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.

Intra-heterogeneity in transcription and chemoresistant property of leukemia-initiating cells in murine Setd2^(-/-) acutemyeloid leukemia

Background:Heterogeneity of leukemia-initiating cells(LICs)is a major obstacle in acute myeloid leukemia(AML)therapy.Accumulated evidence indicates that the coexistence of multiple types of LICs with different pathogenicity in the same individual is a common feature in AML.However,the functional heterogeneity including the drug response of coexistent LICs remains unclear.Therefore,this study aimed to clarify the intra-heterogeneity in LICs that can help predict leukemia behavior and develop more effective treatments.Methods:Spleen cells from the primary Setd2^(-/-)-AML mouse were transplanted into C57BL/6 recipient mice to generate a transplantable model.Flow cytometry was used to analyze the immunophenotype of the leukemic mice.Whole-genome sequencing was conducted to detect secondary hits responsible for leukemia transformation.A serial transplantation assay was used to determine the self-renewal potential of Setd2^(-/-)-AML cells.A limiting-dilution assay was performed to identify the LIC frequency in different subsets of leukemia cells.Bulk and single-cell RNA sequencing were performed to analyze the transcriptional heterogeneity of LICs.Small molecular inhibitor screening and in vivo drug treatment were employed to clarify the difference in drug response between the different subsets of LICs.Results:In this study,we observed an aged Setd2^(-/-)mouse developing AML with co-mutation of Nras^(G12S) and Braf^(K520E).Further investigation identified two types of LICs residing in the c-Kit^(+)B220^(+)Mac-1^(-)and c-Kit^(+)B220^(+)Mac-1^(+)subsets,respectively.In vivo transplantation assay disclosed the heterogeneity in differentiation between the coexistent LICs.Besides,an intrinsic doxorubicinresistant transcriptional signature was uncovered in c-Kit^(+)B220^(+)Mac-1^(+)cells.Indeed,doxorubicin plus cytarabine(DA),the standard chemotherapeutic regimen used in AML treatment,could specifically kill c-Kit^(+)B220^(+)Mac-1^(−)cells,but it hardly affected c-Kit^(+)B220^(+)Mac-1^(+)cells.Transcriptome analysis unveiled a higher activation of RAS downstream signaling pathways in c-Kit^(+)B220^(+)Mac-1^(+)cells than in c-Kit^(+)B220^(+)Mac-1^(-)cells.Combined treatmentwithDAand RAS pathway inhibitors killed both c-Kit^(+)B220^(+)Mac-1^(−)and c-Kit^(+)B220^(+)Mac-1^(+)cells and attenuated disease progression.Conclusions:This study identified two cell subsets enriched for LICs inmurine Setd2^(-/-)-AML and disclosed the transcriptional and functional heterogeneity of LICs,revealing that the coexistence of different types of LICs in thismodel brings about diverse drug response.