Identification of QTLs for Plant Height and Its Components by Using Single Segment Substitution Lines in Rice (Oryza sativa)

QTLs for plant height and its components on the substituted segments of fifty-two single segment substitution lines （SSSLs） in rice were identified through t-test （P〈0.001） for comparison between each SSSL and recipient parent Huajingxian 74. On the 14 substituted segments, 24 QTLs were detected, 10 for plant height, 2 for panicle length, 4 for length of the first internode from the top, 5 for length of the second internode from the top and 3 for length of the third internode from the top, respectively. All these QTLs were distributed on nine rice chromosomes except chromosomes 5, 9 and 11. The additive effect ranged from -4.08 to 3.98 cm, and the additive effect percentages varied from -19.35% to 10.43%.

Gap-free genome assembly and comparative analysis reveal the evolution and anthocyanin accumulation mechanism of Rhodomyrtus tomentosa

Rhodomyrtus tomentosa is an important f leshy-fruited tree and a well-known medicinal plant of the Myrtaceae family that is widely cultivated in tropical and subtropical areas of the world.However,studies on the evolution and genomic breeding of R.tomentosa were hindered by the lack of a reference genome.Here,we presented a chromosome-level gap-free T2T genome assembly of R.tomentosa using PacBio and ONT long read sequencing.We assembled the genome with size of 470.35 Mb and contig N50 of∼43.80 Mb with 11 pseudochromosomes.A total of 33382 genes and 239.31 Mb of repetitive sequences were annotated in this genome.Phylogenetic analysis elucidated the independent evolution of R.tomentosa starting from 14.37MYA and shared a recent WGD event with other Myrtaceae species.We identified four major compounds of anthocyanins and their synthetic pathways in R.tomentosa.Comparative genomic and gene expression analysis suggested the coloring and high anthocyanin accumulation in R.tomentosa tends to be determined by the activation of anthocyanin synthesis pathway.The positive selection and up-regulation of MYB transcription factors were the implicit factors in this process.The copy number increase of downstream anthocyanin transport-related OMT and GST gene were also detected in R.tomentosa.Expression analysis and pathway identification enriched the importance of starch degradation,response to stimuli,effect of hormones,and cell wall metabolism during the f leshy fruit development in Myrtaceae.Our genome assembly provided a foundation for investigating the origins and differentiation of Myrtaceae species and accelerated the genetic improvement of R.tomentosa.

Fine mapping of an adult-plant resistance gene to powdery mildew in soybean cultivar Zhonghuang 24

Powdery mildew(PM),caused by the fungus Microsphaera diffusa,causes severe yield losses in soybean[Glycine max(L.)Merr.]under suitable environmental conditions.Identifying resistance genes and developing resistant cultivars may prevent soybean PM damage.In this study,analysis of F_(1),F_(2),and F8:11 recombinant inbred line(RIL)populations derived from the cross between Zhonghuang 24(ZH24)and Huaxia 3(HX3)indicated that adult-plant resistance(APR)to powdery mildew in the soybean cultivar(cv.)ZH24 was controlled by a single dominant locus.A high-density genetic linkage map of the RIL population was used for fine mapping.The APR locus in ZH24 was mapped to a 281-kb genomic region on chromosome 16.Using 283 susceptible plants of another F2 population,the candidate region was finemapped to a 32.8-kb genomic interval flanked by the markers InDel14 and Gm16_428.The interval harbored five genes,including four disease resistance(R)-like genes,according to the Williams 82.a2.v1 reference genome.Quantitative real-time PCR assays of candidate genes revealed that the expression levels of Glyma.16g214300 and Glyma.16g214500 were changed by M.diffusa infection and might be involved in disease defense.Rmd_B13 showed all-stage resistance(ASR)to PM in soybean cv.B13.An allelism test in the F2 segregating population from the cross of ZH24 × B13 suggested that the APR locus Rmd_ZH24 and the ASR locus Rmd_B13 may be allelic or tightly linked.These results provide a reference marker-assisted selection in breeding programs.

