Genetic dissection and validation of candidate genes for flag leaf size in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Key message

Two major loci with functional candidate genes were identified and validated affecting flag leaf size, which offer desirable genes to improve leaf architecture and photosynthetic capacity in rice.

Abstract

Leaf size is a major determinant of plant architecture and yield potential in crops. However, the genetic and molecular mechanisms regulating leaf size remain largely elusive. In this study, quantitative trait loci (QTLs) for flag leaf length and flag leaf width in rice were detected with high-density single nucleotide polymorphism genotyping of a chromosomal segment substitution line (CSSL) population, in which each line carries one or a few chromosomal segments from the japonica cultivar Nipponbare in a common background of the indica variety Zhenshan 97. In total, 14 QTLs for flag leaf length and nine QTLs for flag leaf width were identified in the CSSL population. Among them, qFW4-2 for flag leaf width was mapped to a 37-kb interval, with the most likely candidate gene being the previously characterized NAL1. Another major QTL for both flag leaf width and length was delimited by substitution mapping to a small region of 13.5 kb that contains a single gene, Ghd7.1. Mutants of Ghd7.1 generated using CRISPR/CAS9 approach showed reduced leaf size. Allelic variation analyses also validated Ghd7.1 as a functional candidate gene for leaf size, photosynthetic capacity and other yield-related traits. These results provide useful genetic information for the improvement of leaf size and yield in rice breeding programs.

     

QTLs linked to leaf epicuticular wax,physio-morphological and plant production traits under drought stress in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Drought stress is the major constraint to rice (Oryza sativa L.) production and yield stability in rainfed ecosystems. Identifying genomic regions contributing to drought resistance will help to develop rice cultivars suitable for rainfed regions through marker-assisted breeding. Quantitative trait loci (QTLs) linked to leaf epicuticular wax, physio-morphological and plant production traits under water stress and irrigated conditions were mapped in a doubled haploid (DH) line population from the cross CT9993-5-10-1-M/IR62266-42-6-2. The DH lines were subjected to water stress during anthesis. The DH lines showed significant variation for epicuticular wax (EW), physio-morphological and plant production traits under stress and irrigated conditions. A total of 19 QTLs were identified for the various traits under drought stress and irrigated conditions in the field, which individually explained 9.6%–65.6% of the phenotypic variation. A region EM15_10-ME8_4-R1394A-G2132 on chromosome 8 was identified for leaf EW and rate of water loss i.e., time taken to reach 70% RWC from excised leaves in rice lines subjected to drought stress. A large effect QTL (65.6%) was detected on chromosome 2 for harvest index under stress. QTLs identified for EW, rate of water loss from excised leaves and harvest index under stress in this study co-located with QTLs linked to shoot and root-related drought resistance traits in these rice lines and might be useful for rainfed rice improvement.     

Genetic architecture of flag leaf length and width in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) revealed by association mapping

Rice flag leaf is the main photosynthetic organ and plays a key role in grain yield. In this study, 106 loci associated with flag leaf length and width were identified using association mapping in a rice mini core collection. Analyzing the phenotypic effects of each allele represented 156 positive and 167 negative alleles, with a few alleles showing inconsistent effect in different environments. Among the 106 loci, 69 were environment-specific loci, 16 were environmentally stable and 21 were environmentally sensitive. Fifteen associated markers were shared by two traits and were designated as pleiotropic markers. According to the frequency distribution of alleles in different variety types, 343 alleles were categorized into three types based on geographic source and usage in breeding. Among these, 156 were used alleles, 138 were unused and 49 were foreign alleles. The results further our understanding of the genetic mechanisms of flag leaf length and width.     

