Bulked Segregant RNA-Seq Analysis of Pollinated Pistils Reveals Genes Influencing Spikelet Fertility in Rice

Prezygotic isolation is important for successful fertilization in rice, significantly affecting yield. This study focused on F_(5:6) generation plants derived from inter-subspecific crosses(Nipponbare × KDML105) with low(LS) and high seed-setting rates(HS), in which normal pollen fertility was observed. However, LS plants showed a reduced number of pollen grains adhering to the stigma and fewer pollen tubes reaching the ovules at 4-5 h post-pollination, compared with HS plants. Bulked segregant RNA-Seq analysis of pollinated pistils from the HS and LS groups revealed 249 and 473 differentially expressed genes(DEGs), respectively. Kyoto Encyclopedia of Genes and Genomes analysis of the HS and LS-specific DEGs indicated enrichment in metabolic pathways, pentose and glucuronate interconversions, and flavonoid biosynthesis. Several of these DEGs exhibited co-expression with pollen development genes and formed extensive clusters of co-expression networks. Compared with LS pistils, enzyme genes controlling pectin degradation, such as OsPME35 and OsPLL9, showed similar expression patterns, with higher levels in HS pistils pre-pollination. Os02g0467600, similar to cinnamate 4-hydroxylase gene(CYP73), involved in flavonoid biosynthesis, displayed higher expression in HS pistils post-pollination. Our findings suggest that OsPME35, OsPLL9, and Os02g0467600 contribute to prezygotic isolation by potentially modifying the stigma cell wall(OsPME35 and OsPLL9) and controlling later processes such as pollen-stigma adhesion(Os02g0467600) genes. Furthermore, several DEGs specific to HS and LS were co-localized with QTLs and functional genes associated with spikelet fertility. These findings provide valuable insights for further research on rice spikelet fertility, ultimately contributing to the development of high-yielding rice varieties.

Avoidance of Linkage Drag Between Blast Resistance Gene and the QTL Conditioning Spikelet Fertility Based on Genotype Selection Against Heading Date in Rice

Previous study showed that a linkage drag between a blast resistance gene Pi25（t） and QTLs conditioning spikelet fertility （qSF-6） and number of filled grains per panicle （qNFGP-6） was detected on the short arm of chromosome 6. A larger population was used for further verification, and the results confirmed the linkage drag between the blast resistance gene and QTL conditioning spikelet fertility, other than QTL conditioning number of filled grains per panicle. Breakdown or avoidance of the linkage drag could be achieved by selection against the genotype background of a heading-date gene （qHD-7） that resided in the region between RM2 and RM214 on chromosome 7. For further validation, two lines with almost identical genotypes on all chromosomal regions except the Pi25（t） region on chromosome 6 were chosen to develop a new population The results showed that qSF-6 could be further subdivided into qSF-6-1 and qSF-6-2. When the genotype of the region between RM2 and RM214 was from rice variety Zhong 156, the linkage drag between Pi25（t） and qSF-6-2 was detected and the allele of qSF-6-2 from rice variety Gumei 2 reduced the spikelet fertility. When the genotype of the region between RM2 and RM214 was from Gumei 2, no linkage drag was detected. This indicates that the linkage drag between the blast resistance gene and the QTL conditioning spikelet fertility could be broken down or avoided under a certain background genotype selection against heading-date and provides a marker aided solution for high level of blast resistance and yield breeding in rice and other crops as well.

Temperature Difference Between the Air and Organs of Rice Plant and Its Relation to Spikelet Fertility

Based on the experiment of measuring panicles and leaves, air temperature, and humidity above the canopy of rice cultivars after heading in 2005 and 2006, we investigated the temperature difference （TD） between the air and organs of rice plant and its relationship with spikelet fertility. The results showed that TDs between the air and organs of rice varied with air temperature, air humidity, and plant type. For similar air humidity, TDs were lower at the air temperature of 28.5℃ than at higher temperature of 35.5℃, whereas for the same air temperature, the TDs decreased as the air humidity increased. TDs were also affected by plant type of the cultivars. Erect panicle cultivars showed higher TDs than those with droopy panicles under similar climatic conditions, and cultivars with panicles above flag leaf （PAFL） had higher TDs than those with panicles below the flag leaf （PBFL）. Cultivars grown in a location with lower air humidity and higher temperature, such as Taoyuan, China, had higher spikelet fertility than those in higher humidity under the similar air temperature during the grain filling stage. This is partially attributed to the larger TDs under the lower humidity. Rowspacing and the ratio of basal-tillering to panicle-spikelet fertilizer showed a significant influence on TD and subsequently on spikelet fertility, suggesting the possibility of increasing spikelet fertility by agronomic management.

Effects of Heat Stress at Vegetative and Reproductive Stages on Spikelet Fertility

Seed-setting rate, yield components and grain quality traits of 169 accessions from an exotic rice germplasm were tested under high temperatures from 40 oC to 45 oC for 6 h during the daytime at the vegetative and reproductive stages, respectively. The results showed that heat stress significantly decreased the seed-setting rate of all the accessions, but the heat stress effects varied among accessions. Based on the decreases in seed-setting rate at high temperatures, N22 was the most tolerant, followed by AUS17, M9962, SONALEE and AUS16. Moreover, the reductions in seed-setting rate and yield under heat stress were more serious at the vegetative stage （45 d before heading） than at the booting stage （15 d before heading）. In addition, heat stress also affected grain quality, especially by conferring chalkiness to most of the accessions, but SONALEE did not change much. The heat-tolerant accessions identified here and the phenotype protocols developed could be used in future genetic studies and breeding programmes focused on heat tolerance.