Rice gene OsUGT75A regulates seedling emergence under deep-sowing conditions

Poor seedling emergence is a challenge for direct seeding of rice under deep-sowing field conditions.Here we reveal that UDP-glucosyltransferase OsUGT75A promotes rice seedling emergence under deepsowing conditions by increasing shoot length.Expression of OsUGT75A was higher in the middle regions of the shoot and in shoots under deep-sowing conditions.Levels of free abscisic acid(ABA)and jasmonates(JA)were higher in shoots of OsUGT75A mutants than in those of wild-type plants,and OsUGT75A mutants were more sensitive to ABA and JA treatments.Reduced shoot length was attributed to higher ABA INSENSITIVE 3(OsABI3)expression and lower JASMONATE-ZIM domain protein(OsJAZ)expression in shoots.Shoot extension by OsUGT75A is achieved mainly by promotion of cell elongation.An elite haplotype of OsUGT75A associated with increased shoot length was identified among indica rice accessions.OsUGT75A acts to increase seedling emergence under deep-sowing conditions.

Magnesium fertilizer application increases peanut growth and pod yield under reduced nitrogen application in southern China

This study investigated the effect of magnesium application on peanut growth and yield under two nitrogen(N)application rates in acidic soil in southern China.The chlorophyll content,net photosynthetic rate and dry matter accumulation of the N-sensitive cultivar decreased under reduced N treatments,whereas no effect was observed on the relevant indicators in the N-insensitive variety GH1026.Mg application increased the net photosynthetic rate by increasing the expression of genes involved in chlorophyll synthesis and Rubisco activity in the leaves during the pegging stage under 50%N treatment,while no effect on the net photosynthetic rate was observed under the 100%N treatment.The rate of dry matter accumulation at the early growth stage,total dry matter accumulation and pod yield at harvest increased after Mg application under 50%N treatment by increasing the transportation of assimilates from stems and leaves to pods in both peanut varieties,whereas no effect was found under 100%N treatment.Moreover,Mg application increased the NUE under 50%N treatment.No improvement of NUE in either peanut variety was found under 100%N treatment,while Mg application under the 50%N treatment can obtain a higher economic benefit than the 100%N treatment.In acidic soil,application of 307.5 kg ha^(-1)of Mg sulfate fertilizer under 50%reduced nitrogen application is a suitable fertilizer management measure for improving carbon assimilation,NUE and achieve high peanut yields in southern China.

Prospects of utilization of inter-subspecific heterosis between indica and japonica rice

The Asian cultivated rice(Oryza sativa L.) grown worldwide is divided into two subspecies, indica and japonica. It is well known that the heterosis of inter-subspecies is usually stronger than that of intra-subspecies. Since the 1970 s, indica hybrid rice, an intra-subspecific hybrid rice, has being widely used in China and even in the world. However, the inter-subspecific hybrid rice between indica and japonica is still unavailable. The major obstacle is the hybrid sterility of the inter-subspecies. In recent decades, the genetic and molecular basis of indica-japonica hybrid sterility was understood more and more clearly. Some breeding approaches for overcoming inter-subspecific hybrid sterility were proposed and used to develop the indicajaponica hybrid rice. The updated understanding will offer new approaches for development of breeding lines for overcoming indica-japonica hybrid sterility, which facilitates developing of inter-subspecific hybrid rice.

Detection of QTLs for Important Agronomic Traits and Analysis of Their Stabilities Using SSSLs in Rice

