记录一篇2019年Wulff实验室发表在NBT上的一篇关于R基因克隆方法——AgRenseq的文献，由于还未吃透，暂做简单记录。
驯化和快速育种导致作物多样性降低，单个R基因导入作物中费时费力而且抗性很快会被病原克服，而同时导入多个R基因则可避免上述情况，但是目前R基因克隆的方法要求分离或突变后代的产生，这对于有着难以分辨性状的近缘野生种属更难实现。GWAS可以关联性状至基因，但是受参考基因组的影响（如目标基因来自外源渗入片段，而参考基因组无此片段），而Kmer-GWAS则可以不依赖于参考基因组而实现。在本文中作者利用Kmer-GWAS与RenSeq技术发明了一种新的快速鉴定克隆R基因的方法——AgRenSeq
AgRenseq: 利用基于Kmer方法的关联分析和R基因富集测序的方法从作物多样性库中发现和克隆R基因。
优点：是一种不依赖参考基因组的GWAS方法，可以直接鉴定到单个NLR基因而不用像其他GWAS方法验证到一个含有多个旁系同源基因的区间。
缺点：结果受NLR捕获能力的影响，而且不能鉴定非典型的NLR基因类型
1. AgRenSeq技术原理
Combining association genetics and R gene enrichment sequencing (AgRenSeq) for R gene cloning
a–c, A genetically diverse panel of accessions (a) is phenotyped with different pathogen races (b), and subjected to RenSeq followed by assembly of the NLR repertoire and extraction of NLR k-mers for each accession (c). d, k-mers are pre-filtered based on the correlation of their presence/absence to the level of resistance or susceptibility in the phenotyped panel. Each pre-filtered k-mer is given a P value based on its ability to predict the phenotype using linear regression, with PCA dimensions as covariates to control for population structure. Phenotypes are color-coded as in b, and the presence and absence of k-mers is indicated by dark gray and white, respectively. e, k-mers are then plotted in an association matrix according to their sequence identity to NLRs from a given accession (x axis) and the measure of their association with phenotype (y axis) (see Fig. 2). A candidate R gene contig is illustrated by a red-dot column.
2.173个Ae. tauschii ssp. strangulata+19个ssp. tauschii品种鉴定Sr基因来验证AgRenseq
Genetic architecture of stem rust resistance in Ae. tauschii.
a, Geographic distribution of Ae. tauschii ssp. strangulata (cyan) and ssp. tauschii (magenta) used in this study. Two accessions from China and two from Pakistan, which fall outside the map, are not shown. ARM, Armenia; AZE, Azerbaijan; GEO, Georgia; IRN, Iran; KAZ, Kazakhstan; SYR, Syria; TJK, Tajikistan; TUR, Turkey; TRK, Turkmenistan; UZB, Uzbekistan. b, Phylogenetic tree displaying Ae. tauschii ssp. strangulata (173 accessions, cyan) and ssp. tauschii (19 accessions, magenta) with an intermediate accession (dark green). Stem rust phenotypes and Sr genotypes are displayed by concentric circles around the tree. c–e, Identification of Sr33, Sr45, Sr46, and SrTA1662 by AgRenSeq using PGT races RKQQC, TTKSK, and QTHJC, respectively. The number of accessions used for each phenotype is provided in Supplementary Table 8. Each dot column on the x axis represents an NLR contig from the RenSeq assembly of a single accession (BW_01005, BW_01077 or BW_01072) containing the respective Sr gene. Each dot on the y axis represents one or more RenSeq k-mers associated with resistance across the diversity panel to the respective PGT race. The association score is defined as the negative log of P value obtained using likelihood ratio test for nested models. Dot columns corresponding to Sr genes are colored red. Dot size is proportional to the number of k-mers associated with resistance.
3.AgRenseq未来的应用
AgRenSeq to engineer disease resistance
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Discovery and cloning of diverse R genes (colored dots on vertical chromosomes) in a germplasm panel (far left) allows the rapid engineering by transformation of a multi-R gene stack with zero linkage drag (R1 to R4, top right) or facilitates incorporation and stacking of R genes into elite lines and reduction of linkage drag (colored bars around R genes) by multiple backcrossing and marker-assisted selection (MAS; bottom right).
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