Success Story 7: Hybrid Purity Test

Genetic purity of parental lines and hybrids is of crucial importance, as one percent reduction in purity of hybrid seed, results in a reduction of about 100 kg/ha in yield of commercial crop. Traditionally genetic purity is done by Grow-out Tests (GOT), based on morphological assay. This method requires space for growing the samples drawn, considerable time till flowering/maturity (one season) and labour for raising the crop. Seed stocks and the investments made are locked up till the results from GOT are received. To overcome these major problems, Directorate of Rice Research (DRR), in collaboration with Centre for Cellular and Molecular Biology (CCMB), Hyderabad has developed a DNA marker based assay (popularly called DNA test) for rapid and reliable estimation of genetic purity of parental lines and hybrids. In this assay, specific target DNA molecules are amplified from a pool of DNA using a pair of oligonucleotide primer sequences that are complementary to the target DNA. The principle of this method for hybrid seed purity testing is that the parents have different alleles at the DNA marker locus being tested and that heterozygosity is an indicator of hybridity. It also implies that the markers being scored should be co- dominant in nature. SSR markers are highly suitable for this purpose in crops like rice as they are abundant, co-dominant and hyperpolymorphic. Purity testing is done with DNA isolated from rice seeds. The DNA isolated from each seed is subjected to PCR using SSR primers, which exhibit unique polymorphism among the parental lines constituting the hybrid (whose seed purity is to be tested). The PCR amplified fragments are separated by agarose gel electrophoresis, stained with ethidium bromide, visualized under UV light and documented (Yashitola et al. 2002). The protocol is explained below in Figure 1. The entire procedure takes only 3-5 hours for completion and any plant material including seed, grain or leaf can be used for DNA isolation and purity analysis.

Detection  of  off  types  in  a  hybrid  seed  lot  using  DNA  marker  based  purity  test. Amplification  of  two  fragments  (characteristic  of  the  two  parental  lines)  is  indicative  of hybridity while only one fragment is amplified from the off types. Ensuring the purity of parental lines used in hybrid seed production is an essential prerequisite for ensuring the purity of rice hybrids. Contamination of the CMS lines with seeds of the maintainer line is  a  serious  cause  for  concern  in  hybrid  rice  production  because,  being  isonuclear  lines, they  cannot  be  distinguished  from  each  other  prior  to  flowering.  A  particular  sequence difference  between  mitochondrial  DNA  from  male  sterile  CMS  lines  and  male  fertile maintainer lines has been discovered recently. Based on this sequence difference, a DNA marker assay based on a mitochondrial microsatellite marker has been designed by DRR, Hyderabad  that  can  be  used  to  reliably  distinguish  between  CMS  and  maintainer  lines. This  assay  can  be  used  to  screen  seed  lots  of  the  CMS  line  for checking  contamination with the maintainer line. Only those seed lots that have the desired level of purity will be retained and used for hybrid seed production. Thus, a major cause for reduction in hybrid seed  purity  can  be  eliminated.  The  assay  test  involves  the  use  of  mitochondrial  SSR markers  as  primers  in  a  Polymerase  Chain  Reaction  (PCR)  assay  wherein  fragments  of different  sizes  are  amplified  in  DNA  samples  from  CMS  lines  and  maintainer  lines (Figure 3) and contaminants can be detected easily.