Map-Based Cloning Of Two Important Genes In Rice Using BSA-seq

Rice produces a number of culms through tillering at the vegetative phase. The numbers of tillers are regarded as the factor determining grain yield since tillers are specialized branches bearing panicles. Panicles are the reproductive organs which are explained by unique structural units known as spikelet and florets. The development of the spikelets and florets at different levels determines panicles viability and the amount of grain a plant yield. The plant viability could be described as either fertile or sterile. Therefore tillering and panicle development are two crucial agronomic factors which determines proper vegetative growth and panicle development which abundantly contribute for grain productivity.In this study two mutants were isolated from tissue culturing regenerated plants namely srtl and pshl-1/2. We mapped and cloned the genes using BSA and whole genome resequencing technology which was fast and efficient method. The results are as follows;1. Sterile and Reduced Tillering 1: a mutant with no tillers and exhibited complete female sterility.(1) The morphological characteristics of srtl are that it had few or hardly any tillers. The results showed that MH86 (indica variety) used as control produced 9.0 ± 3.6 tillers compared to srtl with only 1-4 (0.5 ± 0.7) tillers per plant.(2) The stem of srtl was thick and strong compared to the normal plants. The sectioning of the stem showed well developed vascular bundles in the cross-section which can provide enough nutrients to the plant and the longitudinal sections displayed regular arrangement of the cells.(3) The leaves of the srtl were longer and broad. The leave length and width ratio of normal to srtl were 52.2 ± 3.34:59.5 ±2.1 and 1.55 ± 0.2:2.05 ± 0.1, respectively.(4) srtl was completely sterile. The sterility was attributed to female organ according the results that pollen analysis shows normal gametes using I2KI2, and the reciprocal test cross analysis using srtl pollen in normal panicles had seed setting rate of 9.9 ± 6.8% while normal pollen in srtl panicles produced no seeds.(5) In mapping SRT1 using whole-genome sequencing a major peak of CAAFD was found in the long arm of chromosome 4 approximately (29-34) Mb in the physical map. Using INDEL markers, the target was fine mapped to 231kb.(6) The target gene was identified from 231 kb genomic region with 37 ORFs. LOC_Os04g56780 was found to have allelic variation (wild-type v/s mutant) with a 21 bp deletion. LOC_Os04g56780 encodes the rice ortholog (OsWUS) of Arabidopsis Wuschel (AtWUS).(7) The 21-bp deletion occurs at exon 1 of OsWUS and results in deletion of 7 amino acids in the highly conserved homeodomain. This short deletion make OsWUS losing its main functions, suggesting that the homeobox domain is pivotal for OsWUS function2. Pepper-Shaped husk 1:a mutant with a defective pepper shaped spikelet.(1) The morphological characteristics of pshl had the panicle shorter by 20% and compact compared to wild-type MH86. The spikelet of psh1-1 displayed pepper-like shape, with no difference in lemma but slight difference of the palea compared to wild-type.(2) The seed setting rate of pshl-1 was only 5.8% ± 7.2 less than wild-type 85.2% ± 4.9. The morphology of pshl during heading was observed to be abnormal. Many flowers remained closed with 6.55% ± 5.3 eventually opening while most flowers (84.65% ± 6.7) of wild type opened eventually.(3) The seeds produced by pshl appeared small than wild type. The grain length of (8.7 ± 0.48) and width of (1.975 ± 0.07) of psh1-1 were significantly small than wild type. The shape of the grain of psh1-1 also appeared pepper-shaped and smaller with 1000 seed weight of seeds being 28.67 ± 0.65 compared to wild type (15.0 ± 1.18).(4) Pepper-shaped husk and low fertility trait of psh1-1 were controlled by recessive gene, as observed in the segregating F2 population with a 3:1 ratio.(5) In mapping PSH1 using whole-genome sequencing a major peak of CAAFD was found at the beginning of the short arm of chromosome 4, covering a region of approximately 3Mb (from 0 Mb to 3.0Mb in the physical map). Further linkage analysis narrowed the region to 218 kb.(6) The candidate gene was identified from 218 kb which contained 31 ORFs. The comparison of assembled genomic sequences of different pools of wild-type and psh1, identified one nucleotide substitution of (G to T) in the gene LOC_Os04g01590 resulting to the stop codon (TAA) and terminating the protein prematurely.(7) LOC_Os04g01590 encodes arginase protein. It has six exons and encodes 340 amino acids. Addition to (G to T) substitution in the 3rd exon of pshl-1, another mutant pshl-2 was found another substitution in the 2nd exon of (G to T) which also resulted to a stop codon of (TAG). These results confirmed that the mutated phenotypes of these mutants were caused by mutation in LOC_Os04g01590.(8) Biochemical analysis of the oxidative stress showed no difference between psh1 and wild type.