Development of Chloroplast Genomic Resources for Oryza Species Discrimination

Abstract

Rice is the most important crop in the world as the staple food for over half of the population. The wild species of Oryza represent an enormous gene pool for genetic improvement of rice cultivars. Accurate and rapid identification of these species is critical for effective utilization of the wild rice germplasm. In this study, we developed valuable chloroplast molecular markers by comparing the chloroplast genomes for species identification. Four chloroplast genomes of Oryza were newly sequenced on the Illumina HiSeq platform and other 14 Oryza species chloroplast genomes from Genbank were simultaneously taken into consideration for comparative analyses. Among 18 Oryza chloroplast genomes, five variable regions (rps16-trnQ, trnTEYD, psbE-petL, rpoC2 and rbcL-accD) were detected for DNA barcodes, in addition to differences in simple sequence repeats (SSR) and repeat sequences. The highest species resolution (72.22%) was provided by rpoC2 and rbcL-accD with distance-based methods. Three-marker combinations (rps16-trnQ + trnTEYD + rbcL-accD, rps16-trnQ + trnTEYD + rpoC2 and rpoC2 + trnTEYD + psbE-petL) showed the best species resolution (100%). Phylogenetic analysis based on the chloroplast genome provided the best resolution of Oryza. In the comparison of chloroplast genomes in this study, identification of the most variable regions and assessment of the focal regions of divergence were efficient in developing species-specific DNA barcodes. Based on evaluation of the chloroplast genomic resources, we conclude that chloroplast genome sequences are a reliable and valuable molecular marker for exploring the wild rice genetic resource in rice improvement.

Keywords: DNA barcoding; Oryza; chloroplast genome; sequence divergence; variable markers.

FIGURE 1

Gene map of Oryza chloroplast genome. Genes on the inside are transcribed in the clockwise direction while genes on the outside are transcribed in the counterclockwise direction. Genes in different functional groups are shown in different colors. The thick lines indicate the extent of the inverted repeats (IRa and IRb).

FIGURE 2

Analysis of perfect simple sequence repeats (SSRs) in 18 Oryza chloroplast genomes. (A) Number of SSRs detected in 18 chloroplast genomes. (B) Frequency of identified SSRs in LSC, IR, and SSC regions. (C) Number of SSR types detected in 18 chloroplast genomes.

FIGURE 3

Analysis of repeated sequences in 18 Oryza chloroplast genomes.

FIGURE 4

Phylogenetic tree reconstruction of 25 taxa using maximum likelihood, Bayesian inference, and maximum parsimony methods based on the complete chloroplast genome sequences. ML topology shown with ML bootstrap support value/Bayesian posterior probability/MP bootstrap support given at each node. Nodes with 100 ML-BP/1.0 BI-PP/100 MP-BP are not marked.

FIGURE 5

DNA barcode development. (A) Mean distance of each window using the 18 species data set. (B) The proportion of zero pairwise distances for each species based on the 18 species data set. (C) Mean distance of each window using the AA genome species data set. (D) The proportion of zero pairwise distances for each species based on the AA genome species data set.

FIGURE 6

MP tree for Oryza using the rbcL + matK and rps16-trnQ + rpoC2 + rbcL-accD + trnTEYD + psbE-petL DNA barcode combinations.