Specific Spikelet Fertility as an Indicator of Cold Tolerance Identification at Booting Stage in Rice

By using the parents, Kunmingxiaobaigu, a landrace variety with cold tolerance from Yunnan, China, and Towada, a cold-sensitive variety bred in Japan, as well as their F8 and F9 RIL progenies as test materials, cold tolerance of rice at the booting stage in 2002 and 2003 under four different altitudes （environments）, and the correlationship between spikelet fertility and specific spikelet fertility （spikelet fertility of nine spikelets from the 3rd to the 5th spikelets of three primary branches at the top of each panicle） of single plant were analyzed, The data revealed a significant correlationship between spikelet fertility and specific spikelet fertility with the range from 0.7364 to 0.9011. However, under the cold stress environments, the correlation coefficients were higher than those without cold stress. On the other hand, the panicle exsertion was correlated to both spikelet fertility and specific spikelet fertility. Moreover, the heredity analysis suggested that specific spikelet fertility could replace spikelet fertility as an identification indicator, while panicle exsertion could be used as a partial indicator of cold tolerance identification at the booting stage in rice.

Response of spikelet water status to high temperature and its relationship with heat tolerance in rice

In rice, high-temperature stress(HT) during flowering results in decreased grain yield via a reduction in spikelet fertility;however, the effect of plant water status on spikelet fertility under HT remains unknown. To investigate the relationship between spikelet water status and spikelet fertility under HT, two experiments were performed under temperature-controlled conditions using four genotypes with varying tolerance to HT. Rice plants were exposed to HT for seven consecutive days during the flowering stage under three soil water treatments(soil water potential 0, -20, and -40 kPa), as well as under hydroponic conditions in a separate experiment. HT significantly decreased spikelet fertility, pollen fertility, and anther dehiscence under each of the three water treatments. HT significantly increased the spikelet transpiration rate, and this change was accompanied by a significant decrease in the internal temperature of the spikelets. HT decreased pollen grain diameter in heat-sensitive genotypes. HT had varying effects on the water potential of panicles and anthers but increased anther soluble-sugar concentration. Different aquaporin genes showed different expression profiles under HT, and the expression levels of PIPs for plasma membrane intrinsic proteins and TIPs for tonoplast intrinsic proteins increased in anthers but decreased in glumes.Correlation analyses showed that anther dehiscence and pollen(spikelet) fertility were tightly associated with anther water status, and the expression levels of almost all anther aquaporin genes were significantly correlated with anther dehiscence under HT. In summary, an increased spikelet transpiration rate and decreased internal spikelet temperature were associated with alleviation of the effects of HT in rice genotypes with varying degrees of heat tolerance, and the response of spikelet water status to HT, involving increased total expression of aquaporins and soluble sugar content, thereby improved pollen fertility,anther dehiscence, and spikelet fertility, especially in heat-resistant genotypes. The heat-resistant genotypes N22 and SY63 may adopt different approaches to reduce heat damage.

The wheat sucrose synthase gene TaSus1 is a determinant of grain number per spike

Some haplotypes of the sucrose synthase gene TaSus1 are associated with thousand-grain weight(TGW)in wheat(Triticum aestivum L.).However,no mutations have been identified within the gene to test this association.The effects of TaSus1 on grain number per spike(GNS)also are largely unknown.Our previous genome-wide association study identified TaSus-A1 as a candidate gene controlling fertile spikelet number per spike(FSN).In the present study,we generated two independent mutants for the three TaSus1 homoeologs by CRISPR/Cas9-mediated genome editing.The triple mutants displayed lower FSN,GNS,grain number per spikelet(GNST),and TGW than wild-type plants.In 306 hexaploid wheat accessions,two single-nucleotide polymorphisms in TaSus-A1 contributed differently to GNS.Introgression of the two alleles into a wheat genetic background confirmed their effects.The alleles differed in geographical distribution among the accessions.

Test on Indica-compatibility of Candidate Indicacompatible Japonica Lines in Rice

[Objective] The aim was to breed the indica-compatible japonica lines（ICJLs）.[Method] The indica-compatibility of 5 candidate ICJLs were tested based on the pollen fertility and spikelet fertility of F1 hybrids with 6 indica testers,6 japonica testers and 6 middle testers.[Result] Candidate ICJL G2123,G2417,G2410 and G3005 were grouped into the exceptionally（Specific）indica-compatible lines based on their high indica-compatibility and low japonica-compatibility.Candidate ICJL G2615 was grouped into the non-compatible lines on their low indica-compatibility and low japonica-compatibility.[Conclusion] The testers,fertility identification standards,test methods of pollen fertility and spikelet fertility were discussed when the compatibility of CICJLs was tested.The reason of the indica-compatibility in G2417 was analyzed.

Responses of Yield Characteristics to High Temperature During Flowering Stage in Hybrid Rice Guodao 6

By sowing at different dates during 2005 and 2006 both in paddy fields and greenhouse, a super hybrid rice combination Guodao 6 and a conventional hybrid rice combination Xieyou 46 （as control） were used to analyze the differences in heat injury index, seed setting rate, grain yield and its components. Guodao 6 showed more stable yield and spikelet fertility, and lower heat injury index than Xieyou 46. Further studies indicated that the spikelet sterility is positively correlated with the average daily temperature and the maximum daily temperature, with the coefficients of 0.8604 and 0.9850 （P〈0.05） respectively in Guodao 6. The effect of high temperature injury on seed setting caused by maximum daily temperature was lower than that by average daily temperature during the grain filling stage.