Development of Isogenic Lines of Morphological Markers in Indica Rice

A total of 28 accessions of rice (Oryza sativa L.) germplasms were collected from International Rice Research Institute (IRRI), Philippine and Gene Pool of China National Rice Research Institute (CNRRI). These germplasms were attributed as a Donor Gene Pool, consisting of 27 diverse morphological marker lines with each having a unique valuable trait (gene), and the recurrent parent-Zhefu 802, an early-season indica cultivar in south of China in the 1980s and 1990s. A set of 27 isogenic lines with 2-3 markers on every chromosome were obtained after more than 10 successive backcrosses. Moreover, 15 accessions of double-marker isogenic lines were developed by further pyramiding markers on the same chromosome. Key agronomic traits such as heading date, plant height, tillering number and panicle length in this set of lines were similar to that of Zhefu 802 except the marker traits.

Construction of Near Isogenic Lines for Pericarp Color and Evaluation on Their Near isogenicity in Rice

The inheritance of purple pericarp was studied through genetic analysis using F2, BC1F1 and F3 from a cross between a purple pericarp rice variety Zixiangnuo and a white pericarp rice variety Chunjiangnuo 2. Seven pairs of near isogenic lines （NILs） for pericarp color of rice were further constructed. Genetic analysis indicated that the purple pericarp was controlled by two complementary genes （Pb and Pp）. Agronomic trait analysis and polymorphism analysis using SSR markers on these seven pairs of NILs were used in the evaluation of near-isogenicity. No significant differences in agronomic traits were found except 1000-grain weight between the NILs. The polymorphic SSR markers for the parents were only detected in target segments of the five pairs of NILs different in Pb, which revealed the ideal near isogenicity of them. However, for the two pairs of NILs different in Pp, the polymorphic markers for the two parents were detected as well as in non-targeted segments of chromosomes 11 and 12, respectively.

Establishment of Near Isogenic Lines of Huangzaosi Maize with Resistance against Maize Head Smut

[ Objective ] The paper was to establish the near isogenic lines of Huangzaosi maize with resistance against maize head smut, and to provide basis for its wide application in breeding and production. [Method] Combing with hybrid, backcress and self-cross method, as well as molecular markers, Huangzaosi maize was successfully introduced with head smut resistance, and its near isogenic lines were studied. The characteristics of resistant and susceptible variation and the changes of combining ability of near isogenic lines after backcrossing breeding were observed. [ Result ] The incidence rate of Huangzansi maize in control was 46% ; the incidence rates of the selected 24 near isogenic lines of resistant Huangzaosi were all lower than 10%, and the selected rate was 92.6% ; the combining ability of most near isogenic lines of resistant Huangzaosi maize had no significant difference with that of H^i maize in control; the yields of hybrids prepared by several inbred lines such as M135 and M140 were outstanding, which exceeded the control Zhengdan 958, showing a higher combining ability. [ Conclusion] The phenotype and combining ability of near isngenic lines of Huangzaosi maize with resistance against maize head smut obtained in the test were very close to Huan- gzaosi, and its resistance against maize head smut was greatly increased, thus solving the problems of Huangzaesi without resistance against maize head smut.

Development of branchless watermelon near isogenic lines by marker assisted selection

Pruning is time-consuming and laborious in watermelon cultivation,which can not meet the needs for simplified cultivation in the future.The development of branchless lines will provide important germplasms for breeding watermelon varieties and is an important method for genetic improvement.In this study,the watermelon accession,Wu Cha Zao(WCZ)is a branchless inbred line that carries the branchless gene Clbl,which was used as the donor parent to develop branchless near isogenic lines(NILs).To construct the NILs of Clbl,WCZ crossed with the normal branching watermelon inbred line WT20 which was used as the recurrent parent.The co-segregating markers dCAPS10 and Indel1 with Clbl were used for foreground selection,and a total of 108 SSR markers was selected with good polymorphism between two parental lines for background selection which had relatively uniform distribution across 11 chromosomes.Using these markers to select individuals from the BC_(1)F_(1),BC_(2)F_(1),and BC_(2)F_(2) generations,three NILs with a proportion of recurrent parent genome(PRPG)>99%were finally obtained.The lateral branch and plant height phenotypes did not significantly differ between the NILs and WCZ,indicating that the NILs of Clbl under the genetic background of WT20 has been successfully developed.These results provide ideal materials for further in-depth analysis of the genetic mechanisms of lateral branch development and ideal plant architecture breeding in watermelon.

