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.

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.

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.

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.

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.

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.

Analysis of bulked segregants to identify molecular markers linked with cocoon weight and cocoon shell weight in the silkworm Bombyx mori L

Two silkworm strains viz, B20 A (high cocoon shell ratio) and C.Nichi (low cocoon shell ratio) were sib mated for 10 generations to determine the homozygosis. Both bulked segregant analysis(BSA) and near isogenic lines (NIL) studies were done to identify the RFLP markers closely linked to cocoon shell parameters. Three hundred and fifty two random clones were identified as the low copy number sequence and used for identification of Restriction Fragment Length Polymorphic (RFLP) marker linked to cocoon weight and cocoon shell character. In the bulk segregant analysis, DNA from the parents (B20 A, C.Nichi), F 1 and F 2 progeny of high shell ratio (HSR) and low shell ratio (LSR) were screened for hybridization with the random clones. Polymorphic banding pattern achieved through southern hybridization with different probes indicated the probable correlation of polymorphism with high and low cocoon shell character which are possible landmarks in identifying the putative marker(s) for the cocoon shell character. Out of the 100 probes tried with parents, F 1, F 2 and their bulks, 10 probes were found to be closely linked to cocoon shell characters.

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.

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.

Genetic Identification of a New Small Grain Dwarf Gene in Rice

The plant material used in the study was rice line 162d, a new small grain dwarf mutant. Polymorphic analysis of 221 SSR loci demonstrated that 162d derived from a semidwarf variety Shuhui 162 through mutation, and 162d and Shuhui 162 were just a pair of near isogenic lines. Genetic analysis of F_1 and F_2 populations suggested that dwarfism in 162d was controlled by a single recessive gene. Phenotypic characteristics of the mutant gene were that plant height was about a quarter of normal height, grain size about a quarter of normal size, leaf was short and broad, and seed setting rate was very low, compared with the near isogenic line Shuhui 162. The mutant gene was sensitive to gibberellin (GA_3) treatment and did not located on the region near the centromere of rice chromosome 5, where dl gene located. Therefore, it was concluded that the mutant gene of 162d was a new small grain dwarf gene in rice.

A Major QTL, Ghd8, Plays Pleiotropic Roles in Regulating Grain Productivity, Plant Height, and Heading Date in Rice

Rice yield and heading date are two distinct traits controlled by quantitative trait loci （QTLs）. The dissection of molecular mechanisms underlying rice yield traits is important for developing high-yielding rice varieties. Here, we report the cloning and characterization of Ghd8, a major QTL with pleiotropic effects on grain yield, heading date, and plant height. Two sets of near isogenic line populations were developed for the cloning of GhdS. Ghd8 was narrowed down to a 20-kb region containing two putative genes, of which one encodes the OsHAP3 subunit of a CCAAT-box binding protein （HAP complex）; this gene was regarded as the Ghd8 candidate. A complementary test confirmed the identity and pleiotropic effects of the gene; interestingly, the genetic effect of Ghd8 was dependent on its genetic background. By regulating Ehdl, RFT1, and Hd3a, Ghd8 delayed flowering under long-day conditions, but promoted flowering under short-day conditions. Ghd8 up-regulated MOC1, a key gene controlling tillering and branching; this increased the number of tillers, primary and secondary branches, thus producing 50% more grains per plant. The ectopic expression of Ghd8 in Arabidopsis caused early flowering by 10 d-a situation similar to the one observed by its homolog AtHAP3b, when compared to wild-type under long-day conditions; these findings indicate the conserved function of Ghd8 and AtHAP3b in flowering in Arabidopsis. Our results demonstrated the important roles of Ghd8 in rice yield formation and flowering, as well as its opposite functions in flowering between rice and Arabidopsis under long-day conditions.

Yield-related QTLs and Their Applications in Rice Genetic Improvement

Grain yield is one of the most important indexes in rice breeding, which is governed by quantitative trait loci （QTLs）. Different map- ping populations have been used to explore the QTLs controlling yield related traits. Primary populations such as F2 and recombi- nant inbred line populations have been widely used to discover QTLs in rice genome-wide, with hundreds of yield-related QTLs detected. Advanced populations such as near isogenic lines （NILs） are efficient to further fine-map and clone target QTLs. NILs for primarily identified QTLs have been proposed and confirmed to be the ideal population for map-based cloning. To date, 20 QTLs directly affecting rice grain yield and its components have been cloned with NIL-F2 populations, and 14 new grain yield QTLs havebeen validated in the NILs. The molecular mechanisms＇of＇a continuous/y increasing number of genes are being unveiled, which aids in the understanding of the formation of grain yield. Favorable alleles for rice breeding have been ＇mined＇ from natural cultivars and wild rice by association analysis of known functional genes with target trait performance. Reasonable combination of favorable alleles has the potential to increase grain yield via use of functional marker assisted selection.