BL (Figure 3C). Because the rye segment was shorter than the normal short arm of chromosome 5B, the translocated chromosome of this NIL was more heterobrachial than the normal [iw2]chromosome 5B. The NIL with Hp3 showed a small rye chromosome segment at the tip of the short arm of chromosome 6D, and thus it is designated as T5RL-6DS.6DL (Figure 3D). However, this chromosome could not be detected by C-banding.
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Discussion |
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Genetic Behavior of Marker Genes
Although most genes investigated here had a strong effect on plant morphology, all except Hgb, Bg-C1, and Hp2 segregated normally in the backcrossed and selfed progeny. This indicated that they did not affect the function of the gametophyte. All of the genes that segregated abnormally were those derived from alien species.
The Hp2 gene was originally located on the long arm of chromosome 5R of rye, and the chromosome region including the Hp gene is homologous to the long arm of group 4 chromosomes of Triticeae species (Korzun et al. 1996). However, in the NIL the rye chromosome segment carrying Hp2 replaced the short arm of chromosome 5B (Figure 3C). Consequently the rye chromosome segment could not compensate for the gene function on the short arm of the chromosome 5B. Likewise the rye segments in the NILs of Hp1 and Hp3 do not seem to compensate for the function of the lost segments of chromosome 4B and 6D. However, they were transmitted normally from the male, probably because the translocated segments are smaller than those in the NIL of Hp2.
The NIL of Bg-C1 carried a translocation chromosome between chromosome 1D of wheat and chromosome 1C of Ae. caudata. The translocation breakpoint was located in the interstitial region of the long arm (Figure 3A). Since the secondary source of this gene, P168, spontaneously appeared in the progeny of a hybrid between common wheat and Ae. caudata (Kihara 1951), the translocation probably resulted from a homologous chromosome recombination. Kihara and Tsunewaki (1967) have already reported the preferential transmission of this chromosome. A superior gene(s) in pollen competition must be located somewhere on the alien segment. Ae. caudata and tetraploid Aegilops species carrying the C genome possess gametocidal genes that are transmitted preferentially to the next generation from both the male and female (Endo 1990). However, the gene of the present NIL is not gametocidal because it did not function in the female. In addition, the homologous group (group 1) is different from the groups known to carry gametocidal genes (groups 2, 3, and 4, Tsujimoto 1995.
Pleiotropic Effect of Marker Genes
Before discussing the pleiotropic effect of marker genes it is necessary to consider the degree of heterogeneity of the genetic background between CS and NILs. Most of the NILs were made by eight backcrosses, indicating that only 0.2% of the genes of the donor parent may still exist in the NILs. However, this is an underestimate because genes linked with a marker gene are also selected and remain in the offspring, depending on the distance from the marker gene. Hanson (1959) estimated the length of a chromosome segment that remains heterozygous through recurrent backcrossing. According to his estimation, a region of about 10 cM around the marker genes is not replaced by the CS chromosome region. However, since common wheat has as many as 21 chromosomes, and the genetic length of each chromosome is usually more than 200 cM (Gill et al. 1991b), the 10 cM region around the marker is not so large in the genomes of common wheat. Based on this fact, I will discuss the pleiotropic effect of the marker genes.
Two genes of Rht had a strong pleiotropic effect. It is known that their carriers do not respond to gibberellic acid (Gale and Law 1976). Because gibberellic acid is an important growth hormone, its insensitiveness must affect many physiological reactions. Likewise, q and s1 may play important roles upstream of the physiological pathways, although their physiological functions have remained unsolved. The effect of gene C was also large, but it was restricted to the spike characters, indicating that the gene acts after spike determination. The pleiotropic effect seen in Hp gene carriers may be attributed to an unbalanced chromosomal rearrangement. The NIL of Bg-C1 did not show any pleiotropic effect, though it carries a large chromosome segment of Ae. caudata, suggesting that the translocation resulted from a homologous chromosome recombination and that the segment of chromosome 1C compensates well for the function of the lost chromosome 1D segment. This was possible because the gene order of homologous group 1 chromosomes is the most highly conserved in the evolution of the grass family (Deynze et al. 1995). The other genes that did not show any significant pleiotropic effect probably function at the end of the pathway.
Use of Marked Monosomics in Wheat Genetics
After Sears (1954) produced the monosomic series of CS wheat, it has been widely used to localize genes on a chromosome by monosomic analysis and determine the homologous group of wheat relatives by chromosome substitution. Other useful experimental lines such as ditelocentrics, nullisomic-tetrasomics, alien and homologous chromosome substitution lines, and monosomics of the other varieties of common and emmer wheat were derived from the monosomic lines of CS. Although these are important lines, an accurate technique to count chromosome number is required to maintain and to use the lines in experiments. In addition, since the other chromosome may shift the monosome, the monosome must be checked periodically. Some of these problems could be overcome by providing a marker gene on the monosome of the monosomics.
Of the 13 markers tested, 8 showed morphologic differences in the monosomic (hemizygous) and disomic (homozygous) states. These were monosomic 1A with Hg, monosomic 4A with Rht-B1c, monosomic 4B with Hp1, monosomic 6B with b2, monosomic 2D with C, monosomic 3D with s1, monosomic 4D with Rht-D1c, and monosomic 6D with Hp3 (Table 4, Figure 2). Although the monosomics in these chromosomes usually show similar morphology to normal disomics, in these marked monosomics different expression of the marker genes made it possible to select monosomic plants in the selfed progeny of the monosomics, meaning that no chromosome observation is necessary to maintain these monosomic lines. In addition, chromosome shifts are avoidable. In the F1 between the marked monosomics and other varieties, disomic F1s with the marker character and monosomic F1s without the marker could be distinguished. For this purpose, monosomic 2B with Iw1 and monosomic 2D with Iw2 can be used.
These lines will be particularly useful in the production of homologous chromosome substitution lines in which recurrent backcrossing and recurrent chromosome observation are necessary. In addition, monosomics infrequently produced by pollination of n = 20 pollen to normal egg cell could be avoided by this method.
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Acknowledgments |
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I would like to thank Dr. K. Tsunewaki, Emeritus Professor Kyoto University, who encouraged my starting this study as a student 18 years ago and who allowed me to use his isogenic lines of S-615. I thank Dr. T. Koba, Chiba University, and Dr. T. Sasakuma, Kihara Institute, for providing valuable suggestions for this study, and Ms. T. Yamada, Kihara Institute, for her technical support.
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Footnotes |
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Corresponding Editor: Kendall R. Lamkey
Received August 12, 2000
Accepted January 15, 2001
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References |
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