Discrimination of haploids and doubled haploids/diploids in indica rice: correlation of morphological indicators with molecular markers

Abstract

To obtain immediate homozygosity by androgenesis, the doubled haploid method is often used. As a result, a mapping population was created utilizing rice (Oryza sativa L.) cvs. Mahulata and IR 20 as parents in order to find QTLs/genes for drought tolerance at the vegetative stage. The effectiveness of the doubled haploids (DHs) approach, on the other hand, is largely dependent on the ability to distinguish haploids from diploids among the green regenerants. Although flow cytometry and cytological screening for pollen sterility can be used to identify haploids, these methods are expensive, time-consuming, and need a sophisticated laboratory with highly trained workers. Plant height and other spikelet features have also been used to differentiate haploids from doubled haploids. However, no systematic analysis of several morphological features for distinguishing haploids in doubled haploids has been published to date. As a result, a cost-effective approach for distinguishing haploids from true DHs obtained from anther culture is required. The goal of this work was to identify haploids using morphological features and simple microscopic examinations without the use of chemicals or complex laboratory facilities. The cross between the IR20 and Mahulata yielded a total of 198 anther culture (AC) derived plants. A group of 41 plantlets was chosen as putative haploids based on their shorter height and Cq values using qPCR-based genotyping and finally validated that, in addition to plant height, other morphological traits such as total number of leaves/plant, total number of tillers/plant, and floral characters can be used to successfully identify haploids. We report a variety of morphological signs as indicators of haploid plants, including smaller plants, higher tiller density, narrower and shorter leaf length, and partial exertion of panicle from the flag leaf sheath. Other morphological markers for identifying haploids from DHs include smaller florets and anthers, and small desiccated microspores.

Keywords

• androgenesis
• cq value
• doubled haploids
• haploids
• indica rice
• pearsonꞌs correlation
• principal component analysis
• qpcr