Thematic schema representing main and sub-themes.

Main themes are indicated in darker boxes, with sub-themes in paler boxes.</p

Participant characteristics.

Participant characteristics.</p

Interview topic guide.

(DOCX)</p

Consolidated Criteria for Reporting Qualitative Studies (COREQ) checklist.

(PDF)</p

Identification of Genomic Regions and the <i>Isoamylase</i> Gene for Reduced Grain Chalkiness in Rice

<div><p>Grain chalkiness is an important grain quality related to starch granules in the endosperm. A high percentage of grain chalkiness is a major problem because it diminishes grain quality in rice. Here, we report quantitative trait loci identification for grain chalkiness using high-throughput single nucleotide polymorphism genotyping of a chromosomal segment substitution line population in which each line carried one or a few introduced <i>japonica</i> cultivar Nipponbare segments in the genetic background of the <i>indica</i> cultivar ZS97. Ten quantitative trait loci regions were commonly identified for the percentage of grain chalkiness and the degree of endosperm chalkiness. The allelic effects at nine of these quantitative trait loci reduced grain chalkiness. Furthermore, a quantitative trait locus (<i>qPGC8-2</i>) on chromosome 8 was validated in a chromosomal segment substitution line–derived segregation population, and had a stable effect on chalkiness in a multiple-environment evaluation of the near-isogenic lines. Residing on the <i>qPGC8-2</i> region, the isoamylase gene (<i>ISA1</i>) was preferentially expressed in the endosperm and revealed some nucleotide polymorphisms between two varieties, Nipponbare and ZS97. Transgenic lines with suppression of <i>ISA1</i> by RNA interference produced grains with 20% more chalkiness than the control. The results support that the gene may underlie <i>qPGC8-2</i> for grain chalkiness. The multiple-environment trials of the near-isogenic lines also show that combination of the favorable alleles such as the <i>ISA1</i> gene for low chalkiness and the <i>GS3</i> gene for long grains considerably improved grain quality of ZS97, which proves useful for grain quality improvement in rice breeding programs.</p></div

Bin mapping of grain chalkiness QTLs.

<p>The bin effects on PGC (a) and DEC (b) along the whole genome in the NIP/ZS97 CSSL population. The green and black bars on the x-axis represent the chromosomes. The -log scale of <i>P</i> values are plotted on the y-axis. The dotted lines indicate the threshold with <i>P</i> = 0.01.</p

Suppressed <i>ISA1</i> influences the expressions of starch synthesis related genes in the rice panicle.

<p>Differential expressions of eight starch-related genes between the RNAi line and the control, with the identification numbers based on the rice genome database (<a href="http://www.plantbiology.msu.edu/" target="_blank">http://www.plantbiology.msu.edu</a>); the <i>Actin</i> gene was used as an internal control for the semi-quantitative RT-PCR.</p

Frequency distribution of grain chalkiness in the CSSL population.

<p>(a) The percentage of grain with chalkiness (PGC) and (b) degree of endosperm chalkiness (DEC). The arrows indicate the mean of ZS97.</p

Suppression of <i>ISA1</i> causes chalky grains.

<p>(a) The gene <i>ISA1</i> model showing INDEL and SNP differences between ZS97 and NIP; the exons (rectangles) and introns (lines connecting the exons) are illustrated; the RNAi target domain is indicated by a short bold line above the model. (b) Box plot for PGC levels of 31 NIP type and 73 ZS type varieties at the InDel (the middle line indicates the median, the box indicates the range of the 25th to 75th percentiles of the total data, the whiskers indicate the interquartile range). (c) Quantitative RT-PCR showing the repressed <i>ISA1</i> expression in the RNAi line, where the <i>Actin</i> gene was used as an internal control. The (d) grain appearances and (e) PGC difference in the <i>ISA1</i>-RNAi line and the control. Asterisks (**) indicate mean values are significantly different at <i>P</i> < 0.01.</p

Evaluation of the NILs with contrasting <i>ISA1</i> alleles.

<p>(a) The trial with four sowing dates (A1–A4) in 2010. (b) The trial with three sowing dates (B1–B3) in 2011. ISA-N and ISA-Z represent the <i>ISA1</i> alleles from NIP and ZS97, respectively. Different letters (a, b, c, and d) above the bars indicate significant differences at <i>P</i> < 0.01 using Duncan’s test.</p