Transposition behavior of nonautonomous a <Emphasis Type="Italic">hAT</Emphasis> superfamily transposon <Emphasis Type="Italic">nDart</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Transposable elements (TEs) have a significant impact on the evolution of gene function and genome structures. An endogenous nonautonomous transposable element nDart was discovered in an albino mutant that had an insertion in the Mg-protoporphyrin IX methyltransferase gene in rice. In this study, we elucidated the transposition behavior of nDart, the frequency of nDart transposition and characterized the footprint of nDart. Novel independent nDart insertions in backcrossed progenies were detected by DNA blotting analysis. In addition, germinal excision of nDart occurred at very low frequency compared with that of somatic excision, 0–13.3%, in the nDart1-4(3-2) and nDart1-A loci by a locus-specific PCR strategy. A total of 253 clones from somatic excision at five nDart loci in 10 varieties were determined. nDart rarely caused deletions beyond target site duplication (TSD). The footprint of nDart contained few transversions of nucleotides flanking to both sides of the TSD. The predominant footprint of nDart was an 8-bp addition. Precise excision of nDart was detected at a rate of only 2.2%, which occurred at two loci among the five loci examined. Furthermore, the results in this study revealed that a highly conserved mechanism of transposition is involved between maize Ac/Ds and rice Dart/nDart, which are two-component transposon systems of the hAT superfamily transposons in plant species.     

Functional markers for <Emphasis Type="Italic">xa5</Emphasis>-mediated resistance in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>, L.)

The recent cloning of several agronomically important genes has facilitated the development of functional markers. These markers reside within the target genes themselves and can be used with great reliability and efficiency to identify favorable alleles in a breeding program. Bacterial blight (BB) is a severe rice disease throughout the world that is controlled primarily through use of resistant cultivars. xa5 is a race-specific, recessive gene mediating resistance to BB. It is widely used in rice breeding programs throughout the tropics. Due to its recessive nature, phenotypic selection for xa5-mediated resistance is both slow and costly. Previously, marker assisted selection (MAS) for this resistance gene was not efficient because it involved markers that were only indirectly linked to xa5 and ran the risk of being separated from the trait by recombination. Recently, the cloning of the gene underlying this trait made it possible to develop functional markers. Here we present a set of CAPS markers for easy, quick and direct identification of cultivars or progeny carrying xa5-mediated resistance and provide evidence that these markers are 100% predictive of the presence of the xa5 allele. These markers are expected to enhance the reliability and cost-effectiveness of MAS for xa5-mediated resistance.     

Assessment of genetic diversity of <Emphasis Type="Italic">Saltol</Emphasis> QTL among the rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) genotypes

Eight Saltol quantitative trait locus (QTL) linked simple sequence repeat (SSR) markers of rice (Oryza sativa L.) were used to study the polymorphism of this QTL in 142 diverse rice genotypes that comprised salt tolerant as well as sensitive genotypes. The SSR profiles of the eight markers generated 99 alleles including 20rare alleles and 16 null alleles. RM8094 showed the highest number (13) of alleles followed by RM3412 (12), RM562 (11), RM493 (9) and RM1287 (8) while as, RM10764 and RM10745 showed the lowest number (6) of alleles. Based on the highest number of alleles and PIC value (0.991), we identified RM8094 as suitable marker for discerning salt tolerant genotypes from the sensitive ones. Based upon the haplotype analysis using FL478 as a reference (salt tolerant genotypes containing Saltol QTL), we short listed 68 rice genotypes that may have at least one allele of FL478 haplotype. Further study may confirm that some of these genotypes might have Saltol QTL and can be used as alternative donors in salt tolerant rice breeding programmes.     

Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

Quantitative proteomics analysis of proteins involved in leaf senescence of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Delaying leaf senescence and prolonging the available time for photosynthesis is one of the important approaches to increase grain yield of rice (Oryza sativa L.). Here, iTRAQ-based quantitative proteomics approach was used to comparative analyze the expression profiles of proteins in rice leaves in response to senescence. Totally 5067 proteins were identified. Compared with the proteins in the flag leaves at early stage of grain-filling in rice Liang-You-Pei 9 (LYP9), 240 and 188 proteins were up-regulated and down-regulated in the flag leaves at middle stage of grain-filling, and 387 and 202 proteins were up-regulated and down-regulated in the flag leaves at late stage of grain-filling, respectively. In addition, 39 and 18 identified proteins were constantly up-regulated and down-regulated in the leaves from early to middle and late stages of grain-filling, respectively. Among them, chloroplast chaperonin 10, geranylgeranyl diphosphate reductase, Mg chelatase subunit ChLD, porphobilinogen deaminase, protochlorophyllide reductase B and thioredoxin-like protein CITRX might have involved in the senescence of leaves. This study provided important information for understanding the age-sensitive mechanism of LYP9, and offered a foundation for future studying and improving it.     

Map-based cloning and functional analysis of the chromogen gene <Emphasis Type="Italic">C</Emphasis> in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The chromogen gene C is critical for anthocyanin regulation in rice, and apiculus color is an important agronomic trait in selective breeding and variety purification. Mapbased cloning and in-depth functional analysis of the C gene will be useful for understanding the molecular mechanism of anthocyanin biosynthesis and for rice breeding. Japonica landrace Lijiangxintuanheigu (LTH) has red apiculi and purple stigmas. Genetic analysis showed that red apiculus and purple stigma in LTH co-segregated indicating control by a single dominant gene, or by two completely linked genes. Using 1,851 recessive individuals from two F₂ populations, the target gene OsC was delimited to a 70.8 kb interval on chromosome 6 that contains the rice homologue of the maize anthocyanin regulatory gene C1. When the entire OsC gene and its full-length cDNA cloned from LTH were transformed into japonica cultivar Kitaake with colorless apiculi and stigmas all positive transformants had red apiculi but non-colored stigmas, validating that OsC alone was responsible for the apiculus color and represented the functional C gene. OsC was constitutively expressed in all tissues examined, with strongest expression in leaf blades. These results set a foundation to clarify the regulatory mechanisms of OsC in the anthocyanin biosynthetic pathway.     

Fine genetic mapping and physical delimitation of the lesion mimic gene <Emphasis Type="Italic">spotted leaf 5</Emphasis> (<Emphasis Type="Italic">spl5</Emphasis>) in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Spotted leaf 5 (spl5), a lesion mimic mutant, was first identified in rice (Oryza sativa L.) japonica cv. Norin8 in 1978. This mutant exhibits spontaneous disease-like lesions in the absence of any pathogens and resistance to rice blast and bacterial blight; however, the target gene has not yet been isolated. In the present study, we employed a map-based cloning strategy to finely map the spl5 gene. In an initial mapping with 100 F₂ individuals (spl5/spl5) derived from a cross between the spl5 mutant and indica cv. 93-11, the spl5 gene was located in a 3.3-cM region on chromosome 7 using six simple sequence repeat (SSR) markers. In a high-resolution genetic mapping, two F₂ populations with 3,149 individuals (spl5/spl5) were derived from two crosses between spl5 mutant and two indica cvs. 93-11 and Zhefu802 and six sequence-tagged site (STS) markers were newly developed. Finally, the spl5 gene was mapped to a region of 0.048 cM between two markers SSR7 and RM7121. One BAC/PAC contig map covering these markers’ loci and the spl5 gene was constructed through Pairwise BLAST analysis. Our bioinformatics analysis shows that the spl5 gene is located in the 80-kb region between two markers SSR7 and RM7121 with a high average ratio of physical to genetic distance (1.67 Mb/cM) and eighteen candidate genes. The analysis of these candidate genes indicates that the spl5 gene represents a novel class of regulators controlling cell death and resistance response in plants.     