Single segment substitution lines （SSSLs） each with a single chromosome segment from a donor under the same genetic background as the recipient were developed in rice by advanced backcrossing and molecular marker-assisted selection. Using the SSSLs, the QTLs for the important agronomic traits in rice would be detected under different environmental conditions. Detection of the QTLs controlling 22 important traits in rice was done with 32 SSSLs by the randomized block design in 2-4 cropping seasons. 59 QTLs were detected and distributed on chromosomes 1, 2, 3, 4, 6, 7, 8, 10, and 11, of which 18 QTLs were detected more than twice. Only 30.5% of the QTLs were detected repeatedly in different cropping seasons. Most of the QTLs of important agronomic traits were of little additive effects and instability. The QTLs controlling the traits, such as grain weight, grain length, ratio of grain length to width, and heading date were relatively stable. The stable QTLs usually had larger additive effects and were less affected by environment. The QTLs for the important agronomic traits were detected using the SSSLs in rice with high resolution under different environmental conditions. The instability of the QTLs may be the basis of the variation of rice plants during growth and development. It would be the genetic basis for improving yield and quality in rice cultivars by farming methods.

Target chromosome-segment substitution: A way to breeding by design in rice

Progress in plant breeding depends on the development of genetic resources,genetic knowledge,and breeding techniques.The core of plant breeding is the use of naturally occurring variation.At the beginning of the post-genomic era,a new concept of"breeding by design"was proposed,which aims to control all allelic variation for all genes of agronomic importance.In the past two decades,we have applied a three-step strategy for research on rice breeding by design.In the first step,we constructed a singlesegment substitution line(SSSL)library using Huajingxian 74(HJX74),an elite xian(indica)rice cultivar,as the recipient in which to assemble genes from the rice AA genome.In the second step,we identified a series of desirable genes in the SSSL library.In the third step,we designed new rice lines,and achieved the breeding goals by pyramiding target genes in the HJX74-SSSL library.This review introduces the background,concept,and strategy of breeding by design,as well as our achievements in rice breeding by design using the HJX74-SSSL platform.Our practice shows that target chromosome-segment substitution is a way to breeding by design.

Identification of Multiple Alleles at the Wx Locus and Development of Single Segment Substitution Lines for the Alleles in Rice

The microsatellite markers 484/485 and 484/W2R were used to identify the multiple alleles at the Wx locus in rice germplasm. Fifteen alleles were identified in 278 accessions by using microsatellite class and G-T polymorphism. Among these alleles, （CT）12-G, （CT）15-G, （CT）16-G, （CT）17-G, （CT）18-G and （CT）21-G have not been reported. Seventy-two single-segment substitution lines （SSSLs） carrying different alleles at the Wx locus were developed by using Huajingxian 74 with the （CT）11-G allele as a recipient and 20 accessions containing 12 different alleles at the Wx locus as donors. The estimated length of the substituted segments ranged from 2.2 to 77.3 cM with an average of 17.4 cM.

Strategies on Sample Size Determination and Qualitative and Quantitative Traits Integration to Construct Core Collection of Rice (Oryza sativa)

The development of a core collection could enhance the utilization of germplasm collections in crop improvement programs and simplify their management. Selection of an appropriate sampling strategy is an important prerequisite to construct a core collection with appropriate size in order to adequately represent the genetic spectrum and maximally capture the genetic diversity in available crop collections. The present study was initiated to construct nested core collections to determine the appropriate sample size to represent the genetic diversity of rice landrace collection based on 15 quantitative traits and 34 qualitative traits of 2 262 rice accessions. The results showed that 50-225 nested core collections, whose sampling rate was 2.2%-9.9%, were sufficient to maintain the maximum genetic diversity of the initial collections. Of these, 150 accessions （6.6%） could capture the maximal genetic diversity of the initial collection. Three data types, i.e. qualitative traits （QT1）, quantitative traits （QT2） and integrated qualitative and quantitative traits （QTT）, were compared for their efficiency in constructing core collections based on the weighted pair-group average method combined with stepwise clustering and preferred sampling on adjusted Euclidean distances. Every combining scheme constructed eight rice core collections （225, 200, 175, 150, 125, 100, 75 and 50）. The results showed that the QTT data was the best in constructing a core collection as indicated by the genetic diversity of core collections. A core collection constructed only on the information of QT1 could not represent the initial collection effectively. QTT should be used together to construct a productive core collection.

