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.

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.

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.

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.

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.

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.

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.

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).

GL9 from Oryza glumaepatula controls grain size and chalkiness in rice

Grain size is a key factor influencing grain yield and appearance quality in rice.We identified twelve quantitative trait loci(QTL)for grain length(GL),nine for grain width(GW),and nine for 1000-kernel weight(TKW)using GLU-SSSLs,which are single-segment substitution lines with Oryza glumaepatula as donor parent and Huajingxian 74(HJX74)as recipient parent.Among the QTL,qGL1-2,qGL1-4,qGL9-2,qGW2-2,qGW9-1 and qTKW9-2 contributed to high grain yield.GL9 was identified as a candidate gene for qGL9-2 by map-based cloning and sequencing,and is a novel allele of GS9.The kernel of NIL-gl9was slenderer and longer than that of HJX74,and the TKW and grain yield per plant of NIL-gl9 were higher than those of HJX74.The proportion of grain chalkiness of NIL-gl9 was much lower than that of HJX74.Thus,gl9 increased grain yield and appearance quality simultaneously.Three pyramid lines,NIL-gs3/gl9,NIL-GW7/gl9 and NIL-gw8/gl9,were developed and the kernel of each was longer than that of the corresponding recipient parent lines.The gl9 allele may be beneficial for breeding rice varieties with high grain yield and good appearance quality.

Progress on Transferring Elite Genes from Non-AA Genome Wild Rice into Oryza sativa through Interspecific Hybridization

The progress of research on transferring elite genes from non-AA genome wild rice into Oryza sativa through interspecific hybridization are in three respects, that is, breeding monosomic alien addition lines （MAALs）, constructing introgression lines （ILs） and analyzing the heredity of the characters and mapping the related genes. There are serious reproductive barriers, mainly incrossability and hybrid sterility, in the interspecific hybridization of O. sativa with non-AA genome wild rice. These are the ＇bottleneck＇ for transferring elite genes from wild rice to O. sativa. Combining traditional crossing method with biotechnique is a reliable way to overcome the reproductive barriers and to improve the utilizing efficiency of non-AA genome wild rice.

Comparisons on Genetic Diversity among the Isonuclear-Alloplasmic Male Sterile Lines and Their Maintainer Lines in Rice

Four sets of rice isonuclear-alloplasmic lines including 16 male sterile lines and their maintainer lines were analyzed by using 91 pairs of SSR primers to study the genetic diversity of nuclear genome and their relative relationships. A total of 169 alleles were detected in the 16 lines, with a frequency of polymorphic loci of 53.85% and an average number of alleles per locus of 1.8, and the average gene diversity was 0.228. Four sets of the isonuclear-alloplasmic male sterile lines shared 146 identical alleles, corresponding to 86.39% of the total alleles; meanwhile, there are 23 different alleles among the tested materials, being 13.61% of the total alleles. On average, 78.70% identical alleles and 21.30% different alleles of the total alleles were detected between the isonuclear-alloplasmic male sterile lines and their maintainer lines. There were 53.85% identical alleles and 46.15% different alleles of the total alleles among the homozygous allonucleus male sterile lines. The fingerprints were established for some male sterile lines and maintainer lines. All the materials tested were divided into three groups at the 0.2 genetic distance based on the cluster analysis. Eight lines of Huanong A and Huayu A （including Huanong B and Huayu B） were in the first group, four lines of Kezhen A （including Kezhen B） in the second group, and four lines of Zhenshan 97A （including Zhenshan 97B） in the third group. For the isonuclear-alloplasmic male sterile lines, the similarity coefficient between Y （Yegong） type and WA （wild abortive） type or between CW （Raoping wild rice） and WA type reached 87-98%.

Megasporogenesis and Megagametogenesis in Autotetraploid Indica/Japonica Rice Hybrid

Autotetraploid indic, a/japonica rice hybrid combines both the advantages of polyploidy and the heterosis between indica and japonica rice. Embryo sac abortion is an important factor influencing spikelet fertility in autotetraploid dce hybrid. To clarify the cytological mechanism of embryo sac abortion, the megasporogenesis and megagametogenesis in an autotetraploid japonicaAndica hybrid were examined by the whole-mount eosin B-staining confocal laser scanning microscopy （WE-CLSM） technique. Abnormalities were observed from the megasporocyte stage to the mature embryo sac stage. The degeneration of the tetrad cells and the functional megaspore was the characteristic of abnormalities during megasporogenesis. Abnormal small embryo sacs and disordered number of nuclei were frequently observed during embryo sac development. Some interesting phenomena, such as two functional megaspores, the diplospory-like megasporocyte, and five-nucleate embryo sac were found. The abnormalities that occurred during female gametophyte development resulted in more than five types of abnormal embryo sacs （i.e. embryo sac degeneration, embryo sac without female germ unit, embryo sac without egg apparatus, embryo sac with abnormal polar nuclei and abnormal small embryo sac） in autotetraploid japonica/ndica hybdd. Embryo sac fertility was lower in diploid japonica/ndica hybdd than in autotetraploid japonicaAndica hybrid although many abnormal phenomena were observed in autotetraploid hybrid.

基因组倒位变异参与塑造了水稻遗传和表型的多样性

Genomic structural variations affected widely gene function and morphological traits in plants.Chromosomal inversions(INVs),as an important form of structural variation,can be large and extend to megabases in length[1],and form the genetic basis of local adaptation and ecotypic differentiation in sunflowers[2],Boechera stricta[3],monkeyflowers[4],mimetic butterflies[5]and ruffs[6],sex determination in nine-spined stickleback[7]and cancer and neurodevelopmental disease in human[8].At present,structural variants,and in particular,INVs,remain largely uncharacterized in plants despite their importance for local adaptation in a variety of species[1],with only a few studies exploring INVs polymorphisms at the population level.Hence,it remains unclear whether adaptive INVs can directly shape the genetic basis for diverse phenotypes in plants,especially in domesticated crops.

Decoding the microbiome for sustainable agriculture

Root-associated microbiota profoundly affect crop health and productivity.Plants can selectively recruit beneficial microbes from the soil and actively balance microbe-triggered plant-growth promotion and stress tolerance enhancement.The cost associated with this is the root-mediated support of a certain number of specific microbes under nutrient limitation.Thus,it is important to consider the dynamic changes in microbial quantity when it comes to nutrient condition-induced root microbiome reassembly.Quantitative microbiome profiling(QMP)has recently emerged as a means to estimate the specific microbial load variation of a root microbiome(instead of the traditional approach quantifying relative microbial abundances)and data from the QMP approach can be more closely correlated with plant development and/or function.However,due to a lack of detailed-QMP data,how soil nutrient conditions affect quantitative changes in microbial assembly of the root-associated microbiome remains poorly understood.A recent study quantified the dynamics of the soybean root microbiome,under unbalanced fertilization,using QMP and provided data on the use of specific synthetic communities(SynComs)for sustaining crop productivity.In this editorial,we explore potential opportunities for utilizing QMP to decode the microbiome for sustainable agriculture.