rspcr什么时候用Introgression of a functional epigenetic OsSPL14WFP allele into elite indica rice genomes greatly improved panicle traits and grain yield

Increased rice production per unit area is the mandate to feed the increasing population and to achieve global food security in the near future. However, the genetic gain in rice yield potential has stagnated for several decades, especially in the tropical rice-growing regions of Asia^20,21. A recent report by Dingkuhn et al.²¹ reflects the stagnation in yield potential, in which simultaneous yield evaluation of 12 elite irrigated rice cultivars, including old and current varieties in the Philippines, showed that the current best tropical rice varieties do not have higher yield potential than some old popular varieties developed in the 1970s and 1980s. In addition, although many yield QTLs/genes are cloned in rice, those genes have not been reported to increase the grain yield of indica rice cultivars in the field⁶. Recently, one gene, SPIKLET NUMBER (SPIKE), originating from a tropical japonica landrace has shown a high possibility to increase yield potential in indica rice backgrounds²². The identification of new yield-enhancing genes, critical evaluation of cloned genes, and the application of effective alleles in actual breeding programs are expected to improve the genetic yield potential of elite rice cultivars. In this study, we presented a method for allele evaluation and allele application for breeding. Interactions between the QTLs/genes and genetic backgrounds and between the QTLs/genes and environment have been reported in many vegetables and cereals^23,24,25,26. For the clear evaluation of the effect of yield-enhancing alleles, the same yield-positive alleles of Gn1a and OsSPL14 genes were tested in different indica cultivar backgrounds and in two cropping seasons. Moreover, to exclude the influence of other donor DNA segments, the alleles’ effects were compared at similar genome status except for the target locus by using segregating BC₃F₂ progenies, which were derived from a single heterozygous BC₃F₁ plant. Also, background genotyping by high-density SNP markers provided clearer information on the genome reconstitution of parents in the breeding lines than the conventional low-density SSR markers. Our evaluation method may produce solid data on allele mining effects and will be applicable for evaluating other trait-related genes.

In previous reports, the Gn1a^Habataki (type 3) allele significantly increased GNPP by 21% and 28~37% in temperate japonica cultivar (Koshihikari and Sasanishiki) backgrounds, respectively^13,15. In addition, the Gn1a-indica allele increased grain yield in Kongyu 131²⁷ with elite japonica background. However, in our study, the Gn1a-type 3 allele derived from lines Habataki, ST12, and ST6 was not effective in several indica cultivar backgrounds that had the Gn1a-type 2 allele. These results indicate that the Gn1a-type 2 allele has the same functionality with the Gn1a-type 3 allele although three SNPs were found in the promoter region between the two alleles¹⁹. As reported in a previous study¹³, the 16-bp deletion found in both alleles might be directly associated with GNPP rather than the three SNPs. However, the expression level of Gn1a in young panicles needs to be compared among the three allele types in a further study. The allele of the Gn1a gene with high GNPP was originally identified from indica-type variety Habataki¹³. Habataki is a Japanese high-yielding variety derived from a cross between two Korean Tongil-type breeding lines, Milyang 42 and Milyang 25²⁸. More than 90% of the Tongil genome is derived from indica parents²⁹. The pedigree of Habataki²⁸ suggests that variety Habataki might have much indica genomes, probably IR8 (Green Revolution variety) and IR24 developed by IRRI, which were widely distributed in East Asian countries, and those were also used as breeding sources of the modern indica rice cultivars³⁰. This information suggests that the Gn1a^Habataki allele might originate from high-yielding indica lines and the allele might be continuously selected in indica rice breeding programs. This might be the cause of a yield plateau in modern indica rice cultivars. Consistent with this, out of our 12 recipient cultivars, four had exactly the same allele as Habataki at the sequence level¹⁹ and another seven indica varieties had the Gn1a-type 2 allele, while the other one had the Gn1a-type 1 allele. The yield-positive Gn1a allele was also found in Chinese high-yielding indica varieties 93–11 and Teqing²⁶. Hence, most of the elite indica varieties might already have yield-positive Gn1a alleles (type 2 or type 3), and these indica alleles will be effective in many japonica cultivars having the Gn1a-type 1 allele, as shown in previous reports. Gn1a together with Gn1b (another QTL near the Gn1a locus in Habataki) exhibited an additive effect in GNPP by ~45% in a Koshihikari background¹³. In line YP15–752, we found that the edge of the short arm of chromosome 1 (~5.5 Mb segment) containing both Gn1a and Gn1b loci was derived from Habataki (Fig. 2B). But, GNPP did not increase significantly in this line, suggesting that the Gn1b allele might also be the same between Habataki and the background of YP15–752 of IRRI 146.

