High-density Genetic Linkage Map Construction And QTLs For Important Traits On Litchi

Genetic map is an effective auxiliary breeding tool.Higher quality genetic maps can effectively perform QTL precision mapping studies.In this study,the genome sequencing and resequencing data of litchi was analyzed,the InDel locus were detected,and a part of the locus were selected to develop a homozygous co-dominant molecular marker for hybrid progeny identification.The designed primers were subjected to polymorphism detection in the parental material of the hybrid combination to determine homozygous co-dominant molecular markers,which is suitable for true hybrid identification in specific hybrid combinations.Then,the real hybrid identification was carried out,and a batch of true hybrid populations for map construction was obtained.The differences of hybridization efficiency between different hybridization methods and different hybrid combinations were compared.Using’Ziniangxi(ZNX)as the female,’ Wuheli(WHL)as the male,a mapping population with 250 hybrid F1 individuals was used to construct the high-density genetic map of litchi.The 13 traits,such as the internode length,the length of the compound leaf,the chlorophyll content,the photosynthetic rate,and the diameter of the anthracnose lesions,were measured and tested for normality.The 6 traits with normal distribution were subjected for QTL scanning.The main results are as follows:1.A total of 218 pairs of InDel primers were designed and 111 pairs of primers were found to be polymorphic at least in two cultivars.the polymorphic marks can distinguish the hybrid parents:’ Baitangying(BTY)’,’ Dadingxiang(DDX)’,’ Guizaoli(GZL)’,’ Feizixiao(FZX)’,’Sanyuehong(SYH)’,’Xinqiumili(XQML)’,’Yutanmili(YTML)’,’WHL’ and ’ZNX’.2.Identification F1 individuals of 20 hybrid combinations,such as ’ZNX X ’WHL’,’ZNX’X ’FZX’,’ZNX’ × ’ SYH’,a total of 1880 true hybrids were obtained among 2800 individual plants.The average hybrid ratio is 67.14%.The hybrid ratio between different hybrid combinations is various.The hybrid ratio of ’YTML × ’SYH is highest and that of ’XQM L’×’ GZL’ is lowest,indicating that the affinity of different parents is different.The efficiency of pollination methods was carried out on the hybrid combination of ’ BTY ’ x ’ FZX ’.The hybrid ratio is not significant between net chamber assistant pollination(NCAP)and artificial assistant pollination(AAP),while the time of NCAP is shorter than AAP.The results showed that NCAP is an efficient cross breeding method.3.The genetic map was constructed with 250 F1 individuals of ’ZNX × ’WHL as the mapping population.The DNA of each plant was treated by HaeⅢ and Hpy 166Ⅱ enzymes.And then the fragments with 314-514 bp were recovered and sequenced.A total of 1415.69 M reads(302.56 Gb)were obtained.SNP marker development and mutation detection were performed among the parents and offsprings by GATK and samtools.A total of 11,255,471 SNP markers were identified.4.According to the separation ratio in the offspring,the markers were typed,3,789,874 were successfully typed,of which 3,353,448 markers could be used for following analysis.Filtering criteria:1 Parental sequencing depth 22X or less fragment;2 Sequencing quality below Q30 Fragment;3 integrity below 80%;4 partial segregation markers(chi-square test P<0.01);5 in the parents showed homozygous markers.A total of 10,710 markers were obtained for genetic map construction.Eight of individuals were identified as pseudo hybrids by abnormal marker typing and were removed.Using software of Joinmap 4.0 to construct genetic map,set MLOD>5,10710 markers assign to 15 linkage groups.The total distance of the genetic map of litchi is 1564.45 cM with an average map distance of 0.15 cM.It is the highest genetic map of litchi so far.5.In 2017 and 2018,the traits of chlorophyll content,anthracnose diameter,leaf shape index and specific leaf weight of litchi were measured.The trait data were tested normality.The outliers,the value is greater than 3 standard deviations,were removed.Six traits(petiole length,internode length,compound leaf spindle length,chlorophyll content,leaf shape index,and anthrax lesion diameter)obeyed the normal distribution in 2017.QTL mapping of each traits was performed using MapQTL 5.0.A total of 29 QTL intervals were detected,which including 5447 genes.10 traits follow the normal distribution in 2018,namely:petiole length,compound leaf spindle length,chlorophyll content,leaf shape index and so on.A total of 19 QTL intervals were detected,which including 4006 genes.6.It was showed that the spindle length had 2 coincident QTL intervals between two years,while shape index of the compound leaf had 6 coincident QTL intervals.These 8 QTL intervals contained 1181 genes.By of function annotation analysis of genes in QTL intervals,a few genes associated with the trait were selected as candidate genes.In this study,we were developed a batch of InDel markers in litchi.The hybrid F1 offspring of 20 cross combinations were identified,and the average hybrid ratio is 60.19%.The high-density genetic map of litchi was constructed with a total map distance of 1560.45 cM and an average map distance of 0.15 cM.This genetic linkage map was the highest density genetic map in litchi.In 2017 and 2018,QTL mapping of 6 and 10 traits of lychee were carried out,respectively.A total of 29 and 19 QTL intervals were respectively identified.Several candidate genes in QTL intervals were selected according to the results of lychee genome annotation.