Iron homeostasis in tropical <Emphasis Type="Italic">indica</Emphasis> rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars having contrasting grain iron concentration

Iron homeostasis was studied in two tropical indica rice cultivars viz. Sharbati (high Fe) and Lalat (low Fe) having contrasting grain Fe concentration. Plants were hydroponically grown with 5 concentrations of Fe (0.05, 2, 5, 15, 50 mg L^?1) till maturity. The effect of incremental Fe treatment on the plant was followed by analyzing accumulation of ferritin protein, activities of aconitase enzyme, enzymes of anti-oxidative defense and accumulation of hydrogen peroxide and ascorbic acid. Plant growth was adversely affected beyond 15 mg L^?1 of Fe supplementation and effects of Fe stress (both deficiency and excess) were more apparent on the high Fe containing cultivar Sharbati than the low Fe containing Lalat. Level of ferritin protein and aconitase activity increased up to 5 mg L^?1 of Fe concentration. Lalat continued to synthesize ferritin protein at much higher Fe level than Sharbati and the cultivar also had higher activities of peroxidase, superoxide dismutase and glutathione reductase. It was concluded that the tolerance of Lalat to Fe stress was because of its higher intrinsic ability to scavenge free radicals of oxidative stress for possessing higher activity of antioxidative enzymes. This, together with its capacity to sequester the excess Fe in ferritin protein over a wider range of Fe concentrations made it more tolerant to Fe stress.     

<Emphasis Type="Italic">compact shoot and leafy head 1</Emphasis>, a mutation affects leaf initiation and developmental transition in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The shoot apical meristem (SAM) produces lateral organs in a regular spacing (phyllotaxy) and at a regular interval (phyllochron) during the vegetative phase. In a Dissociation (Ds) insertion rice population, we identified a mutant, compact shoot and leafy head 1 (csl1), which produced massive number of leaves (∼70) during the vegetative phase. In csl1, the transition from the vegetative to the reproductive phase was delayed by about 2 months under long-day conditions. With a reduced leaf size and severe dwarfism, csl1 failed to produce a normal panicle after the transition to reproductive growth. Instead, it produced a leafy panicle, in which all primary rachis-branches were converted to vegetative shoots. Phenotypically csl1 resembled pla mutants in short plastochron but was more severe in the conversion of the reproductive organs to vegetative organs. In addition, neither the expression nor the coding region of PLA1 or PLA2 was affected in csl1. csl1 is most likely a dominant mutation because no mutant segregant was observed in progeny of 67 siblings of the csl1 mutant. CSL1 may represent a novel gene, which functions downstream of PLA1 and/or PLA2, or alternatively functions in a separate pathway, involved in the regulation of leaf initiation and developmental transition via plant hormones or other mobile signals.     

The opposite roles of <Emphasis Type="Italic">OsmiR408</Emphasis> in cold and drought stress responses in <Emphasis Type="Italic">Oryza sativa</Emphasis>

MiR408 is a conserved miRNA family in plants. Although AtmiR408 is generally regarded as participating in stress responses, it still remains obscure whether OsmiR408 modulates tolerance to environmental stress. In the current study, expression of Pre-OsmiR408 and OsmiR408 was found to be induced by cold stress, but repressed by drought stress in the rice cultivar “Kongyu 131”. By comparing the wild type and OsmiR408 transgenic lines, we found that OsmiR408 overexpression conferred enhanced cold tolerance at both the early seedling stage and the young seedling stage. On the other hand, the OsmiR408 transgenic lines exhibited decreased drought tolerance, which is further verified by greater water loss. We also predicted the putative target genes of OsmiR408 and verified the decreased expression of seven targets in OsmiR408 transgenic lines, including four phytocyanins and three atypical target genes. Among them, Os09g29390, a phytocyanin gene, and Os01g53880, an auxin responsive Aux/IAA gene, were down-regulated by cold treatment, which is opposite to the cold-induced expression of OsmiR408. Taken together, our results suggest opposite roles of OsmiR408 in plant responses to cold and drought stresses.     

