Fine Mapping Of QRN5a,a QTL Associated With Root Number Under K Deficiency At The Seedling Stage In Rice(Oryza Sativa L.)

Potassium（K）is an indispensable mineral constituent involved in diverse physiological processes required for plant growth and development.K deficiency and depletion in rice occurs frequently,resulting in limited growth,agronomical loss,and reduced yield.At present,the molecular mechanism for low K（LK）tolerance remains poorly understood.To investigate the genetics of LK tolerance in the seedling stage of rice;quantitative trait locus（QTL）analysis,fine mapping,and gene expression patterns analysis of candidate genes was performed in this study.In the first part of this study,a set of high throughput genotyped chromosome segment substitution lines（CSSLs）comprises of 75 lines derived from the cross between Zhonghui9308（ZH9308,susceptible to LK）and XieqingzaoB（XQZB,tolerant to LK）was used to identify QTL for the shoot and root traits at the seedling stage.The experiment was conducted in hydroponic culture to explore the QTL of five seedling traits（root length,root number,root dry weight,shoot dry weight,and total dry weight）under two K conditions,LK and normal K（NK）and their ratio（LK/NK）for relative traits.A total of five QTLs were identified on four chromosomes（3,4,5,and 6）with positive allelic effects from XQZB for root length（RL）and root number（RN）and negative allelic effects for shoot dry weight（SDW）and root dry weight（RDW）.Two QTLs,qRN5a and qSDW4,were detected under LK and three QTLs,qRL6,qRN5b,and qRDW3,were identified under LK/NK ratio explaining 11.81%to 13.07%of total phenotypic variation.Among the identified QTLs,the QTL qRN5a and qRN5b associated with RN on chromosome 5 to be the most effective because these QTL were detected under two K levels（LK and LK/NK）in the same chromosomal location（in the marker InD78）with a positive additive effect.Therefore,qRN5a was selected for further study including fine mapping and map-based cloning,which may be used in molecular marker-assisted breeding.In the second part of this study,one line of the CSSLs,CSSL35,which carries a segment of chromosome 5 from XQZB in the genetic background of ZH9308,was selected as starting material to validate,fine map and evaluate the effect of qRN5a on seedling RN and other seedling root traits.CSSL35 showed a significantly higher RN than ZH9308 under LK at the seedling stage of rice.To confirm the existence of QTL qRN5a,a secondary F₂（BC₅F₂）population obtained from the cross of CSSL35 and genetic background parent ZH9308 was used.qRN5a was mapped between the markers InD78 and RM18472 with an interval of 1023-kb on the long arm of chromosome 5 using the F₂（BC₅F₂）population.Then fine map,candidate gene analysis and gene expression of qRN5a were performed to understand the molecular basis of K deficiency tolerance.To fine map the qRN5a,a large F_2:3(BC₅F_2:3)population obtained from the cross between CSSL35 and ZH9308 was constructed.High-resolution linkage analysis narrowed down the qRN5a locus to a 48.8-kb genomic interval flanked by markers A99and A139.Seven putative candidate genes were annotated in the delimited region of which three genes（Os05g0346700,LOC＿Os05g27980,and LOC＿Os05g28000）had nonsynonymous single nucleotide polymorphisms in the coding region between the two parents.Expression analysis suggests that LOC＿Os05g27980,which encodes a LATERAL ORGAN BOUNDARIES domain-containing protein,is a positive regulator of RN with LK tolerance and is the most likely candidate gene for qRN5a.Moreover,it was also observed that qRN5a promotes the expression of OsIAA23 and represses the OsHAK5 expression in root tissues to promote root initiation in CSSL35 under LK conditions by qRT-PCR assay.Moreover,the qRN5a locus from XQZB will be very useful for marker-assisted selection to develop a K deficiency tolerant cultivar with an improvement of the root system architecture in rice.