Distribution and Genetic Analysis of Dwarfing Gene Rht-D1b in Chinese Bread Wheat Cultivars and Lines
Songjie Yang and Shigui Liu
Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, School of Life Science, Sichuan University, Chengdu, China
Corresponding author: Shigui Liu
Key Laboratory of Bio-resources and Eco-environment of Ministry of Education, School of Life Science, Sichuan University, Chengdu, China
E-mail: not available
Abstract
PCR-based marker for the major semi-dwarfing gene Rht-D1b derived from the Japanese cultivar ‘Norin 10’ was used to detect 421 cultivars and lines from 8 of 10 wheat zones in China. The marker was validated by testing nine selected cultivars and lines. Detection of this wheat collection showed that the semi-dwarfing gene Rht-D1b tended to be distributed non-random in geographically divergent breeding programs. The distribution frequency of the semi-dwarfing Rht-D1b gene among these wheat cultivars and lines over the whole country was 43.5% on average. The highest was 63.6% in Northern spring wheat zone and the lowest was 13.3% in Northeastern spring wheat zone. The distribution frequency of the semi-dwarfing Rht-D1b gene for the other wheat zones was 37.9% in Northern winter wheat zone, 55.6% in Yellow & Huai River Facultative winter wheat zone, 32.4% in Middle & Low Yangtze Valley winter wheat zone, 33.3% in Southwestern winter wheat zone, and 12.5% in Northwestern spring wheat zone and 25.0% in Xinjiang winter-spring wheat zone, respectively.
Introduction
The control of plant height in cereals is known to be complex because of its polygenic and subject to environmental effects. Tall wheat cultivars and lines(Triticum aestivum 2n=6x=42) are more prone to lodging, particularly when grown in favorable environments whereas semi-dwarf cultivars are shorter, less prone to lodging (Ahmad et al., 2002). Reducing height has both directly improved the plant’s ability to divert available resources into grain rather than straw and has also improved lodging resistance. But it is known that most height genes with this ability have been difficult to be detected, resulting in the vast majority of the world’s semi-dwarf wheat crop cultivars having their height reduction determined primarily by one of two major dwarfing genes Rht-B1b and Rht-D1b derived from the old Japanese cultivar ‘Norin 10’(Worland and Sayers, 1995). During the past half century, the wheat plant height of China-grown commercial cultivars and lines has been steadily reduced from over 107.9cm down to the current height of around 90cm(Xu X.B. et al. 2001). The application of dwarfing genes to reduce plant height improving yield potential has been one of the major strategies in breeding modern, high yielding bread wheat cultivars (Gale and Youssefian, 1985).
Under optical circumstances in China, the two semi-dwarfing genes Rht-B1b and Rht-D1b (new nomenclature after Borner et al. 1996) can combine height reductions of approximately 23% with similar levels of increased yield (Borner et al., 1993; Flintham et al., 1997; Wang S.H. et al., 2001). These two GA-insensitive dwarfing genes are probably present in around 90% of the world’s semi-dwarf wheat crop and were responsible for the worldwide ‘green revolution’ in wheat cultivation(Worland et al., 1998). It is therefore of up-most importance that alternative GA-responsive dwarfing genes are studied and that where those offer potential to improving crop yields. Molecular markers should be generated to enable breeders to recognize and select for these genes in their segregating populations (Worland et al., 1998).
Ellis (2002) developed PCR-based markers specific for the Rht-B1b and Rht-D1b semi-dwarfing genes in wheat and in the sense these markers could be described as ‘perfect markers’. What is more, Ellis presented evidence that validated the specificity of the markers-a range of selected wheat cultivars of known Rht status being correctly genotyped by the markers.