Integrating transcriptomic and proteomic analyses of photoperiod-sensitive in near isogenic maize line under long-day conditions

As a short-day plant species, maize requires an optimal photoperiod for inducing reproductive growth. However, there is a lack of information regarding photoperiod-induced changes in maize mRNA and protein levels. In this study, a photoperiod-insensitive maize inbred line and its near isogenic photoperiod-sensitive line were used. By integrating RNAbased transcriptomic and iTRAQ LC-MS/MS-based proteomic approaches, we generated a comprehensive inventory of the transcripts and proteins with altered abundances in response to a long photoperiod(LP) during growth stage transitions. We detected 22 000 transcripts in RNA-sequence runs and 5 259 proteins from an iTRAQ-based analysis. A weak correlation between mRNA-and protein-level changes was observed, suggesting the LP-induced transition between maize growth stages is largely regulated post-transcriptionally. Differentially expressed genes influenced by LP conditions were associated with several regulatory processes in both maize inbred lines, especially phosphate ion transport and the circadian rhythm. Additionally, 31 transcripts and six proteins related to photoperiodic flowering in maize were identified by comparing transcriptomic and proteomic data. This transcriptomic and proteomic analysis represents the first comprehensive and comparative study of gene/protein-level changes occurring in photoperiod-sensitive and-insensitive maize inbred lines during growth stage transitions under LP conditions.

Dissection of QTLs for Yield Traits Using Near Isogenic Lines Derived from Residual Heterozygous Lines in Rice

Three residual heterozygous lines (RHLs) carrying heterozygous segments in the intervals RM587–RM225, RM204–RM6119 and RM6119–RM402 on the short arm of rice chromosome 6, respectively, were selected from a rice population derived from an RHL for the interval RM587–RM402. Ten maternal homozygotes, 10 paternal homozygotes and 20 heterozygotes were selected from each of the F2 populations derived from the three RHLs. The three sets of near isogenic lines (NILs) were grown to detect the grain yield per plant, number of panicles per plant, number of filled grains per panicle and 1000-grain weight. With analysis on the phenotypic differences among the three genotype groups in each NIL set and those among overlapping chromosome segment substitution lines, three QTLs for number of filled grains per panicle and two QTLs for grain yield per plant were resolved. They were located in the intervals ranging from 0.66 Mb to 2.49 Mb. The additive effect was higher than the dominance effect at each locus. The allele for increasing the trait value was derived from the paternal parent at qNFGP6-1, and from the maternal alleles at other QTLs. Based on the present study, an approach for constructing new genetic resource to facilitate fine mapping of QTLs in rice was proposed.

Identification of AFLP Markers Linked to Leaf Rust Resistance Genes Using Near Isogenic Lines of Wheat

The present investigation was undertaken to find molecular markers linked to leaf rust resistance genes, Lr9 and Kharchia local mutant KLM4-3B. Preliminary AFLP analysis was carried out with different stocks, a survey of primer combinations with different selective nucleotide indicated that for each primer combination, the number of scorable loci ranged from 34 to 123. Only a limited primer combination used in the set of parental and near isogenic lines showed a high level of polymorphism for AFLP marker. Putative AFLP marker were found to be linked to Lr9, Lr19 and KLM4-3B. The alien genes were readily identified.

Cottonseed Protein, Oil, and Mineral Nutrition in Near-Isogenic <i>Gossypium hirsutum</i>Cotton Lines Expressing Leaf Color Phenotypes under Field Conditions

Information about the effects of phenotype traits on cottonseed protein, oil, and nutrients is scarce. The objective of this research was to investigate the effects of leaf color trait on seed nutrition in near-isogenic Gossypium hirsutum cotton expressing green (G) and yellow (Y) leaf color phenotypes. Our hypothesis was that leaf color can influence the accumulation of nutrients in seeds. Sets of isogenic lines were: DES 119 (G) and DES 119 (Y);DP 5690 (G) and DP 5690 (Y);MD 51ne (G) and MD 51ne (Y);SG 747 (G) and SG 747 (Y). Each NIL set is 98.44 % identical. Parent line SA 30 (P) was used as the control. The experiment was repeated for two years (2014 and 2015). The results showed that, in 2014, seed oil in DES 119 (G) and SG 747 (G) were significantly higher than their equivalent yellow lines. Green lines showed higher content of phosphorus compared with yellow lines. Higher levels of Cu, Fe, Mn, Ni, and Zn were recorded in DES 119 (G) and MD 51ne (G). In 2015, seed protein, oil, C, N, P, B, Cu, and Fe were higher in green lines than in yellow lines. There was a significant correlation between protein and nutrients, and between oil and nutrients in 2015, but not in 2014 as the temperature was warmer in 2015 than in 2014. This research demonstrated that leaf color can alter seed composition and mineral nutrition under certain environmental growing conditions such as temperature.