In this study, two different yield-positive alleles of the OsSPL14 gene were tested in indica backgrounds. One of the donors, japonica variety Aikawa1, showed high GNPP with very low TN (~3 tillers per plant) in an IRRI field in the Philippines. Some of our breeding lines derived from Aikawa1 also exhibited very low TN with some other undesirable agronomic traits, which might be caused by an indica x japonica cross. Therefore, for the critical evaluation of the OsSPL14^Aikawa1 allele, removing the donor genome in the breeding lines is needed for further study. However, a severe reduction in TN (45.3%) was also reported in the NIL OsSPL14^IPA1 in the japonica background of Xiushui 11¹¹. Therefore, breeders need to consider the effect of the OsSPL14^IPA1 allele on the trait tiller number. Unlike Aikawa1, cultivar ST12 produced approximately 12 tillers per plant in the IRRI field. Also, there was no severe reduction in TN in our NILs having the OsSPL14^WFP allele. Thus, the NILs and the newly bred high-yielding lines produced 8.7~11.7 tillers per plant in different backgrounds. However, a minor tendency of a reduction in TN was observed in some backgrounds (Tables 1 and 2; Supplementary Tables S3, S4, and S6). But, this reduction is not of a considerable amount in current breeding programs, and we successfully improved grain yield from the five elite indica backgrounds using the OsSPL14^WFP allele. The OsSPL14^WFP allele significantly increased PBN and SBN, which resulted in high GNPP in all high-yielding lines with an indica background tested across cropping seasons. Similarly, this allele also improved GNPP (51.3%) in Nipponbare with a japonica background¹⁶. These data suggest that the OsSPL14^WFP allele can provide a strong genetic gain in grain production across diverse genetic backgrounds and environments. Recent molecular studies revealed the function of IPA1/WFP/OsSPL14 in both shoot and panicle branching. IPA1/WFP/OsSPL14 transcription factor binds directly to the promoter sequences of the key regulators of tillering, OsTB1, and of panicle architecture, DEP1³¹. In addition, IPA1/WFP/OsSPL14 protein level is regulated by IPA1 INTERACTING PROTEIN1 (IPI1) in a tissue-specific ubiquitination manner and the ipi1 mutant showed both increased tiller number and panicle size³². IPA1/WFP/OsSPL14 together with DWARF 53 protein is the key repressor of the strigolactones signaling pathway which controls tillering in rice³³. These reports suggest that IPA1/WFP/OsSPL14 plays a key role in two major architecture traits, tillering and panicle branching. The newly identified natural epigenetic allele OsSPL14^ipa1–2D having tandem repeat sequences of OsSPL14 promoter region exhibited high GNPP without a tiller reduction¹⁸. Therefore, the application of proper alleles of OsSPL14 can improve plant architecture. Furthermore, the identification of new functional alleles of the known yield-related genes may extend breeders’ choice in attempting to increase yield.

To enhance trait performance using an additive effect of different genes, gene pyramiding in the same background is widely used in MAS breeding programs. In japonica variety Nipponbare possessing the Gn1a-type1 allele, lines combined with the two genes (Gn1a-type 3 and OsSPL14^WFP) showed higher GNPP (~70.4%) than single-gene introgressed lines with Gn1a by 21.5% and with OsSPL14^WFP by 51.3%, respectively¹⁶. However, we could not see a significant additive effect of two genes in indica backgrounds because of the absence of a Gn1a-type 3 effect in Gn1a-type 2 backgrounds (Table 1).