Materials and Methods
Plant materials
In the experiments, a total of 421 genotypes aboratively chosen from 8 of 10 wheat zones in China diverse national breeding programs and widely and commercially grown during the last decade and some still being grown now could be characteristic of wheat growing trend in the country. The pedigree information for most of the cultivars and lines was kindly provided by various breeders or from the works by Zhuang Q.S (2003). All the wheat cultivars and lines along with their pedigree, breeding place, wheat zone and genotype were listed in Table 1.
Template DNA preparation and PCR analysis
High-molecular-genomic DNA was extracted from leaves of 2- to 4-week-old plants grown in the growth chamber. The DNA extraction procedure was a modification described by Sharp et al.(1989). The genomic DNA was diluted in sterile water to a final concentration of 0.02ug/ul. Primer combinations for polymerase chain reaction (PCR) conditions and PCR were fully described by Ellis et al. (2002). PCRs were conducted with a Precision Scientific Genetic Thermal Cycler (MJ Research, US). Amplifications were assayed to running according to the following program: after initial denaturing for 15 min at 95oC, 38 cycles of 30 s at 94oC, 30s at 63 oC, 30s at 72 oC, and a final extension step of 5 min at 72 oC ( personal communication). PCR products were separated on 2% (w/v) agarose gel at 190v. Gels were then stained with ethidium bromide (EB) and visualized and photographed under UV light.
Results and discussion
Validation of PCR markers on selected wheat cultivars and lines
Nine wheat cultivars and lines with unknown Rht genotype but ‘Norin 10’ as a check cultivar carrying two semi-dwarf genes Rht-B1b and Rht-D1b were selected and DNA samples from these cultivars and lines were analyzed by PCR using the Rht-B1a, Rht-B1b, Rht-D1a and Rht-D1b primer markers for ten repetitions, respectively. The cultivars and lines carrying semi-dwarfing gene Rht-B1b gave an amplification product of expected fragments (237bp) with the Rht-B1b primer combination as described in detail by Ellis. At the same time these cultivars and lines didn’t give an amplification product of expected fragments (237bp) with the Rht-B1a primer combination. It could be indicated that these primer combinations were validated for the Rht-B1a, Rht-B1b specific. But while the 421 wheat cultivars and lines were detected, some false positives were on the agarose gels and their frequency got to 14.67%, therefore some doubts were produced for the Rht-B1a, Rht-B1b specific primer combinations. In the present paper, the results of the nine selected and the 421 cultivars and lines detected concerning Rht-B1b dwarfing gene were not listed.
The same status didn’t bring forth to the Rht-D1a and Rht-D1b primer combinations either for the nine selected cultivars and lines or for the 421 wheat cultivars and lines, and the frequency of the false positives was only less than 0.7%. All the detecting results from the 421 wheat cultivars and lines were repeated twice, so it could be sure that the primer combinations of the semi-dwarfing Rht-D1b gene were specific to it (Fig. 1 and Fig. 2).
Countrywide frequency distribution of semi-dwarfing Rht-D1b gene
The PCR-based marker analyses for the semi-dwarfing Rht-D1b gene of the 421 wheat cultivars and lines indicated that the distribution frequency was highly different between 8 of 10 wheat zones in China. Among the 421 cultivars and lines, there were 183 cultivars and lines carrying the semi-dwarfing Rht-D1b gene and their distribution frequency approximately was 43.5%. As for the 4 winter wheat zones(WWZs), Yellow & Huai River Facultative WWZ(YHRF WWZ) had the highest frequency of 55.6%, 12.1% more than countrywide average, while the lowest one was Middle & Low Yangtze Valley WWZ (MLYV WWZ) at a ratio of 32.4%, the other two WWZs of Northern WWZ and Southwestern WWZ were 37.9% and 33.3%, respectively. Conversely among the 4 spring wheat zones(SWZs), Northern SWZ had the highest frequency of 63.6%, 20.1% higher than the countrywide average, however the lowest SWZ was Northeastern SWZ at a ratio of 13.3%, namely, there was only two cultivars and lines carrying the semi-dwarfing Rht-D1b gene. The evidences demonstrated above that the screening of cultivars and lines for the Rht-D1b dwarfing gene tended to be non-random in geographically divergent breeding programs for each wheat zone in China. The causes that gave rise to the different distribution frequency in China’s wheat zones should be: firstly, different dwarf resources used in different wheat zones had created different distribution frequency of Rht-D1b gene. As an example, Youbaomai wheat which had been being widely grown as an elite long before and now is still being used in YHRF WWZ had once caused the higher ratio of the Rht-D1b dwarfing gene; secondly, some wheat zones in China needed and are still needing specially for tall wheat cultivars and lines, for this goal the programs of wheat breeding had to balance the relationships between yield and plant height and other traits. Therefore, sometimes some characters should not be taken into consideration principally, for instance, plant height as a minor character in Northwestern SWZ.
