Construction Of A Genetic Linkage Map In Pyropia Yezoensis And QTL Analysis Of Several Economic Traits Of Blade

Pyropia yezoensis is a marine red alga with high nutritional values and is one of the most important maricultural crops across the world,mainly in Japan,Korea and China.During the cultivation of P.yezoensis,hundreds of tons of nutrients（nitrogen and phosphorus）are removed from the eutrophic seawater by blade harvests every year.However,some problems such as germplasm degeneration,frequent diseases and bad harvests have arisen under the influence of global warming.Therefore,new varieties with higher yield,stronger resistance to abiotic stress and greater ecological adaptability are urgently needed for sustained development of Pyropia industry.Major efforts are underway to increase the production of P.yezoensis by extending genetic diversity and analyzing key traits.However,its genome is relatively unexplored,which affects the efficiency of breeding.The traditional breeding methods of P.yezoensis are based on either observed variations by selecting blades with induced variants,or controlled crosses by selecting blades presenting recombination of desired genes from different parents.However,breeding based on traditional method is usually taking more time and the efficiency is relatively low,and has a limited ability to breed complex traits.Fortunately,progress in molecular genetics has enabled plant breeders to select genotypes directly,thereby accelerating crop improvement.Nowadays,marker-assisted selection（MAS）using molecular markers has become the main direction of plant breeding,during which a genetic linkage map is an inevitable step.It has been successively constructed in dozens of different species of plants and animals and has played important roles in various studies.However,map construction in seaweeds remains in its infancy and has only been reported in five important species,including Laminaria japonica,L.longissima,Ectocarpus siliculosus,Porphyra/Pyropia haitanensis and Undaria pinnatifida.Those maps have been used for quantitative trait locus（QTL）detection of economic traits,mapping of sex-linked loci and large-scale assembly of genome sequence.However,no genetic linkage map for P.yezoensis has been constructed.In order to establish MAS breeding technology and to raise the breeding productivity of Pyropia,a red-type pigmentation mutant Py-HT and a wild-type strain Py-LS were used as maternal and paternal parents,respectively,in an intraspecific cross to establish a mapping population.Only four-color sectored mosaic blades were screened from the F₁blades which developed from the conchospores released from the heterozygous conchocelis.Every selected blade was then cut into four color-sectors along the boundaries of adjacent color-sectors and every color-sector was subsequently cultured individually.A doubled haploid strain was obtained when one of the carpospores was released from a self-fertilized color-sector and developed into a single conchocelis.The DH population was analyzed by sequence-related amplified polymorphism（SRAP）markers and the fluorescent products were separated by high-performed capillary electrophoresis in an DNA analyzer.A medium-density genetic map containing 92 SRAP markers was constructed using Join Map v4.0 software.It was further found that six economically important traits of gametophytic blades were quantitatively inherited traits controlled by multiple genes.By means of genetic mapping,10 minor QTLs associated with five economic traits of blades were detected on the genetic map.The concrete results were as follows:Results 1.Construction of a DH mapping population.Two parental strains of P.yezoensis with different economic traits were crossed in this study.Only carpospores released from the blades of fertilized Py-HT were collected and cultured into single conchocelis colonies.The offspring of HL-4^#conchocelis colony were color-sectored blades with a percentage of 91.88%,indicating that the concocelis was the heterozygote of the cross（Py-HT×Py-WT）.F₁ blades of HL-4^#heterozygous concocelis were cultured subsequently in large scale,and 57 chimeric blades with normal development of all the four color-sectors were screened from more than 3,000 blades.The four color-sectors of each chimeric blade were separated by a razor blade and cultured singly.A DH strain was declared when a homozygous concocelis was developed from a carpospore released from a self-fertilized color-sector.A DH population of P.yezoensis composed of 152 DH strains was constructed by obtaining the homozygous concocelis of every color-sector of 38 four-color sectored blades.In addition,19 other four-color sectored blades in which at least one color-sector of every blade failed to form homozygous concocelis were not used,in order to maintain the integrity of genetic structure of the DH mapping population.The establishment of mapping population laid a foundation for the construction of genetic map.Result 2.Genetic analysis of major economical traits of blades.It was found that all six traits（L50,W50,FW50,LGR,WGR and FWGR）of the blades of DH population were quantitative traits when analyzed by one-sample Kolmogorov-Smirnov test(P_ks>0.05).The mean values of four traits were between their parents,among which W50 was nearer to maternal parent,FW50,LGR and FWGR were nearer to paternal parent.L50and WGR were superiority over their parents.Coefficient of variation of the six traits of DH population was between 21.11%～56.68%,which meant that they had intermediate variability.Highly significantly positive correlations were observed between L50 and W50,L50 and FW50,W50 and FW50,respectively,and between LGR and WGR,LGR and FWGR,WGR and FWGR,respectively.Heritabilities of L50,W50 and FW50 were58.17%,64.00%and 57.64%,respectively.The number of genes controlling the three traits were 6.61,12.63 and 8.09,respectively.The equation of the curve between heritability（y）and the number of genes（x）was y=0.2922x²-4.6533x+76.162（R2=1）.Based on the two estimated values of skewness and kurtosis of the three traits tested,gene interactions were found to be absent for L50 and FW50,respectively;complementary interactions were observed in W50 and WGR,respectively;duplicate interactions were observed in LGR and FWGR,respectively.The