Our combined breeding approach was successful in breeding high-yielding lines. Five selected lines showed higher grain yield than their recipient cultivars as well as current top-yielding tropical rice cultivars such as IRRI 146, IRRI 154, and IRRI 156, which were also included as high-yielding checks in the experiment on yield comparison between old and current tropical varieties by Dingkuhn et al.²¹. Yield improvement of our breeding lines might be possible because of the strong effect of the OsSPL14^WFP allele and other improved agronomic traits driven by phenotypic selection. One remarkable common phenotype among five breeding lines is increased stem diameter (Fig. 4F–G). Similarly, the OsSPL14^ipa1–2D allele also promoted culm diameter¹⁸, suggesting that the OsSPL14^WFP allele might increase stem thickness. Four breeding lines showed increased PH. This trait, providing a little bit larger plant size, might also influence yield increase. Another character, HI, might be involved in yield enhancement. A positive correlation between grain yield and HI was reported in rice^34,35. Overall, our breeding lines showed a higher HI value than the recurrent parents (Table 2). Yield improvement was more remarkable in varieties with high yield potential of Latin America³⁶, including CT5803, CT5805, and IRGA427, suggesting that the genetic combining ability of ST12 was much better with Latin American rice genotypes than IRRI high-yielding indica cultivars. These high-yielding lines need to be tested in Latin American countries in the future. In our breeding program, we could not obtain very good breeding lines in other IRRI variety backgrounds such as IRRI 146, IRRI 154, and IRRI 156. In these backgrounds, GNPP increased but actual yield did not improve significantly because of other negative agronomic traits. Currently, precise molecular breeding is available because of low-cost genomics approaches. To remove undesirable phenotypes, we need to further eliminate the donor DNA segments and/or trim the surrounding sequences of the OsSPL14^WFP gene. In this study, the size of the introgressed DNA segment containing the target locus was higher than 1.6 Mb (1.6–11.0 Mb) (Fig. 2). This can lead to linkage drag in some specific backgrounds. However, for precise MAS, recombinant selection in early breeding generations is required. High-density background genotyping using genome-wide SNP markers or next-generation sequencing techniques provide a precise genome map of the breeding lines and enable us to know the number and size of the chromosome segments derived from the donor line. Eventually, the donor genome rate will be decreased by genotyping of the anchored donor-removing/donor-trimming with additional backcrossing and selfing. Through this process, we can expect actual yield improvement from IRRI top-yielding variety backgrounds.

In terms of rice plant architecture, long, erect, and V-shaped leaves will be beneficial to capture light energy, resulting in high photosynthetic efficiency and high grain yield^37,38,39. Line YP16–40 generated erect V-shaped leaves, elongated flag leaf, and dark green leaves (Supplementary Fig. S5), which were probably caused by parental genome effects or epistasis between the parental genomes. This ideal leaf morphology needs to be tested in a further study to enhance yield.

To improve genetic yield potential, the identification of new yield-enhancing genes is important. Along with this, critical evaluation of the identified genes in several elite backgrounds and pyramiding of effective alleles in the same background are also crucial. Here, we showed a strong possibility to increase rice grain yield in elite indica backgrounds using the OsSPL14^WFP allele. The IPA1/WFP/OsSPL14 gene may emerge as a new Green Revolution gene after the semi-dwarf (sd1) gene because of its importance in expressing improved rice plant architecture⁴⁰. The epigenetic OsSPL14^WFP allele is unique throughout rice germplasm and so this allele will be effective in most rice cultivars, including both indica and japonica types. We believe that this allele upgrades the current stagnant yield potential of tropical indica varieties and also contributes genetic yield enhancement in many favorite local varieties from different countries.