Risheng Chen et al. 2017. BMC Genomics Whole genome sequencing and comparative transcriptome analysis of a novel seawater adapted, salt-resistant rice cultivar – sea rice 86 Risheng Chen et al. 2017. BMC Genomics

Oryza sativa Cultivated rice

Oryza sativa Family Poaceae: grasses, corn, bamboo, wheat….

SR86 New rice cultivar domesticated from a wild strain of rice first found in 1986. Grows in saline-alkaline, infertile soil and marginal lands. Resists submergence and water logging. Acceptable average yield (22 Kg/ha) when growing in extreme environments. Potential to either integrate high yield traits of elite rice cultivars into SR86 or to bring the salinity and submergence tolerance features of SR86 to elite cultivars.

Rationale Most important crop & primary food source for more than half of humanity. Rice production has to increase 25% or more to meet the demands of projected population growth (9 billion by 2050). Need for the identification and breeding of new varieties able to grow in marginal soils and adverse environments. Gathering information about the genetic diversity of O. sativa gene pool is a first step towards this goal.

Sequencing and Assembly Strategy Whole genome of SR86 was sequenced and compared to existing rice genomes. Genomic DNA extracted from fresh root tissue. DNA separated into ~300 bp and ~2000 bp. Libraries prepared with KAPA LTP DNA Library prep kit, and sequenced by whole-genome paired end sequencing using Illumina. Transcriptome seq. for SR68 & R1 (hybrid with low salt tolerance) planted in sea & fresh water. RNA libraries prepared with KAPA Stranded mRNA-Seq Kit and sequenced with Illumina. Raw sequences were mapped to Ensemble MSU6 and gene expression levels were estimated using RSEM v1.2.15 and normalized with the timed mean of M-values. Differentially expressed genes were identified using the edgeR program.

Sequencing and Assembly Strategy Genes showing altered expression with FDR < 0.05 and more than 2-fold changes were considered differentially expressed. Raw sequences were trimmed based on quality and GC content, and aligned to the O. sativa japonica reference genome using BWA software. Variant calling (different nucleotide when compared to reference genome) was performed using Genome Analysis Toolkit and Picard Package. Phylogenetic analyses were based on SNPs (single nucleotide polymorphisms) of 300 rice genomes and the SR86 genome. Genome-wide INDEL markers: 24 INDELs > 28 bp. One for the short arm (p) & one for the middle of the long arm (q) of each chromosome.

Assembly Statistics Length of ~373 Mbp 3,800,137 variants when compared to reference genome, of which 85% (SNPs) and 3.3% (INDELs). When compared to 300 rice genomes, 64,869 variants were unique to SR86. 947 INDELs and 7,233 SNPs were identified as functionally high impact variants (effect on protein structure or function).

Major Results and Conclusions SR68 is a relatively ancient indica subspecies. R1 planted in sea water did not germinate. Significantly different expressed transcripts between R1 & SR68 planted in fresh water was 3,905.

Major Results and Conclusions

Major Results and Conclusions Differentially expressed genes of SR68 planted in sea water (SW) vs fresh water (FW) GENE FAMILY Fraction differentially expressed in SW vs FW (upregulated in SW) POTENTIAL ROLE Pentatricopeptide repeat (PPR) 36/477 (36) Increase tolerance to biotic and abiotic stresses. Peroxidase 51/160 (44) Changes to the cell wall, which leads to salt adaptation. Dirigent 5/49 (4) Improve the response of endodermis to environmental stress. Multi-antimicrobial extrusion (MATE) 16/56 (4) Membrane transporters. Glutathione S-transferases (GST) 37/95 (6) Involved in oxidative stress metabolism & detox reactions (?) NB-ARC & NBS-LRR 27/85 (25) & 17/111 (17) Response to plant diseases (?) Kinesin motor domain 31/43 (31) Transportation with assistance of microtubules. Changes in microtubule dynamics as novel mechanism of salt stress adaptation?

Questions What do you think will be more practical: integrate high yield traits of common rice cultivars into SR86 or to insert salinity and submergence tolerance features of SR86 into common rice cultivars? Do you think it was necessary to compare the SR68 with other 300 rice genomes? (They were able to identified the amount of unique variants for SR68 but did not elaborate on them).