Identification of Molecular Markers Linked to Leaf Rust Resistance Genes in Wheat and Their Detection in the Local Near-Isogenic Line

Sixty-five random amplified polymorphic DNA (RAPD) primers were used for the detection of polymorphism among recipient and donor parents and their isogenic lines linked to leaf rust resistance genes, Lr9 and the resistant gene in kharchia local mutant KLM4-3B. Three primers showed polymorphism among recurrent parent, donor parent and isogenic lines.

Generation of isogenic single and multiplex gene knockout mice by base editing-induced STOP

Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain:the founders usually carry a mixture of genotypes, and mosaicism harboring multiple genotypes.Therefore, it takes a long time to get homozygous mutants. Recently developed base editing(BE) system,which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons(i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sg RNA targeting CD160 and a mixture of sg RNAs targeting VISTA and CD160,respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.

Blue-White Colony Selection of Virus-Infected Isogenic Recipients Based on a Chrysovirus Isolated from Penicillium italicum

Mycoviruses have been found to infect more than 12 species of Penicillium, but have not been isolated from Penicillium italicum(P. italicum). In this study, we isolated and characterized a new double-stranded RNA(ds RNA) virus, designated Penicillium italicum chrysovirus 1(Pi CV1), from the citrus pathogen P. italicum HSPi-YN1. Viral genome sequencing and molecular characterization indicated that Pi CV1 was highly homologous to the previously described Penicillium chrysogenum virus. We further constructed the mutant HSPi-YN1 Dpks P defective in the polyketide synthase gene(pks P), which is involved in pigment biosynthesis, and these mutants formed albino(white) colonies. Then we applied hyphal anastomosis method to horizontally transmit Pi CV1 from the white virus-donors(i.e., HSPi-YN1 mutants) to wild-type recipients(i.e., P.italicum strains HSPi-CQ54, HSPi-HB4, and HSPi-HN1), and the desirable Pi CV1-infected isogenic recipients, a certain part of blue wild-type strains, can be eventually selected and confirmed by viral genomic ds RNA profile analysis. This bluewhite colony screening would be an easier method to select virus-infected P. italicum recipients, according to distinguishable color phenotypes between blue virus-recipients and white virus-donors. In summary, the current work newly isolated and characterized Pi CV1, verified its horizontal transmission among dually cultured P. italicum isolates, and based on these, established an effective and simplified approach to screen Pi CV1-infected isogenic recipients.

The Inheritance of Early Heading in the Rice Variety USSR5

USSR5, a japonica rice variety from the former Soviet Union, is an extremely early maturing rice variety. To elucidate the genetic basis for its early heading, genetic analysis was carried out by crossing it with a set of major gene nearly isogenic lines （NIL） and QTL-isogenic lines. The early heading of USSR5 was attributed to the presence of photoperiod-insensitive alleles at E1 and Se-1 gene, the photoperiod-sensitive inhibitor gene i-Se-1, and the dominant earliness gene Ef-1. Analysis of a backcrossed population （BCIF1） derived from the cross USSR5 x N22 indicated that two quantitative trait loci （QTL） for early heading were located on chromosomes 7 and 8, accounting for 27.4% and 11.2% of the phenotypic variance, respectively, with both early alleles originating from USSRS. From an F2 population of the same cross, early heading QTLs were detected on chromosomes 1, 2, 7, 9, and 10, with individual QTL accounting for between 4.1% and 15.4% of the phenotypic variance. Early heading alleles at four of these five QTLs originated from USSRS. A comparison of chromosomal locations suggests that one of these QTLs may be identical with the known gene Hd4 （E1）. The relationship between the other QTLs and known genes for heading date are not clear. USSR5 is a promising source for propagating earliness for the development of improved early heading rice varieties.