results indicated that L50 and FW50could be selected at early generation by their high heritabilities,less controlling genes and no gene interactions.Besides,indirect selection could be applied between L50,W50and FW50 to improve breeding efficiency of P.yezoensis because of their high correlations between each other.Through the discussion of the genetic relationship and characteristics among the controlling genes of various traits,the important theoretical basis was provided for increasing the breeding ways and improving the efficiency of genetic breeding.Result 3.Exploitation of polymorphic molecular markers.Genomic DNA of wild-type strain（Py-LS）was extracted and determined on illumina Hi Seq 2500（PE125）,through paired-end sequencing of small-size（500 bp）fractionated genomic library.SSRs were identified by MISA,and the primers for selected SSRs were designed using Primer3 program.Some randomly selected primers were validated through PCR amplification and gel analysis first in Py-LS and then polymorphic primers were screened between Py-LS and Py-HT.Indistinguishable PCR products without size difference were further excised and purified for Sanger sequencing,for the purpose of screening polymorphic primers with sequence difference.In total,20,620 SSR-containing sequences were developed from the 7.16 G clean data with 283,606 contigs.Most of them contained tri-and di-nucleotide motifs（95.42%and 2.34%,respectively）,of which GCC was the most abundant nucleotide motif（15.7%）.Based on the identified SSRs,1,253 pairs of primer sets were designed based on their flanking sequence.124 randomly selected primers were validated through PCR and Sanger sequencing,among which 120 primer pairs were successful in PCR amplification in Py-LS and/or Py-HT,and the remaining four primer pairs failed to generate PCR products at various annealing temperatures and Mg²⁺concentrations.In these 120 primer combinations,104 pairs amplified SSRs with the same motifs and repeat times,three pairs with same motifs but different repeat times,and13 pairs with different motifs when compared with those predicted.Furthermore,21primer pairs amplified polymorphic products between Py-LS and Py-HT were screened,among which five pairs were specific to Py-LS,10 pairs were specific to Py-HT,five pairs amplified SSRs with different motifs,and one pair amplified SSRs with different repeat times.The efficient sequencing method allowed the feasible and high-throughput discovery of SSR markers by using genome information.However,these primers could not amplify products with different sizes between Py-LS and Py-HT,which could not meet the requirements for genetic map construction in this study.Therefore,SRAP was used for further study.Result 4.Construction of a genetic linkage map.There were 441 primer combinations obtained after random pairing of 21 forward primers and 21 reverse primers.After screening,four forward primers（Me4,Me7,Me13 and Me19）and three reverse primers（Em6,Em8 and Em10）were labeled with 5’-HEX（hexachloro fluorescein）and paired with ordinary primers to analyze the mapping population.Genotyping by means of capillary electrophoresis directly provided the digitized information of the fragments（e.g.size and peak height）.Data of 79 primer combinations with missing data number less than or equal to four DH strains per primer combination were used for genetic mapping.They amplified 4,570 loci which were polymorphic between the parents and were segregated among 148 DH population.A chi-square test identified 301 loci with normal segregation（P≥0.01）and 96 loci（24.18%）with low-level skewed segregation（0.001≤P<0.01）.The genetic map was constructed using regression analysis and Haldane’s function after a total of 92 loci were assembled into three linkage groups（LGs）at LOD 7.0,7.0 and 4.0.The map spanned 557.36 c M covering 93.71%of the estimated genome,with a mean interlocus space of 6.23 c M.The number of LGs in the genetic linkage map was equal to the chromosome number of the haploid genome（3chromosomes）of P.yezoensis.The length of the three LGs were 206.52 c M,120.03 c M and 230.81 c M,respectively,and the marker numbers were 28,26 and 38,respectively.There were six segregation distortion regions containing 2-3 clustered distorted markers with the same skew directions after compared with the genotypes of the parental strains.These regions may contain segregation distortion related genes.Based on the total data set of the mapping population,the average estimate of expected genome length was597.74 c M.Thus,the map covered 93.71%of the estimated genome of P.yezoensis.Kolmogorov-Smirnov test（α=5%）showed a uniform distribution of the markers along each LG.Therefore,these results indicated that the genetic map could be used for primary mapping of QTLs.Result 5.QTL analysis of economic traits of blades.Based on the phenotypic values of six economic traits of F₁ gametophytic blades of the mapping population and the information of the genetic map,10 QTLs associated with five economic traits of blades were detected.One QTL was for length,one for width,two for fresh weight,two for specific growth rate of length and four for specific growth rate of fresh weight.These QTLs could explain 2.29%-7.87%of the trait variations,indicating that their effects were all minor.LOD values of the 10 QTLs ranged 1.6-2.3,and three of them were≥2.0.Eight of the 10 QTLs had positive values of additive effect,indicating that their favorable alleles originate from the maternal parent.The remaining two QTLs had negative Add values,indicating that their favorable alleles originate from the paternal parent.The interval of confidence（IC）was 3.0-7.0 c M in seven QTLs and 15.0-20.0 c M in the other three QTLs,in which four QTLs whose IC were less than 5.0 c M could be used for further fine mapping of QTL.Six QTLs in the present study with distances less than 1.0 c M from the nearest markers could be further used for MAS breeding.In conclusion,the first DH mapping population and SRAP genetic linkage map constructed in the present study provided a framework for linkage analysis and QTL detection in P.yezoensis and other monoecious Pyropia species.By saturating the map and validating these QTLs,functional markers could be identified or converted for future marker-assisted breeding.