Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-03-28
2003-03-04
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S023100, C536S029200
Reexamination Certificate
active
06528265
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method of using DNA sequences for assessing seed purity. More specifically, the invention relates to a DNA sequences having homology to rice mitochondrial DNA and being unique to Wild Abortive (WA) cytoplasm containing cytoplasmic male sterile lines of rice, and the use of these sequences in a Polymerase Chain Reaction (PCR) assay to distinguish Male Sterile (CMS) lines of rice from their cognate Male Fertile Maintainer Lines. This invention relates to a method for ensuring the purity of cytoplasmic male sterile lines of rice using DNA based markers
BACKGROUND OF THE INVENTION
Hybrid vigor is the phenomenon by which the progeny of a cross between two inbred lines has higher yield potential than either one of the parents. Hybrids can yield upto 10-30% more than the best variety and are a favored option for increasing yield.
Rice is a major cereal crop all over the world; and in many parts of Asia it is the staple part of the diet. It has been estimated that in a number of Asian countries like India rice yields must double by the year 2025 to meet the demands of the increasing population (Hossain, 1996. In Khush (ed) Rice Genetics III, Proc. Third Intl. Rice Genet. Symp., Los Banos Manila, the Philippines. Oct. 16-20, 1995. International Rice Rearch Institute, Manila, the Philippines). As has been demonstrated in the People's Republic of China, where almost fifty percent of the area under rice cultivation is covered by hybrids, the widespread cultivation of hybrid rice is a readily available option for increasing yield. By comparison, in countries like India the area under hybrid rice is less than 1% of the total area under rice cultivation. This illustrates the tremendous potential for increasing the area under hybrid rice cultivation and it is expected that the market for hybrid rice seeds will increase in a number of rice growing countries, including in India.
The most widely used system for hybrid rice production is the three line system (FIG.
1
). The three lines include: 1. a male sterile, female fertile line called the Cytoplasmic Male Sterile (CMS) line because it carries a male sterility conferring mutation in the cytoplasmic component of the genome, 2. a maintainer line and 3. a restorer line; the maintainer and restorer lines are male fertile as well as female fertile. The CMS and Maintainer lines are practically identical with respect to the nuclear component of the genome (and are often referred to as iso-nuclear lines) but differ from each other with respect to the cytoplasmic component of the genome. The male sterility of the CMS line is maternally inherited and is most likely due to a mutation in the mitochondrial DNA. The CMS line, being female fertile, can be propagated by fertilization with pollen emanating from the Maintainer line. Since the cytoplasmic component of the genome is not transferred through pollen, the progeny of such a cross would inherit the cytoplasm only from the CMS line and would therefore be male sterile. The nuclear component of the genome of the progeny would also be identical to that of the CMS line, even though half of it is inherited from the Maintainer line, as there is no difference between these two lines with respect to this component of the genome.
The hybrid seeds are produced in a cross of the CMS line with another inbred parental line, called the Restorer line, which as indicated above is Male fertile and Female fertile. In this cross the CMS line serves as the female parent while the Restorer line is the male parent. The Restorer line also carries a Rf (restorer of fertility) gene/s in it's nuclear genome which will restore male fertility to a plant whose cytoplasm has been inherited from the CMS line. The hybrid seeds produced in the cross depicted in
FIG. 1
would therefore be fertile. The CMS and Restorer lines are appropriately chosen such that the hybrids exhibit sufficient hybrid vigor (or heterosis) to produce substantially higher yields than inbred varieties.
The vast majority (90% or more) of the rice hybrids that are currently under commercial cultivation in the world derive their cytoplasm from a single source (Yuan, 1995. Hybrid rice seed production technology, Directorate of Rice Research, Hyderabad, India). This cytoplasm, called the WA (wild abortive) cytoplasm, was discovered in a wild rice plant in China. Subsequently, this cytoplasm has been crossed into several different nuclear genetic backgrounds by repeated back-crossing using the recurrent parent as the male donor. In this manner several different CMS lines have been developed, each of which has in turn been crossed with different Restorer lines to develop a number of hybrids, all of which share the same WA cytoplasm.
It is important to maintain the purity of hybrids as any impurities therein would reduce the expected yield. It has been estimated that for every one-percent impurity in the hybrid seed the yield reduction is to the tune of 100 Kg per hectare (Mao et al, 1996 In Virmani, S. S., E. A. Siddiq, and K. Muralidharan (eds) Advances in Hybrid Rice Technology. Proc. Third Intl. Symp. on Hybrid Rice, Directorate of Rice Research, Hyderabad, India). The Indian seed act prescribes that, for hybrid rice, the expected purity should be 98% (Verma, 1996. Seed Tech. News 24:1-4); in the People's Republic of China it is mandated that the purity of hybrid rice should be at least 96% (Wengui Yan, 2000. U.S. Pat. No. 6,066,779). In order to ensure the required levels of hybrid seed purity, the parental lines that are employed in hybrid seed production should have a very high (almost 99%) level of purity.
One of the common admixtures that occur during hybrid seed production is that of Maintainer lines with those of the CMS lines. As these are iso-nuclear lines, it is very difficult to distinguish between these lines based on morphological criteria i.e. other than male sterility which can only be judged at the time of flowering. DNA markers that distinguish the CMS and maintainer lines can be developed and applied at the seedling level for the practical detection of seeds of Maintainer lines that occur as contaminants within stocks of the CMS lines. DNA Markers based on the use of the Polymerase Chain Reaction would be ideally suited for this purpose as they are much more efficient for handling large numbers of samples than hybridization based methods like Restriction Fragment Length Polymorphisms.
The Polymerase Chain Reaction is based on the use of short oligonucleotide sequences as primers for the enzymatic amplification of DNA sequences that occur between two appropriately spaced primer binding sites on the target DNA. The PCR works most reproducibly when the oligonucleotide primers are designed on the basis of a knowledge of the target DNA sequence. Protocols have been developed for PCR which are based on the use of short (8-10 bases long), randomly designed oligonucleotide primers (Williams et al, 1990; Nucleic acids research 18: 6531-6535). These primers are not based on a knowledge of the target DNA sequence and kits containing large numbers of these randomly generated primers are now commercially available. The DNA markers that are developed by this method are known as Randomly Amplified Polymorphic DNA (RAPD) Markers. Because a large number of primers are available, genetic polymorphisms can be detected by this method, even within closely related lines. However, the reproducibility of RAPD markers is poor due to the short length of the oligonucleotide (RAPD) primers and possibly also due to the lack of the required degree of specificity for the target. This severely limits the practical application of RAPD markers, as diagnostic markers for distinguishing different genotypes.
RAPD markers that distinguish CMS (WA cytoplasm) and Maintainer lines of rice have been described (Jena and Pandey 1999; Hybrid Rice Newsletter. 2: 13-14). However, the low reproducibility of these markers has made it practically impossible to apply them in a routine manner for distinguishing CMS and Maintainer l
Jamir Yashitola
Sonti Ramesh V.
Horlick Kenneth R.
Strzelecka Teresa
LandOfFree
DNA markers for assessing seed purity and method of using... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with DNA markers for assessing seed purity and method of using..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and DNA markers for assessing seed purity and method of using... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3031799