Research on Relationship between Recovery Ability after Drought Stress-rewatering and Water Use Efficiency of Winter Wheat Varieties

The study aims at exploring the possibility of using the recovery ability af- ter drought stress-rewatering at vegetative growth stage as the evaluating index in water use efficiency （WUE） of winter wheat varieties. ＇Jing 411 ＇, ＇Jinmai 47＇ and their 34 near isogenic lines （NILs） were used as test materials. Semi-automatic rainproof shelter and the percolating pools were used for simulating drought treat- ment. After suffering severe drought stress, winter wheat crops were rewatered at early jointing stage. The biomass accumulation after rewatering was determined as recovery ability index. In the meanwhile, plant height in the end of vegetative growth stage was measured, and WUE of varieties/lines was also determined. Thereafter, the differences in recovery ability, plant height and the population WUE, together with the correlation between recovery ability and population WUE were analyzed, respectively. The results showed that there were significant differences in recovery ability among some varieties/lines. The recovery ability was affected by both geno- type and environment, and the interaction existed in these two factors. Significant differences existed in plant height and population WUE among the 34 NILs along with their parents. There was a significantly positive correlation between recovery ability and plant height of varieties/lines. Recovery ability and plant height were very significantly and positively correlated with population yield WUE respectively. The re- sults indicated that recovery ability after drought stress-rewatering could be used as an evaluating index of population WUE under drought condition.

Fine Mapping and Candidate Gene Analysis of Resistance Gene R_(SC3Q) to Soybean mosaic virus in Qihuang 1

Soybean mosaic virus （SMV） disease is one of the most destructive viral diseases in soybean （Glycine max （L.） Merr.）. SMV strain SC3 is the major prevalent strain in huang-huai and Yangtze valleys, China. The soybean cultivar Qihuang 1 is of a rich resistance spectrum and has a wide range of application in breeding programs in China. In this study, F1, F2 and F2：3 from Qihuang 1×nannong 1138-2 were used to study inheritance and linkage mapping of the SC3 resistance gene in Qihuang 1. The secondary F2 population and near isogenic lines （nILs） derived from residual heterozygous lines （RhLs） of Qihuang 1×nannong 1138-2 were separatively used in the ifne mapping and candidate gene analysis of the resistance gene. Results indicated that a single dominant gene （designated RSC3Q） controls resistance, which was located on chromosome 13. Two genomic-simple sequence repeat （SSR） markers BARCSOYSSR_13_1114 and BARCSOYSSR_13_1136 were found lfanking the two sides of the RSC3Q. The interval between the two markers was 651 kb. Quantitative real-time PCR analysis of the candidate genes showed that ifve genes （Glyma13g25730, 25750, 25950, 25970 and 26000） were likely involved in soybean SMV resistance. These results would have utility in cloning of RSC3Q resistance candidate gene and marker-assisted selection （MaS） in resistance breeding to SMV.

Characterization of flgK gene and FlgK protein required for H pylori Colonization-from cloning to clinical relevance

AIM： To characterize the role of flgK and its protein product in Hpylori colonization. METHODS： The PCR cloning method identified the flgK gene. An isogenic flgK mutant was constructed by gene replacement and confirmed by Southern blot analysis and PCR analysis. The recombinant FlgK protein （r-FlgK） was purified. Electron microscopy （EM） was applied to demonstrate the flagella of H pylori. An in vitro motility test was assessed in semisolid medium. The densities of H pylori colonization with either the wild-type strain or its flgK mutant were compared among BALB/c mice with or without pre-immunization with r-FlgK. The serological responses to r-FlgK were analyzed for 70 clinical patients with different densities of H pylori colonization. RESULTS： From a duodenal ulcer strain, the flgK gene was cloned and it contained 1821 bp, with a 95.7% identity to the published sequences. No flagella were observed under EM for the mutant strain, which had a loss of motility. Hpylori density was lower in the BALB/c mice inoculated by the mutant or with pre-immunization with r-FlgK compared to unimmunized mice or mice inoculated by the wild-type strain （P 〈 0.05）. In the H pylori-infected patients, the serological responses to r-FlgK were uniformly low in titer.CONCLUSION： FlgK encoded by flgK is important for flagella formation and H pylori motility. Deficiency in FlgK or an enhanced serological response to r-FlgK can interfere with Hpylori colonization. FlgK of Hpylori could be a novel target for vaccination.

Fine Mapping of qTGW3-1,a QTL for 1000-Grain Weight on Chromosome 3 in Rice

The QTL qTGW3-1 was located on chromosome 3 of rice （Oryza sativa L.） and associated with the 1 000-grain weight （TGW） according to the result of our earlier study. With the objective of fine mapping of this locus, we developed a F2 population consisting of 3 428 plants derived from the cross between TGW-related near isogenic line DL017 （BC3F4 generation of GSL 156×Nipponbare） and the recurrent parent Nipponbare. Using six microsatellites, this QTL was delimited between RM5477 and RM6417. Markers MM 1455 and MM 1456 within this region were used for further mapping of this QTL. Finally, qTGW3-1 was fine-mapped into a 89-kb interval between RM5477 and MM1456, which locates in the BAC clone AC107226 harboring five putative candidate genes.