Salinity tolerance of japonica and indica rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) at the seedling stage

In order to identify the degree of salinity tolerance of the indica and japonica rice groups, 10 varieties were tested under saline and non-saline conditions. Twelve-day-old seedlings were grown in normal culture solution, then initially salinized at an electrical conductivity (EC) of 6 dS/m for 4 days, and finally salinized at an EC of 12 dS/m for the next 14 days. The growth parameters, and Na and K absorption in the shoot were measured to characterize the tolerance level of the two rice groups. Reduction in all growth parameters of tolerant varieties was significantly lower in indica varieties than in japonica varieties. Tolerant indica varieties were good Na excluders, absorbed high amounts of K, and maintained a low Na/K ratio in the shoot. Tolerant japonica varieties also absorbed less Na but were not as good excluders as indica varieties. Shoot K concentration alone did not show any relationship to salinity tolerance. These results indicate that, for all parameters measured, the tolerance level of indica was higher than that of japonica.     

Effect of nitrogen-deficiency on midday photoinhibition in flag leaves of different rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) cultivars

Effects of nitrogen (N)-deficiency on midday photoinhibition in flag leaves were compared between two contrastive Japanese rice cultivars, a traditional japonica cultivar with low yield, cv. Shirobeniya (SRB), and a japonica-indica intermediate type with high yield, cv. Akenohoshi (AKN). Both cultivars were grown under high-N and low-N conditions. At midday, low-N supply resulted in more intensive reductions in net photosynthetic rate, stomatal conductance, maximal quantum yield of photosystem II (PSII) and quantum yield of PSII electron transport in SRB than in AKN, indicating that SRB was more strongly photoinhibited than AKN under low-N condition. At midday, the low-N plants of two cultivars showed higher superoxide dismutase (SOD) activities than the high-N plants. However, ascorbate peroxidase (APX) activity was maintained in AKN but significantly decreased in SRB under low-N condition (N-deficiency). In contrast, hydrogen peroxide (H₂O₂) content in SRB significantly increased under low-N condition, indicating that the susceptibility to midday photoinhibition in the low-N plants of SRB is related to the increased H₂O₂ accumulation. It is suggested that the midday depression in photosynthesis may be a result of oxidative stress occurring in the low-N plants in which antioxidant capacity is not enough to cope with the generation of H₂O₂. Therefore, H₂O₂-scavenging capacity could be an important factor in determining the cultivar difference of midday photoinhibition in flag leaves of rice under low-N condition.     

Physiological and proteomic analysis of mycorrhizal <Emphasis Type="Italic">Pinus massoniana</Emphasis> inoculated with <Emphasis Type="Italic">Lactarius insulsus</Emphasis> under drought stress

This study aimed to investigate physiological and protein expression alterations of mycorrhizal Pinus massoniana Lamb. inoculated with Lactarius insulsus in response to drought stress. The P. massoniana seedlings were inoculated with L. insulsus (Li group) and ectomycorrhized fungal-free filtrate (control, CK group), respectively. After two and a half years, all the plants were exposed to a simulate drought condition without water for 21 days. The soil relative water content (SRWC), wilting degree (WD) and wilting rate (WR) of the plants were measured and root proteome was analyzed based on two-dimensional gel electrophoresis (2-DE), respectively at four time points as 0, 7, 14 and 21 days during the whole drought period. Finally, the electrospray ionization mass spectrometry (ESI-MS) was used to identify the differentially expressed proteins (DEPs) between Li and CK groups. The SRWC was higher, while WR and WD were lower in Li group, compared with that in CK group. Based on 2-DE and ESI-MS, 22 DEPs were identified between Li and CK groups during drought stress. Among them, four proteins had the annotated information in relevant databases, including 1,4-benzoquinone reductase, PSCHI4, ribosomal protein L16 (RPL16) and AINTEGUMENTA-like (AIL) protein. Mycorrhizal P. massoniana inoculated with L. insulsus achieved an enhanced drought resistance as compared to the non-mycorrhizal, and the altered protein expressions such as 1,4-benzoquinone reductase, PSCHI4, RPL16, and AIL might contribute to the improved resistance under drought stress.