Abnormal Structure of Embryo Sac in Autotetraploid Rice

The structures of mature embryo sacs in 13 genetic stock lines of autotetraploid rice （Oryza sativa L.）, including indica, japonica and javanica, were studied by using the whole-mount stain-clearing laser scanning confocal microscopy （WCLSM）. Among the 13 autotetraploid rice, the majority of ovaries possess normal polygonum-type embryo sacs, while a few ovaries were characterized by abnormal embryo sacs. The abnormalities of embryo sacs could be classified into six categories, i. e. no female germ unit, abnormal polar nuclei, embryo sac degeneration, no egg apparatus, small embryo sac and ＇double set＇ of embryo sacs. The frequency of abnormal embryo sac in japonica （26.6%） was higher than that in indica （19.34%）. In addition, the major abnormalities in each autotetraploid line varied, suggesting that the abnormalities may be related to the genotypes of the varieties.

Construction and Testing of a Primary Microsatellite Database of Major Rice Varieties in China

Sixty-three major inbred varieties and parental lines of major F1 hybrids used in the commercial rice production in China were assayed with rice microsatellites screened in a previous study and additional microsatellites on four chromosomes. A set of 24 markers was selected and proposed for its application in the variety identification of rice, which are distributed on all the 12 rice chromosomes with 2 markers on each chromosome. The 63 major varieties and parental lines, as well as 41 major F1 hybrids, were genotyped with the markers. Alleles detected in each line at each marker locus were verified. By matching marker genotypes of corresponding F1, maternal and paternal lines of hybrid rice, high reliability of the maternal lines was verified, data on the paternal lines were modified, and a false hybrid was removed. A database containing genotype data of 103 major rice vadeties and parental lines at the 24 marker loci was constructed and analyzed.

Global Analysis of Cytosine Methylation and Proteome Under Cold Treatment in Brassica napus

Cytosine methylation/demethylation plays pivotal roles in regulating gene expression at a genome-wide level. However, limited reports are available to reveal correlating changes of cytosine methylation and proteomic expression in Brassica napus so far. Therefore, in the present study, global cytosine methylation and proteome were analysed in B. napus after cold treatment by methylation-sensitive amplified polymorphism （MSAP） and two-dimensional protein electrophoresis technology （2-DE）. The results showed that the lowered genome-wide DNA methylation status was revealed after cold treatment, and about 0.88% of discrepancy in DNA methylation was detected between the non-flowering and flowering plants after cold treatment. Moreover, the 52 significantly up-regulated proteins emerged in comparison with the 36 down-regulated proteins, as well as the 14 proteins exclusively detected in the flowering plants. Intriguingly the 8 specifically expressed proteins in the non-flowering plants disappeared in the flowering plants with cold treatment. Therefore, these present data proved that the correlating changes of cytosine methylation and proteomic expression were evidenced under cold treatment in B. napus.

Cytological Behavior of Hybridization Barriers Between Oryza sativa and Oryza officinalis

Oryza officinalis is one of the important wild species in the tertiary gene pool of Oryza sativa. It has a number of elite genes for rice breeding in resistance or tolerance. However, breeding barriers are so serious that the gene transfer is much difficult by sexual cross method between O. sativa and O. officinalis. Characteristics of the breeding barriers were systemically studied in this paper. When both the diploid （AA, 2n=2x=24） and autotetraploid （AAAA, 2n=4x=48） cultivated rice were crossed as maternal parents with O. officinalis （CC, 2n=2x=24）, none F1 hybrid seeds were obtained. The young hybrid ovaries aborted at 13-16 d after pollinations （DAP）. By rescuing hybrid embryos, in vitro F1 plantlets were obtained in 2x×2x combinations with the crossabilities lower than 0.5%. Lower rates of double-fertilization and abnormal development of hybrid embryo and endosperm were mainly observed in both combinations of 2x×2x and 4x×2x. Free endosperm nuclei in hybrid degenerated early at 1 DAP in a large scale. Almost no normal endosperm cells formed at 3 DAP. Development of a lot of embryos ceased at globularor pear-shaped stage as well as some degenerated gradually. The hybrid plantlets were both male and female sterility. Due to the abnormal development, a diversity of abnormal embryo sacs formed in hybrids, and hybrid pollen grains were typically abortive. It showed that conflicts of genome A and C in hybrid induced abnormal meioses of meiocytes.

