Development of InDel Markers for Brassica rapa Based on a High-resolution Melting Curve

Brassica rapa is one of the most important leafy vegetable crops with large cultivated area in China.To increase the availability of DNA markers in B.rapa,we developed insertion-deletion(InDel)markers utilizing high-resolution melting(HRM)curve analysis.We designed primers for 252 InDels(≥3 bp)evenly distributed in the genome and tested gene polymorphisms with eight accessions.In total,208 markers were specifically amplified,and 148 InDels with polymorphism were genotyped successfully using HRM.We further analyzed the correlation with InDel size,GC number,and predicted the difference in Tm values(Tm)using 208 markers with specific amplification.We found that the success rate of InDel markers was correlated with the GC number of InDel and the predicted-Tm,but not clearly correlated with the length of InDel.When the GC number within InDel was≥8,the successful rate exceeded 90.0%.When the predicted-Tm reached 0.5°C,the success rate was greater than 90.0%,and when it was≥0.6°C,the rate climbed to 100.0%,indicating their role as the optimal parameter for successful development of an applicable InDel marker.The polymorphic InDel markers can be easily genotyped using HRM.They are of great value in genetic analysis,construction of linkage map,and molecular marker-assisted selection in B.rapa.

Identification of new cotton fiber-quality QTL by multiple genomic analyses and development of markers for genomic breeding

Cotton fiber is one of the main raw materials for the textile industry.In recent years,many cotton fiber quality QTL have been identified,but few were applied in breeding.In this study,a genome wide association study(GWAS)of fiber-quality traits in 265 upland cotton breeding intermediate lines(GhBreeding),combined with genome-wide selective sweep analysis(GSSA)and genomic selection(GS),revealed 25 QTL.Most of these QTL were ignored by only using GWAS.The CRISPR/Cas9 mutants of GhMYB_D13 had shorter fiber,which indicates the credibility of QTL to a certain extent.Then these QTL were verified in other cotton natural populations,5 stable QTL were found having broad potential for application in breeding.Additionally,among these 5 stable QTL,superior genotypes of 4 showed an enrichment in most improved new varieties widely cultivated currently.These findings provide insights for how to identify more QTL through combined multiple genomic analysis to apply in breeding.

Genome-wide analysis of Rf-PPR-like(RFL)genes and a new InDel marker development for Rf1 gene in cytoplasmic male sterile CMS-D2 Upland cotton

Background: Cytoplasmic male sterility in flowering plants is a convenient way to use heterosis via hybrid breeding and may be restored by nuclear restorer-of-fertility(Rf) genes. In most cases, Rf genes encoded pentatricopeptide repeat(PPR) proteins and several Rf genes are present in clusters of similar Rf-PPR-like(RFL) genes. However, the Rf genes in cotton were not fully characterized until now.Results: In total, 35 RFL genes were identified in G. hirsutum, 16 in G. arboreum, and 24 in G. raimondii. Additionally,four RFL-rich regions were identified; the RFL-rich region in Gh05 is the probable location of Rf-PPR genes in cotton and will be studied further in the future. Furthermore, an insertion sequence was identified in the promoter sequence of Gh05 G3392 gene in the restorer line, as compared with the CMS-D2 line and maintainer lines. An InDel-R marker was then developed and could be used to distinguish the restorer line carrying Rfl from other genotypes without the Rf1 allele.Conclusion: In this study, genome-wide identification and analysis of RFL genes have identified the candidate Rf-PPR genes for CMS in Gossypium. The identification and analysis of RFL genes and sequence variation analysis will be useful for cloning Rf genes in the future and also for three-line hybrid breeding in cotton.

Development of New InDel Marker to Detect Genotypes of Rf-1a Conferring Fertility Restoration of BT-Type Cytoplasmic Male Sterility in Rice

Restorer line breeding is an important approach to enhance the heterosis and improve the yields of japonica hybrid rice. To improve the selection efficiency of restorer lines for BT-type cytoplasmic male sterility （CMS） in japonica rice, a functional marker InDeI-Rf-la based on the difference of nucleotide sequence in Rf-la locus between BT-type CMS lines and restorer lines was developed to detect the genotypes of different rice materials. Conventional indica rice varieties, restorer and maintainer lines without 574 bp deletion could restore the fertility for BT-type CMS in japonica rice. By contrast, most conventional japonica rice varieties except Aichi 106 and Yijing 12, with genotype of rf-larf-la showed the 574 bp deletion maintained sterility for BT-type CMS lines. To further verify the effect of genotyping detection in Rf-la locus, this marker was also used to amplify the genomic DNA in different japonica rice restorer lines, CMS lines, hybrids and F2 segregation population, and three genotypes in Rf-la locus could be distinguished distinctly. Therefore, the marker InDeI-Rf-la could be widely used for genetic id^ntifio.~tinn ~nd m^rkp.r-~.~.~i^fp.d .~.tAr.tinn （MA.~ in hr~=dinn i^nnnir~ r^fnr~=r lin==~