Photosynthesis and Dry Matter Accumulation in Different Chlorophyll-Deficient Rice Lines

Three different chlorophyll-deficient rice isogenic lines chl,fgl and pgl,and their recurrent parent zhefu802 （zf802） were used to study effects of leaf color on photosynthesis,dry matter accumulation,yield,and quality in early season indica rice.Analysis showed that the chlorophyll （Chl.） a/b ratio of isogenic lines chl-8,pgl and fgl was 5.35,10.00 and 15.46,respectively,among them,line fgl had higher leaf area index （LAI）,higher net photosynthetic rate and higher grain-filling rate than its recurrent parent zf802 at the later period of grain filling stage;while LAI,net photosynthetic rate and dry matter accumulated in lines chl-8 and pgl were lower than in zf802.Differences were found in the grain yield and quality among chlorophyll deficient isogenic lines,lines fgl,chl-8 and zf802 had similar grain yield,which was significantly higher than that of pgl;the highest milling quality was observed in isogenic line fgl,with relatively high protein content.This study showed that isogenic line fgl would become a unique material for the development of high yield rice with high grain quality because of its slow aging process and relative steady grain-filling rate.

Identification of loci and candidate genes controlling kernel weight in barley based on a population for which whole genome assemblies are available for both parents

Kernel weight(KW), together with kernel number per unit area, determines yield of cereal crops. Here,two barley recombinant inbred lines(RILs) populations with a shared parent were used to identify loci controlling KW. One is Baudin/AWCS276(BA) for which a linkage map was available. Several largeeffect QTL controlling KW were detected in this population. Another is Morex/AWCS276(MA). A linkage map with 5273 makers formed 1454 clusters, was constructed by the genotyping by sequence(GBS) data of 201 RILs from this population. A single marker was selected to represent each of the clusters and the linkage map constructed with these markers covers a total length of 1022.4 c M with an average interval of approximately 0.7 cM between loci. Three of the large-effect loci controlling KW(located on 2 HL, 6 HL,and 7 HL, respectively) identified from the BA population were also detected in the MA population under different environments. The locus on 6 HL was detected in each of the experiments conducted for both populations thus was selected for developing near isogenic lines(NILs). Apart from KW, the two isolines for each pair of the putative NILs obtained showed no significant difference for any of the morphological characteristics assessed. The average difference in KW between the isolines for all the NILs obtained was about 15% based on assessments under both glasshouse and field conditions. Taken advantage that high quality genome assemblies for both Morex and AWCS276 are available, we identified candidate genes underlying two of the three loci based on an orthologous analysis. The NILs developed and the candidate genes identified in this study should facilitate the cloning and functional analysis of genes regulating KW in barley.

Development of Heat Tolerant Two-Line Hybrid Rice Restorer Line Carrying Dominant Locus of OsHTAS

In order to create novel germplasm resources for breeding heat tolerant variety,we transferred a dominant allele OsHTAS,previously characterized and cloned from a high-temperature tolerant local variety HT54,which was collected from the rice production area of southern China,into a high-temperature sensitive intermediate breeding line HT13 through six rounds of successive backcross by using marker-assisted selection.The molecular analysis showed that the recovery of genetic background of a resultant near isogenic line(NIL),MHT13,was around 99.8%.The OsHTAS gene introduced in the MHT13 expressed normally in the HT13 genetic background,mediating heat tolerance and phenotype similar to those of the donor parent HT54.The major agronomic traits of MHT13 resembled those of the recurrent parent HT13.Moreover,MHT13 had high general combining ability and its rice quality reached the grade 3 standard of edible high-quality rice issued by Ministry of Agriculture of the People’s Republic of China,which greatly improved its application value in rice production.

Effect of Dominant Semi-Dwarf Gene on Plant Height and Its Related Traits and Sensitivity to Gibberellic Acid in Rice

Six pairs of tall and dwarf near-isogenic lines derived from a dominant semi-dwarf mutant （Y98149） were selected to study height expression and sensitivity to gibberellic acid （GA3）. The lengths of the 4-5th internode, the 3rd, 2nd, 1st internodes from the top and the panicle length in the six dwarf near isogenic lines were 97.2%, 53.3%, 65.1%, 61.9% and 94.7% of those in the six tall ones, respectively, indicating that the dominant semi-dwarfing gene significantly inhibited the internode elongation. Moreover, Y98149 （mutant type） was more sensitive to GA3 than Y98148 （wild type）, and had a lower GA3 concentration in plant, about 78% of Y98148.