Genetic diversity
The results from PCR amplification of 421 wheat genotypes using Rht-D1b specific primer pairs and the pedigrees of most of the 421 cultivars and lines were summarized in Table 1(some unprovided because of their original notes not being found or other reasons), which showed that the correlation between the semi-dwarfing Rht-D1b gene based on the specific primer combinations and the pedigrees of the cultivar and lines was highly significant. The results of the cultivar analyses with the Rht-D1b-specific primers described above would suggest that the distribution frequency of the semi-dwarfing Rht-D1b gene in countrywide breeding programs be more likely due to selection than chance. The main areas of distribution for the Rht-D1b gene were throughout Northern SWZ and the YHRF WWZ.
The semi-dwarfing Rht-D1b gene mainly derived from two dwarf resources (Jia J Z, 1992): (1) cultivars characteristic of Huixianhong and Youbaomai wheat carrying the semi-dwarfing Rht-D1b GA-insensitive gene (Fig. 3). (2) cultivars characteristic of ‘Norin10’ and ‘Sweon 86’ carrying two semi-dwarfing Rht-B1b and Rht-D1b GA-insensitive genes(Fig. 4). But some cultivars and lines derived from Taigu male sterile lines were found also carrying the Rht-D1b GA-insensitive gene (cultivars and lines such as Jinghe 951, Jingnong 8318, Yuandong 971, Yumai 50 and so on listed in Table 1). Therefore the semi-dwarfing Rht-D1b gene credited with playing major roles in improving wheat yields was categorized into three classes: some from introduction from abroad, some from Chinese old landlaces and some from Taigu nuclear male sterile lines.
Conclusion
Genetic analyses of the 421 cultivars and lines and 10 validation repetitions for the nine selected cultivars and lines and the discussion above demonstrated that the utility of the marker based on the PCR could be used not only as a diagnostic marker to the dwarfing gene Rht-D1b which, until now, has been difficult to be detected in breeding populations but also to be studied in genetic analysis. Therefore, it could be concluded that diverse parents could be selected on the basis of the ‘perfect marker’. With the availability of a rich collection of cultivars and lines recently made available through collaborative efforts and individual efforts elsewhere, this would certainly become the markers of choice in the future for a variety of studies and molecular-assisted selection.
Acknowledgements
We wish to express gratitude to Dr. David Bonnett for his providing PCR reaction condition. We would like to thank my teachers and classmates who acted as reviewers and advisors.
References
Ahmad M. & Sorrells ME (2002) Distribution of microsatellite alleles linked to Rht8 dwarfing genes in wheat. Euphytica 123: 235-240.
Bonjean, A.P. & W.J. August(2001). The world wheat book-A history of wheat breeding. Intercept, Londres- Paris-New York.
Borner A, Worland A.J, Plaschke J, Erika Schumann &.Law C.N (1993) Pleiotropic effects of genes for reduced height (Rht) and day-length insensitivity (Ppd) on yield and its components for wheat genes in middle Europe. Plant Breeding, 111: 204-216.