Fine Mapping of QTLs for Stigma Exsertion Rate from Oryza glaberrima by Chromosome Segment Substitution

Stigma exsertion is an important trait for outcrossing ability in rice.Stigma exsertion rate(SER)of male sterile lines(MSLs)is a key factor affecting F1-seed production in hybrid rice.In this study,seven QTLs for SER were detected on five chromosomes using a set of single-segment substitution lines(SSSLs)derived from O.glaberrima.Three of the QTLs were mapped in the estimated intervals of 92.5–333.0 kb.qSER-5 was located in a substitution segment of 92.5 kb.qS ER-1 b and qS ER-8 b were respectively limited to 333.0 kb and 107.5 kb by secondary substitution mapping.qSER-1 b and qSER-3 had bigger additive effects of 11.5%and 11.9%,respectively,while the other five QTLs had smaller additive effects from 5.7%to 8.6%.Open reading frames were identified in the regions of qS ER-5 and qSER-8 b in O.sativa and O.glaberrima genomes.Fine mapping of the QTLs laid a foundation for the cloning of genes,and QTLs for SER will be used to develop MSLs with strong ability of outcrossing.

RNA-Seq Study Reveals AP2-Domain-Containing Signalling Regulators Involved in Initial Imbibition of Seed Germination in Rice

A number of internal signals are required for seed germination.However,the precise signalling responses in the initial imbibition of seed germination are not yet fully understood in rice.In this study,the RNA sequencing(RNA-Seq)approach was conducted in 8 h imbibed seeds to understand the signalling responses in the initial imbibition of rice seed germination.A total of 563 differentially expressed genes(DEGs)with at least 4-fold change were identified in 8 h imbibed seeds compared to dry seeds.MapMan analysis revealed that the majority of signalling response-related DEGs were hormone-and transcription factor-related genes,in which the largest number of DEGs belong to the AP2-domain-containing regulators,and their expressions were significantly induced in the initial imbibition of seed germination in rice.Moreover,at least five AP2-domain-containing transcription factor OsDREBs were identified in the initial imbibition of rice seed germination,and the expressions of 251 DEGs were putatively regulated by OsDREBs through the dehydration-responsive element(DRE)cis-element assay.It suggested that the OsDREBs might play important roles in the regulation of initial seed imbibition in rice.The identified genes provide a valuable resource to study the signalling regulation of seed germination in the future.

Comparative Embryological Studies on Infertility of Interspecific Hybridizations Between Oryza sativa with Different Ploidy Levels and O. officinalis

As maternal parents, diploid （L202-2x） and autotetraploid （L202-4x） of Oryza sativa cv. L2O2 were crossed with O. officinalis. Embryo development and fertilization in these two crosses were comparatively studied. There were no mature hybrid seeds obtained because all the hybridized spikelets died 30 days after pollination. The main reasons for no seed set were abnormal fertilization and development of the embryos and endosperms in the interspecific hybrids. There were doublefertilization, egg cell single-fertilization and non-fertilization in these crosses. Although 59.45% and 54.87% of hybrid embryos produced in the crosses of L202-2x/O. officinalis and L202-4x/O. officinalis, respectively, hybrid embryos ceased to develop or degenerated and plenty of free endosperm nuclei were in disaggregating state without developing cellular endosperms three days after pollination. Besides, some embryological differences in these two crosses were found, that is, the rate of double-fertilization and total rate of doubleand single-fertilization in L202-2x/O. officinalis were higher than those in L202-4x/O. officinalis. The embryo and endosperm of hybrids developed more slowly, and embryos and free endosperm nuclei were more severely degenerated in L202-4x/O. officinalis than in L202-2x/O. officinalis. Five days after pollination, a few of embryos in L202-2x/O. officinalis developed into pear-shaped ones, however, embryos in L202-4x/O. officinalis were all degenerated. Therefore, it is more difficult to obtain interspecific hybrids by wide crosses between autotetraploid of O. sativa and O. officinalis.