Development and application of molecular markers for TSW(thousand-seed weight)related gene BnaGRF7.C02 in Brassica napus

Brassica napus L(rapeseed)is one of the most important oil crops with large cultivated area in China.Seed size and seed weight play crucial roles for yield and harvest.In this study,a type of 15 bp-deletion in BnaGRF7.CO2 coding region was identified through sequence alignment of BnaGRF7.C02 in 42 rapeseed varieties,and associ-ation analysis indicated that the 15 bp-deletion was related to the rapeseed Thousand-Seed Weight(TSW)phenotype.Furthermore,we developed two InDel markers to identify this 15 bp InDel.The tissue-specific expression patterns showed that BnaGRF7.C02 prominently expressed in the late stage of seed development.These findings may assist in InDel markers-based breeding efforts to select higher TWS varieties and improve the crop yield of B.nqpus.

Stable major QTL on chromosomes A07 and A08 increase shelling percentage in peanut(Arachis hypogaea L.)

Peanut is a major oilseed and food legume.Shelling percentage(SP),closely associated with seed yield,is a trait whose improvement is a major goal of peanut breeding.In this study,a mapping population(Xuhua 13×Zhonghua 6)was used to map quantitative trait loci(QTL)controlling SP in four environments.Two stable major QTL for SP were mapped on both SSR-and SNP-based genetic maps.q SPA07.1 on chromosome A07 explained up to 31.7%of phenotypic variation,and q SPA08.2 on chromosome A08 explained up to 10.8%.Favorable alleles of q SPA07.1 and q SPA08.2 were derived from the female and male parents,respectively.Eight recombinant inbred lines(RILs)carrying both favorable alleles showed superiority in SP over the two parents in all environmental trials.A combination of the two favorable alleles using the linked markers was verified to increase SP by~5%in the RIL population and by~3%SP in diverse peanut cultivars.q SPA07.1 and q SPA08.2 were delimited to respectively a 0.73-Mb interval harboring 96 genes and a 3.93-Mb interval harboring 238 genes.Respectively five and eight genes with high expression in pods,including enzymes and transcription factors,were assigned as candidate genes for q SPA07.1 and q SPA08.2.These consistent major QTL provide an opportunity for fine mapping of genes controlling SP,and the linked markers may be useful for genetic improvement of SP in peanut.

Development of genome-wide insertion/deletion markers in rice based on graphic pipeline platform

DNA markers play important roles in plant breed- ing and genetics. The Insertion/Deletion （InDel） marker is one kind of co-dominant DNA markers widely used due to its low cost and high precision. However, the canonical way of searching for InDel markers is time-consuming and labor- intensive. We developed an end-to-end computational solution （InDel Markers Development Platform, IMDP） to identify genome-wide InDel markers under a graphic pipeline environment. IMDP constitutes assembled genome sequen- ces alignment pipeline （AGA-pipe） and next-generation re- sequencing data mapping pipeline （NGS-pipe）. With AGA-pipe we are able to identify 12,944 markers between the genome of rice cultivars Nipponbare and 93-11. Using NGS-pipe, we reported 34,794 InDels from re-sequencing data of rice cultivars Wu-Yun-Geng7 and Guang-Lu-Ai4. Combining AGA- pipe and NGS-pipe, we developed 2o5,659 InDels in eight japonica and nine indica cultivars and 2,681 InDels showed a subgroup-specific pattern. Polymerase chain reaction （PCR） analysis of subgroup-specific markers indicated that the precision reached 90% （86 of 95）. Finally, to make them available to the public, we have integrated the InDels/markers information into a website （Rice InDel Marker Database, RIMD, http：I/2o2.12o.45.71/）. The application of IMDP in rice will facilitate efficiency for development of genome-wide InDel markers, in addition it can be used in other species with reference genome sequences and NGS data.

Development of Insertion and Deletion Markers for Bottle Gourd Based on Restriction Site-associated DNA Sequencing Data

Bottle gourd is an important cucurbit crop worldwide. To provide more available molecular markers for this crop, a bioinformatic approach was employed to develop insertion–deletions(In Dels) markers in bottle gourd based on restriction site-associated DNA sequencing(RAD-Seq)data. A total of 892 Indels were predicted, with the length varying from 1 bp to 167 bp. Single-nucleotide In Dels were the predominant types of In Dels. To validate these In Dels, PCR primers were designed from 162 loci where In Dels longer than 2 bp were predicated. A total of 112 In Dels were found to be polymorphic among 9 bottle gourd accessions under investigation. The rate of prediction accuracy was thus at a high level of72.7%. DNA fingerprinting for 4 cultivars were performed using 8 selected Indels markers, demonstrating the usefulness of these markers.