Borner A, Plaschke J, Korzun V & A.J. Worland(1996). The relationships between the dwarfing genes of wheat and rye. Euphytica 89: 69-75.
Borner, A., M. Roder & V. Korzun, 1997. Comparative molecular mapping of GA insensitive Rht loci on chromosomes 4B and 4D of common wheat (Triticum aestivum L.) . Theor Appl Genet 95: 1133-1137.
Ellis M.H., Spielmeyer W, Rebetzke GJ, Richards RA (2002) “perfect” markers for the Rht-B1b and Rht-D1b dwarfing genes in wheat..Theor. Appl. Genet. 105:1038-1042.
Flintham J.E., Börner A, Worland AJ & Gale M D(1997) Optimizing wheat grain yield: effects of Rht (gibberellin-insensitive) dwarfing genes. J. of Agri. Sci.128: 11-25.
Gale M.D.& Youssefian (1985) Dwarfing genes in wheat. In: Russell GE(ed). Progress in plant breeding. Butterworths and Co. London. Pp1-35.
Jia J.Z., Ding S.K., Li Y.H. and Zhang H. (1992). Study on main dwarfing genes and their sources of Chinese bread wheat. Chinese agricultural science, 25:1-5.
Jin S.B. (1996). Chinese bread wheats. Chinese agricultural press, Beijing.
Korzun. V., Roder MS, Ganal MW, Worland AG, Law CN (1998) Genetic analysis of the dwarfing gene (Rht8) in wheat. Part I.Molecular mapping of Rht8 on the short arm of chromosome 2D of bread wheat (Triticum aestivum L.). Theor. Appl Genet.96: 1104-09.
McIntosh, R.A.(1988). A catalogue of gene symbols for wheat. pp1225-1323. In: T.E. Miller & R.M.D. Koebner(Eds). Proc. 7th Int. Wheat Genet. Symp. Cambridge, England. Bath Press, Bath, Avon.
McIntosh R.A., Hart GE, Devos KM, Gale MD & Rogers WG(1998). Catalogue of gene symbols for wheat. In: Proc 9th Int Wheat Genet Symp, Saskatoon, Saskatchewan, Canada 2-7 August. Volume 5, Saskatoon: University Extension Press, Univ. of Saskatchewan, Canada.
Pestsova, E., M.W. Ganal & M.S. Roder(2000). Isolation and mapping of microsatellite markers specific for the D genome of bread wheat. Genome 43: 689-697.
Sharp P J, Chao S, Gale M D (1989) The isolation, characterization and application in Triticeae of a set of wheat RFLP probes identifying each homologous chromosome arm. Theor Appl Genet. 78: 342-348.
Wang S.H, Meng F.H, Yang L, Liu B.H (2001) Effects of dwarfing genes on different
agronomical characters in bread wheat. Wheat crop journal 24:5-9.
Worland AJ., Korzun V., Roder MS, Ganal MW and Law CN (1998). Genetic analysis of the dwarfing gene Rht8 in wheat. Part II. The distribution and adaptive significance of allelic variants at the Rht8 locus of wheat as revealed by microsatellite screening. Theor. Appl Genet 96: 1110-20.
Worland, A.J. & E.J. Sayers, 1995. Rht(B. dw), an alternative allelic variant for breeding semi-dwarf wheat varieties. Plant Breeding 114: 397-400.
Xu X.B, Zhang A.M, Li X.H, Sun Y.T (2001). Application of dwarf resources and study advance of dwarfing genes in bread wheat. Neuc-agronomy journal 15:188-192
Zhou Y. He Z.H. Zhang G.S. Xia L.Q. Chen X.M. Zhang L.P. and Chen F(2003). Identification of Rht8 dwarf gene distribution by microsatellite marker in Chinese Wheat. Crop Journal 29(6): 810-814.
Zhuang Q.S.(2003). Chinese wheat improvement and pedigree analysis. Beijing. China. pp 562-681