Detection of Distorted Segregation in Genotype of Pollen Calli Derived from Hybrid F_1 of Cultivated Rice (Oryza sativa L.) Using SSR Markers

S-a, S-b and S-c are three loci for F1 pollen sterility in cultivated rice （Oryza sativa L.）. Taichung 65 （T65） is all Sj/Sj at these three loci, while its F1 pollen sterile near-isogenic lines, TISL2 （S-b）, TISL4 （S-a） and TISL5 （S-c） is Sj/Sj according to their respective sterility locus. Using SSR molecular marker to detect the segregation of the allele Si and Sj in pollen calli population induced from different hybrid F1, which have different pollen sterility locus, showed that the segregation of allele Si and Sj was distorted. The distorted direction of pollen calli population in vitro was not the same as F2 population in vivo. The quantities of pollen callus carrying Sj were much more than that of carrying Siat S-a and S-c locus, the ratio of Si and Sj were 1：4.81 and 1：1.96 respectively. But the opposite tendency was observed at S-b locus, the ratio of Si and Sj being 1：0.35. At the same time, all these results were undisturbed by either culture medium or culture period.

Fine mapping and genetic analysis of resistance genes,Rsc18,against soybean mosaic virus

Soybean mosaic virus(SMV) affects seed quality and production of soybean(Glycine max(L.) Merr.) worldwide.SC18 is one of the dominant SMV strains in South China,and accession Zhonghuang 24 displayed resistance to SC18.The F_(1),F_(2) and 168 F_(11) recombinant inbred lines(RILs) population derived from a hybridization between Zhonghuang 24(resistant,R) and Huaxia 3(susceptible,S) were used in this study.According to the segregation ratios of the F_(2) generation(3 R:1 S) and the recombinant inbred lines(RILs) population(1 R:1 S),one dominant locus may regulate the resistance to SC18 in Zhonghuang 24.By using composite interval mapping(CIM),Rsc18 was mapped to a 415.357-kb region on chromosome 13.Three candidate genes,including one NBS-LRR type gene and two serine/threonine protein type genes,were identified according to the genetic annotations,which may be related to the resistance to SC18.The q RT-PCR demonstrated that these genes were up-regulated in the R genotype compared to the control.In conclusion,the findings of this research enhanced the understanding about the R genes at the Rsc18 locus.Moreover,our results will provide insights for designing molecular markers to improve marker-assisted selection and developing new varieties with resistance to SC18.

High-Quality de novo Genome Assembly of Huajingxian 74, a Receptor Parent of Single Segment Substitution Lines

Rice(Oryza sativa L.)is grown nearly worldwide and provides the staple food for more than half of the global population(Luo et al,2017).The genomes of several cultivated rice varieties including Nipponbare(NPB)(Kawahara et al,2013;Sakai et al,2013),IR64(Tanaka et al,2020),93-11(Zhang et al,2018)and R498(Du et al,2017)at chromosome level,and Minghui 63 and Zhenshan 97(Zhang et al,2016)at scaffold level have been assembled,annotated and released,among which the R498 and NPB genomes are widely used as reference genomes in rice research.However,there are thousands of rice cultivars,landraces and wild rice varieties in the world with dramatically different genetic backgrounds,and the genomes of native rice varieties in South China,which is one of the major rice production areas in China,have not been de novo assembled.Huajingxian 74(HJX74)is an indica rice variety bred in South China Agricultural University,Guangdong Province with widely environmental adaptability and high yield(www.ricedata.cn/variety/varis/602548.htm).HJX74 exhibits significant phenotypic and genetic differences from those varieties whose whole genomes have been properly sequenced and assembled(Fig.1).