US 12,215,393 B2
Method and application for rapid and accurate chromosomal location of economic traits in laver
Yunxiang Mao, Qingdao (CN); Xinzi Yu, Qingdao (CN); Fanna Kong, Qingdao (CN); and Min Cao, Qingdao (CN)
Assigned to Ocean University of China, Qingdao (CN); and Hainan Tropical Ocean University, Hainan (CN)
Filed by Ocean University of China, Qingdao (CN); and Hainan Tropical Ocean University, Hainan (CN)
Filed on Feb. 25, 2021, as Appl. No. 17/185,866.
Claims priority of application No. 202010122553.X (CN), filed on Feb. 27, 2020.
Prior Publication US 2021/0269887 A1, Sep. 2, 2021
Int. Cl. C12Q 1/6895 (2018.01); C12Q 1/6827 (2018.01); C12Q 1/686 (2018.01)
CPC C12Q 1/6895 (2013.01) [C12Q 1/6827 (2013.01); C12Q 1/686 (2013.01)] 9 Claims
 
1. A method for rapid and accurate chromosomal location of traits in laver, comprising following steps: marking and distinguishing different genotype regions of thallus in layer developed from meiotic tetrad by color difference of pigment mutants, inducing and releasing monospores based on asexual reproduction of monochromatic single-genotype sectors of leaves for obtaining complete leaves with same genotype in batch, forming offspring Color-sectored Monospore Developed population to evaluate and measure a commonality of target genetic trait; using a genome of one of the wild-type parents as a reference genome and performing QTL-seq analysis on a collection of offspring exhibiting extreme phenotypes of offspring Color-sectored Monospore Developed population by SNP or #InDel markers; and combining Kompetitive Allele Specific PCR and RNA-seq methods to predict location of target trait genes or major QTL;
wherein the QTL-seq analysis comprising following steps:
i. in the offspring Color-sectored Monospore Developed population of hybrid of wild-type×pigment mutants, mixing a quantity of offspring Color-sectored Monospore Developed individuals according to their traits to form a pair of extreme trait offspring pool with same individual quantity;
ii. performing whole-genome sequencing of wild-type parents, pigment mutants and offspring pool; filtering sequenced data, calling and filtering SNP and/or InDels and annotating SNP and/or InDels;
iii. extracting SNP or InDels which are homogenic in each parent but different between two parents; using genotype of the wild-type parents as a reference to calculate Δ(SNP/InDel-index) of offspring pool; determining candidate genomic region related to mutant trait;
wherein number of individual quantity in each extreme trait offspring pool is ≥24;
analyzing wild-type pool and red mutant pool of the offspring Color-sectored Monospore Developed population of cross combination of wild-type RZ×red mutant HT by QTL-seq to locate red trait locus of layer leaf within 4.9 Mb; and
further narrowing candidate interval to 1.42 Mb by Kompetitive Allele Specific PCR; combining with RNA-seq method to determine potential candidate gene for controlling red coloring in HT is Pv08429.